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folding of proteins into organized three - dimensional structures capable of fulfilling a biological function is determined by the sequence of amino acids and is believed to proceed hierarchically . the emergence of secondary - structure elements constitutes an early event in the chronology of folding , which prefaces the ultimate collapse into well - defined , compact , functional entities . formation of stretches of secondary structure , the elementary bricks of the protein scaffold , therefore , represents an important milestone on the folding pathway , and a convenient framework to investigate the basic physical principles that underlie protein folding notably how do elements of secondary structure nucleate and further propagate into an ordered structure , and to what extent is the organization of the peptide chain collective . understanding this key biological process at the theoretical level has greatly benefited from the recent development of novel , dedicated computer architectures and the unbridled race to model larger proteins over longer time scales . brute - force simulations have now reached a cruising speed that can fold proteins as large as 100 amino - acid residues over tens to hundreds of microseconds , still at the price of substantial computational effort . a number of unbiased , all - atom simulations in explicit solvent have proven successful to illuminate the hierarchical nature of folding , shedding light on the possible pathways that connect a random coil to a functional three - dimensional structure . substantially shorter importance - sampling simulations relying on simpler models consisting of short , organized peptide segments can , however , provide valuable insight into the physical and evolutionary principles that govern the intricate conformational transition of a disordered protein chain into a properly folded one . among suitable candidates of secondary - structure elements for biased simulations are -helices , the most prevalent motif observed in proteins , stabilized by intramolecular interactions , notably through the formation of a hydrogen bond between the carbonyl moiety of the ith residue and the amino moiety of the i+4th residue . owing to its noteworthy propensity to form -helices , alanine has been the amino acid of predilection in theoretical investigations of conformational equilibria in short peptide segments . alanine - rich peptides flanked by titratable residues have also been utilized abundantly at the experimental level to decipher the transition pathway from a disorganized chain to a nascent chain to an ultimately folded -helix . in particular , they were at the center of a controversy on the existence of 310-helices , a secondary - structure motif arising from the formation of hydrogen bonds between the ith and the i+3-th residues of the peptide chain , conjectured to act as an observable intermediate in the conformational transition toward the -helical state . turning to importance - sampling simulations naturally raises the question of an appropriate choice of a transition coordinate , capable of describing folding of the peptide chain into a well - ordered secondary structure . even for appreciably short segments , this choice remains an intricate problem , deeply rooted in the large number of degrees of freedom that vary concurrently as the peptide chain evolves toward its native , organized conformation . much of this intricacy lies in the multidimensionality of the true reaction coordinate , thwarting naive attempts to resort to a limited number of geometric variables , often of low collectivity . fruitful application of collective - variable - based methods rests in large measure upon the fragile hypothesis of time scale separation of slow degrees of freedom , in connection with the reaction coordinate , as well as all other hard , fast degrees of freedom . mapping the free - energy landscape that underlies the folding of a short peptide , therefore , ultimately reduces to either selecting a few relevant collective variables or throwing into the model a plethora of order parameters to describe the multidimensional transition space . the daunting nature of this task explains why biased simulations of complex , intertwined conformational changes remain scarce . in the present contribution , we revisit the paradigmatic capped decamer of alanine , henceforth referred to as deca - alanine . deca - alanine has served on various occasions as a methodological proof of concept , in particular in nonequilibrium work simulations in conjunction with the jarzynski identity , and equilibrium free - energy calculations relying upon the application of a time - dependent bias . notwithstanding their rudimentary character , model peptides like deca - alanine offer valuable thermodynamic and kinetic information on folding , under the assumption that a reasonable , nonambiguous transition coordinate can be designed which is necessarily subservient to the length of the peptide chain . they also help shed light on common shortcomings of importance - sampling simulations of low - dimensionality , notably hidden barriers in orthogonal space , and have proven useful for devising remedies . beyond their undeniable utility in methodological developments , they are also sufficiently simple to serve as models of the nascent chain in more realistic biological applications , like the coupled folding translocation occurring in the secy complex . here , we extend the exploration of reversible extension of deca - alanine in vacuo by examining how the aqueous environment reshapes the free - energy landscape that underlies folding . we find that the range of conformational states explored in water is much greater than in a vacuum , making end - to - end distance a highly degenerate transition coordinate . however , by adding a second coordinate describing the helicity of deca - alanine , we demonstrate that its folding pathway in water is more intricate than in a vacuum . simulations of deca - alanine ( ala10 ) were performed using the 104-atom compact helical model used by park et al . , capped with an acetylated n - terminus and amidated c - terminus , as a starting state , with all hydrogens defined explicitly . for simulations in explicit water , the visualization and analysis program vmd was used to place deca - alanine in a cube of 10 850 tip3p water molecules with dimensions 70 70 70 for a total of 32 659 atoms . molecular dynamics simulations were carried out using namd 2.9 with the charmm all - atom force fields ( charmm22/cmap and charmm36 ) . the temperature was fixed at 300 k using langevin dynamics ; the pressure was kept constant at 1 atm using the langevin piston method . the equations of motion were integrated using the respa multiple time - step algorithm with a time step of 2 fs used for all bonded interactions , 2 fs for short - range nonbonded interactions , and 4 fs for long - range electrostatic interactions . long - range electrostatic interactions were calculated using the particle - mesh ewald method . bonds involving hydrogen atoms were constrained to their equilibrium length , employing the rattle algorithm . pmfs were calculated using both adaptive biasing forces ( abf ) and umbrella sampling ( us ) with the weighted histogram analysis method ( wham ) , utilizing the collective variables ( colvars ) module of namd 2.9 . two reaction coordinates were defined : ( ) the distance from the carbonyl carbon of the backbone of the first residue to the carbonyl carbon of the last residue and ( ) the -helical content of all 10 alanine residues as defined in the colvars module of namd . the colvar is calculated using a scoring function for the backbone i , i + 4 hydrogen bonding , and the dihedral angles compared to that of a pure -helix , normalized between 0 and 1 . the default parameters for the colvar as defined in the colvars module were used in all simulations . for two - dimensional pmfs in water , replica - exchange molecular dynamics was utilized with us ( remd - us ) to increase the sampling efficiency of the entire conformational space . integration of the 2d pmf to obtain a 1d pmf was calculated according to the following equation:1where = ( kbt ) , kb is the boltzmann constant , t is the temperature , w(x , y ) is the 2d pmf , w(x ) the corresponding 1d pmf , and ( xc , yc ) is an arbitrary point in the collective - variable space . along the end - to - end distance coordinate , 20 us windows centered at = 12.5 , 13.5 , ... , 31.5 were used with a force constant of 5.0 kcal / mol for each window . along the -helical content coordinate , nine us windows centered at = 0.1 , 0.2 , ... , 0.9 were used in vacuum and 17 us windows centered at = 0.1 , 0.15 , ... , 0.9 were used in water with a force constant of 500.0 kcal/mol for each window . in vacuum , us windows were simulated for 510 ns per window for one - dimensional pmfs and 15 ns per window for two - dimensional pmfs . in water , us windows were simulated for 5 ns per window for one - dimensional pmfs and 20 ns per window for two - dimensional pmfs . the first 12 ns were not included in the pmf calculations to ensure the system was in equilibrium . all abf simulations used a threshold of 500 samples ( fullsamples ) prior to the application of the bias , unless noted otherwise . starting states along each reaction coordinate were generated either using steered molecular dynamics ( smd ) or from one - dimensional unrestrained abf trajectories . to examine the efficacy of our methods , we first determined the pmf of deca - alanine in vacuum using the end - to - end distance reaction coordinate , denoted . using both the us and abf approaches ( see methods ) , we calculated the pmf with the charmm22/cmap and charmm36 force fields . the two approaches yield nearly identical free - energy profiles for both force fields ( figure 1 ) . examination of the simulation trajectories shows that both methods produced only the accordion - like folding / refolding mechanism as shown in figure 2 , where unfolding begins at one end of the peptide and propagates to the other end , suggesting a cooperative folding mechanism . one - dimensional pmf of ala10 in vacuum using the distance from the n - terminus carbonyl carbon to the c - terminus carbonyl carbon as the reaction coordinate . red lines represent calculations using abf , and blue lines represent calculations using us , with solid lines utilizing the charmm36 force field and dashed lines utilizing the charmm22/cmap force field . unfolding mechanism of ala10 was generated using smd by pulling on the c - terminus c while keeping the n - terminus c fixed . three snapshots of the peptide are shown in various stages of the smd simulations , drawn using the licorice representation for all atoms and a cartoon representation for the backbone structure , where the thick ribbon represents those residues which are in an -helical state . the top image represents the initial , minimized crystal structure of ala10 used as the starting state . the middle image represents an intermediate state in which the peptide is partially extended while the remaining portion of the peptide is still in an -helical state . the results of the charmm36 force field agree quite well with previously reported pmfs . there is only one stable conformation around = 14.2 , which corresponds to the pure -helical state , and there is an energy barrier of 25 kcal / mol from the helical to extended state ( figure 1 ) . while the charmm22/cmap force field does yield a minimum near the -helical state , the entire pmf is shifted by 2 toward lower end - to - end distances , and the energy barrier between helical and extended states is slightly higher ( 30 kcal / mol ) . the corrections to the charmm22/cmap force field added to the charmm36 force field are evident in the difference in the folding pmfs for ala10 . since charmm36 reproduces the expected free - energy minimum for the -helical state in a vacuum and significantly improves agreement with helix - formation experiments , from here on we used solely the charmm36 force field . the abf and us simulations of ala10 were then repeated in explicit water . both methods yield a relatively flat pmf , compared to the vacuum pmf , along most of the reaction coordinate ( figure 3 , thick , solid lines ) . the trajectories reveal that there is no longer only the folding / refolding mechanism seen in vacuum ; instead , the peptide transitions between extended states and compact , but nonhelical , states . figure 5 shows the prevalence of low - helical , compact states in water as opposed to vacuum , which are contaminating the pmf at low end - to - end distances . one - dimensional pmf of ala10 along the end - to - end distance of the peptide calculated using abf ( top ) and us ( bottom ) . top graph : no additional restraints ( thick , solid line ) , no restraints with 50 000 fullsamples ( thick , dashed line ) , 8 - radial confinement ( thin , dashed line ) , 10 - radial confinement plus antihairpin restraint ( thin , dotted - dashed line ) . bottom graph : no additional restraints ( thick , solid line ) , 10 - radial confinement ( thin , dashed line ) , 10 - radial confinement plus antihairpin restraint ( thin , dotted - dashed line ) . the ala10 peptide is shown in the licorice representation with the backbone -helical content represented in orange by a cartoon representation . scatter plot of states from abf simulations in vacuum ( top ) and water ( bottom ) . top graph : scatter plot of states from 50 ns abf simulation in vacuum using the charmm36 force field . bottom graph : scatter plot of states from 100-ns abf simulation in water with 10 - radial confinement plus antihairpin restraint . in order to enforce the -helical folding / refolding mechanism observed in vacuum , multiple additional restraints were imposed . first , the peptide backbone was confined to a cylindrical tube of radius 10 centered along the end - to - end distance vector . this confinement , as well as a smaller tube of radius 8 , failed to prevent the formation of compact nonhelical states , and the pmfs produced were either unchanged or inconsistent ( figure 3 , dashed lines ) , likely because convergence was not achieved . an additional antihairpin restraint , which prevents the backbone c s from passing one another in relation to the end - to - end distance vector , was also insufficient to produce the simple folding / refolding mechanism ( figure 3 , dotted - dashed lines ) . the persistent formation by ala10 of these nonhelical , compact states from extended states reveals a more dynamic folding process than that seen in a vacuum . indeed , previous simulations of ala10 have shown that the disordered and extended states are much more soluble in water than the -helix . instead of running the 1d us simulations longer , because the prevalence of compact , nonhelical states makes it difficult to determine when convergence will be achieved , we switched to a 2d description to ensure adequate sampling of these additional states . to examine the effects of compact , nonhelical states on the free energy of ala10 folding , we calculated a two - dimensional pmf using us , with -helical content as a second reaction coordinate . we first verified this 2d description by calculating the pmf in vacuum with umbrella sampling ( figure 6 , top ) . there is still only one minimum in the pure -helical state , as was seen in the 1d pmf . in addition , we calculated the least free - energy path , which finds the path of least free - energy difference between two local minima on a 2d free - energy surface , from the -helical state to an extended state . there is close agreement between this path ( figure 6 , top inset ) and the 1d pmf ( figure 1 ) , suggesting that the folding / refolding mechanism observed in the 1d biased simulations is in fact the primary mechanism of folding for ala10 in vacuum . two - dimensional pmf of ala10 in vacuum ( top ) and water ( bottom ) using end - to - end distance and -helical content as the two reaction coordinates . green line represents the least free - energy path from the -helical state to the extended state . the inset shows the pmf along the least free - energy path , as projected onto the end - to - end distance coordinate . on the basis of the successful application to the vacuum case , a 2d remd - us ( see methods ) simulation was performed for ala10 in water . trough has appeared along a family of extended , nonhelical states ( figure 6 , bottom ) . these states are also of free energy comparable to the pure -helix , differing by less than 1 kcal / mol , and the energy barrier between the helical and extended states is now less than 4 kcal / mol . the least free - energy path explores a wider range of extended states before refolding compared with the vacuum case . one notable feature of the 2d pmf is the appearance of bands in the free energy along lines of constant helical content , which were presumed to be indications of poor overlap between neighboring windows when implementing the wham algorithm . the poor overlap was confirmed by plotting the histograms ( data not shown ) , and thus , the number of windows along the coordinate was increased from 9 to 17 ( see methods ) . however , the bands still remained as seen in figure 6 , bottom graph . these can be seen more explicitly in the pmf along the least free - energy path , which shows five distinct local minima between the -helical state and the fully extended state ( figure 6 , bottom inset ) . there is less than 1 kcal / mol difference between pmfs calculated for 15 ns per window and 20 ns per window for the entire range of our reaction coordinates , which suggests that the pmfs are converged . to validate the path as well as our choice of reaction coordinates , we also made a rough estimate of the committor distribution for the free - energy maximum . fifty separate conformations were each used to initiate 50 10-ps simulations ( 2500 simulations and 25 ns in total ) with random velocities . the committor was judged to be progressing to the extended state or retreating to the helical state based on the values of and ( see figure 6 , bottom ) although full commitment to either minimum would require time scales at least 100 as long ( see below ) . the resulting distribution is peaked at 0.5 , with some bias toward values greater than 0.5 ( see figure s1 ) . overall , the behavior of the committor near the barrier suggests that it is representative of a true transition state . equilibrium simulations of deca - alanine in water were performed starting from different states for 50 ns each to validate the 2d pmf and to better understand the folding pathway . starting from a state that is near the -helical minimum observed in the 2d pmf , in equilibrium the peptide initially samples the region around the minimum . as the simulation progresses , the peptide begins to unfold roughly along the least free - energy path , and similar bands as seen in the pmf also appear in the histogram , despite no biasing being applied ( figure 7 ) . the protein folds and refolds until finally becoming fully extended near the end of the 50-ns simulation . histograms of 50 ns equilibrium simulations for an -helical ( top ) and extended ( bottom ) starting states . starting from an extended state , ala10 explores the range of extended and compact nonhelical states predicted by the free - energy trough seen in the 2d pmf . the peptide eventually folds into an -helix near the end of the simulation in much the same manner as in the unfolding case . examination of the hydrogen bonding of ala10 with itself and with water during the equilibrium simulations reveals that the transition between helical and extended states occurs in 5 ns with the formation or breaking of 4 peptide peptide hydrogen bonds , with a corresponding decrease or increase in water peptide hydrogen bonds , respectively ( figure s2 ) , in agreement with the results of ozer et al . peptide hydrogen bonds from the remd - us trajectories , with an increase of 810 hydrogen bonds from folded to extended states ( figure s3 ) . based on the success of the two previous equilibrium simulations , we ran 20 additional simulations to get better sampling of the folding mechanism : 10 starting from the -helical minimum and 10 starting from the extended minimum . with only two exceptions , all initially extended states also sampled the helical state during the 50 ns simulations , while all initially -helical states sampled the extended states as well . the histograms for these simulations ( figure s4 ) are practically identical to one another , demonstrating the reproducibility of the conformational equilibria predicted by the pmfs . examination of the hydrogen bonding between the ith residue and the i+4th residue for the simulations in which folding was observed reveals some cooperativity at the n - terminus , where the formation of the ala1ala5 hydrogen bond initiates a propagation of hydrogen bond formation toward the c - terminus as the peptide folds from an extended state to an -helical state ( figure s5 , left graphs ) . in contrast , the c - terminus exhibits less cooperativity , with unfolding typically beginning at the c - terminus and propagating in the opposite direction of folding in the majority of our simulations ( figure s5 , right graphs ) . on average , the n - terminal hydrogen bonds persist longer than those on the c - terminal side while the protein is in a folded conformation . these results are consistent with a previous study which observed that the n - terminus of ala10 slightly favors the -helical conformation over the -sheet conformation , whereas the opposite was observed for the c - terminus . we do observe , however , cases in which the n - terminal hydrogen bonds were broken while the c - terminal hydrogen bonds remained intact ( figure s5 , bottom right graph ) , although these occurred much less frequently . the remd - us trajectories of ala10 in water yields a similar preference for n - terminal hydrogen bond formation ( figure s6 ) near the -helical state . the n - terminal hydrogen bonds also persist for a longer portion of the unfolding pathway ( figure 6 , bottom graph ) than the c - terminal hydrogen bonds . in addition , we observed very little 310-helical ( i , i + 3 ) hydrogen bonding for both the remd - us and equilibrium trajectories ( data not shown ) , confirming that the 310-helix is not a folding intermediate . thus , the folding pathway consists of the breaking or formation of -helical hydrogen bonds and not the rearrangement of those hydrogen bonds into a 310-helical structure . after validation of the free energy minima observed in the 2d pmf of ala10 folding in water by equilibrium simulations , we integrated out the coordinate according to eq 1 ( see methods ) to generate a 1d pmf along the distance coordinate ( figure 8) . this integrated pmf still yields the free - energy minimum observed for ala10 in vacuum around = 14.3 . the main difference between the integrated pmf in figure 8 and the pmf in vacuum ( figure 1 ) is in the unfolding region ( > 15 ) , with the pmf reduced by more than 20 kcal / mol in the extended state . this reduction is comparable to that seen in the previous unrestrained 1d pmfs calculated for ala10 ( see figure 3 ) . however , by ensuring that ala10 more fully explores its entire conformational space through biasing of the additional reaction coordinate , two free energy minima are revealed in the compact states and extended states , respectively , that were not found by biasing of the reaction coordinate alone . the appearance of these new minima supports the suggestion that the previous 1d pmfs had not yet converged . calculation of the free - energy difference between these two minima establishes that the compact state is slightly favored over the extended states ( g = 0.4 kcal / mol ) , with compact states defined as 16.75 , i.e. , below the peak of the energy barrier between the minima . one - dimensional pmf of ala10 in water calculated by integration of the 2d pmf using eq 1 . the free energy of folding for ala10 in vacuum has been used as a benchmark for many free - energy calculation methods , using the end - to - end distance ( ) of the peptide as the reaction coordinate . this choice of reaction coordinate implicitly presumes a reversible , accordion - like folding / refolding of the peptide . we observed using both us and abf that in vacuum , this presumption is indeed correct and only the simple folding / unfolding mechanism predicted is found . in the presence of water , however , the folding / unfolding mechanism is much more complex , and biasing the refolding of ala10 back into an -helix from an extended state is nontrivial . using multiple biasing methods us and abf we discovered that the water - solvated ala10 will explore an extended range of compact , nonhelical states before refolding back into an -helix . these compact , nonhelical states appeared to be contaminating the low- region of the 1d pmfs calculated from the two biasing methods creating a relatively flat pmf compared to the pmf calculated for ala10 in vacuum ( figure 3 ) . calculation of 2d pmfs for the entire ( , ) collective - variable space revealed a new free - energy minimum in a family of extended states not observed in vacuum , along with the -helical minimum that was originally observed in vacuum . in water , the free energy of the extended states decreased significantly compared to in vacuum , with the -helical state less than 1 kcal / mol lower in energy than the extended states a decrease from more than 20 kcal / mol observed in vacuum . a barrier of 4 kcal / mol between the two energy minima is also observed in the pmf . previous studies of ala10 in water have also shown a decrease in the free - energy difference between extended and helical states . for example , abf was applied to a zwitterionic form of ala10 with charged termini to calculate a 1d pmf in water , using the average length of the i , i + 4 hydrogen bonds of the backbone as a reaction coordinate . that pmf shows a comparable free - energy barrier of 5 kcal / mol between the extended and helical states , with the relative energies differing by 1 kcal / mol . the decrease in the free - energy difference was not as pronounced in other studies utilizing adaptive smd and us applied to the end - to - end distance of ala10 with neutral termini . additionally , neither study discovered the free - energy minimum in the extended states . levy et al . found that in hydrophilic environments , the -helix is actually destabilized relative to -sheet conformations , due to their high conformational entropy compared to that of the -helix . although we observed a minimum in extended states rather than -sheet conformations , the extended states are similarly entropically favored over the -helical state and extend into the range of compact , nonhelical states ( figure 6 ) . as a check on our 2d pmf , we performed 50 ns equilibrium simulations of ala10 in water starting from -helical and extended states ( figure 7 ) . furthermore , transitions between the two minima demonstrated cooperativity at the n - terminus and noncooperativity at the c - terminus , as expected . although alanine is used as a model for protein folding since it has the highest helix propensity of all amino acids , the folding mechanism for ala - based peptides is still not very well understood . experiments studying short ala - based peptides have led to inconclusive or contradictory observations for the stability of the -helical state in water . rohl et al . observed that ala - based peptides are the only stable helix formers in water for the 20 common amino acids . they postulated that reducing the extent of solvation of the coiled backbone could increase stabilization of the helix . experiments performed by blondelle et al . showed that peptides of the form ac kyank nh2 ( 10 n 14 ) coexisted as a -sheet and -helix to varying extents . however , it was later observed that the stability of the helix in these peptides was due to the solubility of the flanking lys residues , and not the intrinsic helix propensity of ala . this was followed up by spek et al . stating that although the increase in -content of kank is an artifact of the flanking lys residues , ala is intrinsically -helix stabilizing . so , although there is some discrepancy for the stability of secondary structures for ala - based peptides , the evidence suggests that these peptides do not solely exist as an -helix in solution as one might suspect based on its strong preference for the helical state in vacuum . instead they exist in some combination of secondary structures , including -helices and -sheets . indeed , more recently , nmr data for short polyalanine peptides ( ala37 ) show that these peptides exist primarily as polyproline ii ( ppii ) helix - like structures with very little population of the -helical structure . our results detail the stability , or lack thereof , of the -helix for short polyalanine peptides in solution . however , one should always be aware of the limitations of the force fields utilized in md simulations . best et al . examined a range of force fields and observed that they overemphasized the -helix structure compared with nmr coupling parameters for the backbone ( , ) dihedrals . by reweighting the force fields based on these ( , ) coupling parameters , they were able to yield good agreement with the population of the -helix , -sheet , and ppii structures seen in the nmr data . although there was better agreement with nmr for unblocked ( ionic or zwitterionic ) termini than with blocked ( neutral ) termini , by using the reweighting for unblocked peptides , blocked peptides were found to yield -content of 1223% . similar results were found using agadir , an empirical nmr - based algorithm that determines the -helical propensity of a peptide based on sequence , which yields a helical propensity of 15% for ac ala10nh2 in water at 300 k. in this study , we have used the most recent iteration of the charmm force field , charmm36 , introduced in 2012 . one of the major improvements of charmm36 is the correction of the -helical bias introduced into charmm22 by the backbone ( , ) dihedral cmap potential . this improvement was achieved by capturing the many - body effects not present in the original cmap potential . the cmap potential was optimized to match nmr data for ala5 and ac-(aaqaa)3-nh2 in solution , and the side - chain 1 and 2 the result is a better balance between secondary structures , particularly the -helix and -sheet , addressing the problem posed originally by best et al . in 2008 . in particular , the helical fraction of ac-(aaqaa)3-nh2 produced by charmm36 more closely matches experiments than other force fields ( a reduction from 95% for charmm22/cmap to 21% for charmm36 ) , as well as improved cooperativity for -helix and -sheet formation . thus , the helical fraction of ala10 , as well as its folding mechanism , determined using charmm36 should also have better agreement with experimental results , particularly when compared with charmm22 and charmm22/cmap , which were utilized in previous unfolding simulations of ala10 in water . how the balance between conformational changes in the presence of other peptides and proteins occurs remains uncertain . while it is known that macromolecular crowding can have a significant effect on protein folding in vivo , a recent study on dipeptide aggregation demonstrated that simulations still have some difficulty reproducing such effects quantitatively finally , our work emphasizes the challenge and necessity of choosing relevant reaction coordinates to fully characterize a particular system . for ala10 , the end - to - end distance is no longer sufficient to capture the diversity of conformations explored in water , thus making it a highly degenerate reaction coordinate ( figure 4 ) . contributions from compact , nonhelical states can produce a pmf that does not reveal what one intuitively expects it to , namely the accordion - like folding / refolding mechanism shown in figure 2 . by tracking the -helical content of ala10 during biased folding simulations , we found many states accessible to the peptide in water that were inaccessible in vacuum . these states arise due to ala10 s increased flexibility in water , where a loss of intrapeptide hydrogen bonds is compensated by an increase in peptide - water hydrogen bonds ( figures s2 , s3 ) . reaction coordinates suitable for ala10 in vacuum , therefore , may not be suitable in water . since recent studies of ala10 folding in water have only used the end - to - end distance to characterize the folding pathway , they observe that the preference for the -helical state is still significant in water compared to the extended states . however , we have shown that the helical and extended states are of comparable stability ( |g| < 1 kcal / mol ) , with both states transitioning from one to the other within the course of 50 ns equilibrium simulations .

the determination of the folding dynamics of polypeptides and proteins is critical in characterizing their functions in biological systems . numerous computational models and methods have been developed for studying structure formation at the atomic level . due to its small size and simple structure , deca - alanine is used as a model system in molecular dynamics ( md ) simulations . the free energy of unfolding in vacuum has been studied extensively using the end - to - end distance of the peptide as the reaction coordinate . however , few studies have been conducted in the presence of explicit solvent . previous results show a significant decrease in the free energy of extended conformations in water , but the -helical state is still notably favored over the extended state . although sufficient in vacuum , we show that end - to - end distance is incapable of capturing the full complexity of deca - alanine folding in water . using -helical content as a second reaction coordinate , we deduce a more descriptive free - energy landscape , one which reveals a second energy minimum in the extended conformations that is of comparable free energy to the -helical state . equilibrium simulations demonstrate the relative stability of the extended and -helical states in water as well as the transition between the two states . this work reveals both the necessity and challenge of determining a proper reaction coordinate to fully characterize a given process .

Introduction Methods Results Discussion

formation of stretches of secondary structure , the elementary bricks of the protein scaffold , therefore , represents an important milestone on the folding pathway , and a convenient framework to investigate the basic physical principles that underlie protein folding notably how do elements of secondary structure nucleate and further propagate into an ordered structure , and to what extent is the organization of the peptide chain collective . in particular , they were at the center of a controversy on the existence of 310-helices , a secondary - structure motif arising from the formation of hydrogen bonds between the ith and the i+3-th residues of the peptide chain , conjectured to act as an observable intermediate in the conformational transition toward the -helical state . much of this intricacy lies in the multidimensionality of the true reaction coordinate , thwarting naive attempts to resort to a limited number of geometric variables , often of low collectivity . fruitful application of collective - variable - based methods rests in large measure upon the fragile hypothesis of time scale separation of slow degrees of freedom , in connection with the reaction coordinate , as well as all other hard , fast degrees of freedom . mapping the free - energy landscape that underlies the folding of a short peptide , therefore , ultimately reduces to either selecting a few relevant collective variables or throwing into the model a plethora of order parameters to describe the multidimensional transition space . in the present contribution , we revisit the paradigmatic capped decamer of alanine , henceforth referred to as deca - alanine . deca - alanine has served on various occasions as a methodological proof of concept , in particular in nonequilibrium work simulations in conjunction with the jarzynski identity , and equilibrium free - energy calculations relying upon the application of a time - dependent bias . notwithstanding their rudimentary character , model peptides like deca - alanine offer valuable thermodynamic and kinetic information on folding , under the assumption that a reasonable , nonambiguous transition coordinate can be designed which is necessarily subservient to the length of the peptide chain . here , we extend the exploration of reversible extension of deca - alanine in vacuo by examining how the aqueous environment reshapes the free - energy landscape that underlies folding . we find that the range of conformational states explored in water is much greater than in a vacuum , making end - to - end distance a highly degenerate transition coordinate . however , by adding a second coordinate describing the helicity of deca - alanine , we demonstrate that its folding pathway in water is more intricate than in a vacuum . simulations of deca - alanine ( ala10 ) were performed using the 104-atom compact helical model used by park et al . for simulations in explicit water , the visualization and analysis program vmd was used to place deca - alanine in a cube of 10 850 tip3p water molecules with dimensions 70 70 70 for a total of 32 659 atoms . two reaction coordinates were defined : ( ) the distance from the carbonyl carbon of the backbone of the first residue to the carbonyl carbon of the last residue and ( ) the -helical content of all 10 alanine residues as defined in the colvars module of namd . for two - dimensional pmfs in water , replica - exchange molecular dynamics was utilized with us ( remd - us ) to increase the sampling efficiency of the entire conformational space . along the end - to - end distance coordinate , 20 us windows centered at = 12.5 , 13.5 , ... , 31.5 were used with a force constant of 5.0 kcal / mol for each window . along the -helical content coordinate , nine us windows centered at = 0.1 , 0.2 , ... , 0.9 were used in vacuum and 17 us windows centered at = 0.1 , 0.15 , ... , 0.9 were used in water with a force constant of 500.0 kcal/mol for each window . starting states along each reaction coordinate were generated either using steered molecular dynamics ( smd ) or from one - dimensional unrestrained abf trajectories . to examine the efficacy of our methods , we first determined the pmf of deca - alanine in vacuum using the end - to - end distance reaction coordinate , denoted . the two approaches yield nearly identical free - energy profiles for both force fields ( figure 1 ) . one - dimensional pmf of ala10 in vacuum using the distance from the n - terminus carbonyl carbon to the c - terminus carbonyl carbon as the reaction coordinate . three snapshots of the peptide are shown in various stages of the smd simulations , drawn using the licorice representation for all atoms and a cartoon representation for the backbone structure , where the thick ribbon represents those residues which are in an -helical state . the middle image represents an intermediate state in which the peptide is partially extended while the remaining portion of the peptide is still in an -helical state . there is only one stable conformation around = 14.2 , which corresponds to the pure -helical state , and there is an energy barrier of 25 kcal / mol from the helical to extended state ( figure 1 ) . while the charmm22/cmap force field does yield a minimum near the -helical state , the entire pmf is shifted by 2 toward lower end - to - end distances , and the energy barrier between helical and extended states is slightly higher ( 30 kcal / mol ) . the corrections to the charmm22/cmap force field added to the charmm36 force field are evident in the difference in the folding pmfs for ala10 . since charmm36 reproduces the expected free - energy minimum for the -helical state in a vacuum and significantly improves agreement with helix - formation experiments , from here on we used solely the charmm36 force field . both methods yield a relatively flat pmf , compared to the vacuum pmf , along most of the reaction coordinate ( figure 3 , thick , solid lines ) . the trajectories reveal that there is no longer only the folding / refolding mechanism seen in vacuum ; instead , the peptide transitions between extended states and compact , but nonhelical , states . figure 5 shows the prevalence of low - helical , compact states in water as opposed to vacuum , which are contaminating the pmf at low end - to - end distances . one - dimensional pmf of ala10 along the end - to - end distance of the peptide calculated using abf ( top ) and us ( bottom ) . in order to enforce the -helical folding / refolding mechanism observed in vacuum , multiple additional restraints were imposed . first , the peptide backbone was confined to a cylindrical tube of radius 10 centered along the end - to - end distance vector . an additional antihairpin restraint , which prevents the backbone c s from passing one another in relation to the end - to - end distance vector , was also insufficient to produce the simple folding / refolding mechanism ( figure 3 , dotted - dashed lines ) . to examine the effects of compact , nonhelical states on the free energy of ala10 folding , we calculated a two - dimensional pmf using us , with -helical content as a second reaction coordinate . there is still only one minimum in the pure -helical state , as was seen in the 1d pmf . in addition , we calculated the least free - energy path , which finds the path of least free - energy difference between two local minima on a 2d free - energy surface , from the -helical state to an extended state . two - dimensional pmf of ala10 in vacuum ( top ) and water ( bottom ) using end - to - end distance and -helical content as the two reaction coordinates . green line represents the least free - energy path from the -helical state to the extended state . the inset shows the pmf along the least free - energy path , as projected onto the end - to - end distance coordinate . one notable feature of the 2d pmf is the appearance of bands in the free energy along lines of constant helical content , which were presumed to be indications of poor overlap between neighboring windows when implementing the wham algorithm . these can be seen more explicitly in the pmf along the least free - energy path , which shows five distinct local minima between the -helical state and the fully extended state ( figure 6 , bottom inset ) . to validate the path as well as our choice of reaction coordinates , we also made a rough estimate of the committor distribution for the free - energy maximum . equilibrium simulations of deca - alanine in water were performed starting from different states for 50 ns each to validate the 2d pmf and to better understand the folding pathway . starting from a state that is near the -helical minimum observed in the 2d pmf , in equilibrium the peptide initially samples the region around the minimum . as the simulation progresses , the peptide begins to unfold roughly along the least free - energy path , and similar bands as seen in the pmf also appear in the histogram , despite no biasing being applied ( figure 7 ) . starting from an extended state , ala10 explores the range of extended and compact nonhelical states predicted by the free - energy trough seen in the 2d pmf . the peptide eventually folds into an -helix near the end of the simulation in much the same manner as in the unfolding case . examination of the hydrogen bonding of ala10 with itself and with water during the equilibrium simulations reveals that the transition between helical and extended states occurs in 5 ns with the formation or breaking of 4 peptide peptide hydrogen bonds , with a corresponding decrease or increase in water peptide hydrogen bonds , respectively ( figure s2 ) , in agreement with the results of ozer et al . based on the success of the two previous equilibrium simulations , we ran 20 additional simulations to get better sampling of the folding mechanism : 10 starting from the -helical minimum and 10 starting from the extended minimum . examination of the hydrogen bonding between the ith residue and the i+4th residue for the simulations in which folding was observed reveals some cooperativity at the n - terminus , where the formation of the ala1ala5 hydrogen bond initiates a propagation of hydrogen bond formation toward the c - terminus as the peptide folds from an extended state to an -helical state ( figure s5 , left graphs ) . in contrast , the c - terminus exhibits less cooperativity , with unfolding typically beginning at the c - terminus and propagating in the opposite direction of folding in the majority of our simulations ( figure s5 , right graphs ) . after validation of the free energy minima observed in the 2d pmf of ala10 folding in water by equilibrium simulations , we integrated out the coordinate according to eq 1 ( see methods ) to generate a 1d pmf along the distance coordinate ( figure 8) . this integrated pmf still yields the free - energy minimum observed for ala10 in vacuum around = 14.3 . the main difference between the integrated pmf in figure 8 and the pmf in vacuum ( figure 1 ) is in the unfolding region ( > 15 ) , with the pmf reduced by more than 20 kcal / mol in the extended state . however , by ensuring that ala10 more fully explores its entire conformational space through biasing of the additional reaction coordinate , two free energy minima are revealed in the compact states and extended states , respectively , that were not found by biasing of the reaction coordinate alone . calculation of the free - energy difference between these two minima establishes that the compact state is slightly favored over the extended states ( g = 0.4 kcal / mol ) , with compact states defined as 16.75 , i.e. the free energy of folding for ala10 in vacuum has been used as a benchmark for many free - energy calculation methods , using the end - to - end distance ( ) of the peptide as the reaction coordinate . this choice of reaction coordinate implicitly presumes a reversible , accordion - like folding / refolding of the peptide . in the presence of water , however , the folding / unfolding mechanism is much more complex , and biasing the refolding of ala10 back into an -helix from an extended state is nontrivial . these compact , nonhelical states appeared to be contaminating the low- region of the 1d pmfs calculated from the two biasing methods creating a relatively flat pmf compared to the pmf calculated for ala10 in vacuum ( figure 3 ) . calculation of 2d pmfs for the entire ( , ) collective - variable space revealed a new free - energy minimum in a family of extended states not observed in vacuum , along with the -helical minimum that was originally observed in vacuum . in water , the free energy of the extended states decreased significantly compared to in vacuum , with the -helical state less than 1 kcal / mol lower in energy than the extended states a decrease from more than 20 kcal / mol observed in vacuum . a barrier of 4 kcal / mol between the two energy minima is also observed in the pmf . previous studies of ala10 in water have also shown a decrease in the free - energy difference between extended and helical states . for example , abf was applied to a zwitterionic form of ala10 with charged termini to calculate a 1d pmf in water , using the average length of the i , i + 4 hydrogen bonds of the backbone as a reaction coordinate . that pmf shows a comparable free - energy barrier of 5 kcal / mol between the extended and helical states , with the relative energies differing by 1 kcal / mol . the decrease in the free - energy difference was not as pronounced in other studies utilizing adaptive smd and us applied to the end - to - end distance of ala10 with neutral termini . additionally , neither study discovered the free - energy minimum in the extended states . although we observed a minimum in extended states rather than -sheet conformations , the extended states are similarly entropically favored over the -helical state and extend into the range of compact , nonhelical states ( figure 6 ) . as a check on our 2d pmf , we performed 50 ns equilibrium simulations of ala10 in water starting from -helical and extended states ( figure 7 ) . furthermore , transitions between the two minima demonstrated cooperativity at the n - terminus and noncooperativity at the c - terminus , as expected . although alanine is used as a model for protein folding since it has the highest helix propensity of all amino acids , the folding mechanism for ala - based peptides is still not very well understood . experiments studying short ala - based peptides have led to inconclusive or contradictory observations for the stability of the -helical state in water . however , it was later observed that the stability of the helix in these peptides was due to the solubility of the flanking lys residues , and not the intrinsic helix propensity of ala . indeed , more recently , nmr data for short polyalanine peptides ( ala37 ) show that these peptides exist primarily as polyproline ii ( ppii ) helix - like structures with very little population of the -helical structure . similar results were found using agadir , an empirical nmr - based algorithm that determines the -helical propensity of a peptide based on sequence , which yields a helical propensity of 15% for ac ala10nh2 in water at 300 k. in this study , we have used the most recent iteration of the charmm force field , charmm36 , introduced in 2012 . how the balance between conformational changes in the presence of other peptides and proteins occurs remains uncertain . while it is known that macromolecular crowding can have a significant effect on protein folding in vivo , a recent study on dipeptide aggregation demonstrated that simulations still have some difficulty reproducing such effects quantitatively finally , our work emphasizes the challenge and necessity of choosing relevant reaction coordinates to fully characterize a particular system . for ala10 , the end - to - end distance is no longer sufficient to capture the diversity of conformations explored in water , thus making it a highly degenerate reaction coordinate ( figure 4 ) . by tracking the -helical content of ala10 during biased folding simulations , we found many states accessible to the peptide in water that were inaccessible in vacuum . these states arise due to ala10 s increased flexibility in water , where a loss of intrapeptide hydrogen bonds is compensated by an increase in peptide - water hydrogen bonds ( figures s2 , s3 ) . reaction coordinates suitable for ala10 in vacuum , therefore , may not be suitable in water . since recent studies of ala10 folding in water have only used the end - to - end distance to characterize the folding pathway , they observe that the preference for the -helical state is still significant in water compared to the extended states . however , we have shown that the helical and extended states are of comparable stability ( |g| < 1 kcal / mol ) , with both states transitioning from one to the other within the course of 50 ns equilibrium simulations .

diabetic retinopathy is a leading cause of adult blindness and is the most common complication of diabetes . it affects more than 90% of people with diabetes , ultimately leading to retinal edema , neovascularization , and , in some patients , vision loss [ 1 , 2 ] . systemic control of blood glucose can slow down the progression of diabetic retinopathy but fails to stop or reverse clinical signs of it [ 3 , 4 ] . hence understanding the molecular pathways governing the pathophysiology of dr and targeting them is essential to the prevention of catastrophic visual loss arising from vision - threatening complications of diabetic retinopathy such as macular edema , vitreous hemorrhage , and tractional retinal detachment . melanocortins are endogenous peptides that possess a wide range of biological activities , including inhibition of leukocyte activation , promotion of inflammation resolution , and the ensuing tissue protection [ 512 ] . these effects on the immune response are brought about by five distinct melanocortin receptors , termed from mc1 to mc5 , which are ubiquitously expressed except for the mc2 which is localised to the adrenal glands . within the eye , mc3 , mc4 , and mc5 are expressed in the inner neural retinal layers [ 14 , 15 ] , with mc3 and mc4 expression being reported also in the layer of retinal ganglion cells [ 14 , 15 ] . mc5 alone has been detected in the neural outer plexiform layer , whilst mc1 and mc5 are detected in retinal pigment epithelial cells [ 16 , 17 ] . work is limited to the most common melanocortin peptide , -melanocyte stimulating hormone ( -msh ) , which activates all mc receptors ( except mc2 ) , controls the development and neurotrophism of the ocular tissues [ 1820 ] , and exerts protective effects on the retinal vascular endothelial cells [ 21 , 22 ] . the present study aimed at establishing the efficacy of melanocortin peptides in the prevention of dr and characterizing the mc subtypes engaged . we made use of a mouse model of stz - induced dr , an experimental system suitable for replicating the early signs of nonproliferative dr , such as loss of retinal pericytes and capillaries , thickening of the vascular basement membrane and increased vascular permeability . using a combination of biochemical and functional analyses , streptozotocin was purchased from sigma - aldrich ( city , country ) , mtii from bachem ltd . ( saffron walden , essex , uk ) , and shu9119 from phoenix pharmaceuticals ( karlsruhe , germany ) . other compounds were supplied ( bms-470539 , agrp ) or synthetized ( pg-901 , pg20n ) by professor grieco ( university of naples federico ii ) . all compounds were stored at 20c before use and dissolved in sterile pbs , ph 7.4 . this study was performed according to the guidelines of the ethic committee for animal experiments at the second university of naples . c57bl/6 mice ( harlan , italy ) aged 7 to 10 weeks were rendered diabetic with one intraperitoneal injection of stz ( 65 mg / kg , sigma - aldrich , st . louis , mo , usa ) freshly dissolved in 10 mm citrate buffer ( ph 4.5 ) . development of diabetes ( defined by blood glucose greater than 250 mg / dl ) was verified 1 week after the stz injection ( glucometer elite xl ; bayer corp . , blood glucose levels were checked intermittently throughout the study in order to confirm the maintenance of the diabetic condition . c57bl/6 mice were divided into 8 groups ( n = 10 animals per group ) , labelled consecutively from 1 to 10 to repeat the fluorescein angiography ( fag ) to the same animal at each time point considered . mice were randomised into the following experimental groups : ( 1 ) nondiabetic mice ; ( 2 ) diabetic mice ; ( 3 ) diabetic mice treated with intravitreal injection of the mc1 receptor agonist bms-470539 ; ( 4 ) diabetic mice treated with intravitreal injection of the mixed mc3-mc4 receptor agonist mtii ; ( 5 ) diabetic mice treated with mc1 receptor antagonist agouti related protein ( agrp ; ) ; ( 6 ) diabetic mice treated with intravitreal , mc5 agonist pg-901 ; ( 7 ) diabetic mice treated with intravitreal mc3-mc4 receptor antagonist shu9119 , ; ( 8) diabetic mice treated with intravitreal mc5 receptor antagonist pg20n , . in all cases , animals were monitored over a 16-week period for the development of diabetes , with specific analyses at weeks 8 , 12 , and 16 when fluorescein angiography was conducted . at the end of each time course the animals were sacrificed and the eye ball was displaced forward by placing curved forceps around the posterior part and cut in two halves . on one half of each eye the cornea was cut using a sharp blade or scalpel , and the retina was squeezed through the cut together with residual pigment epithelium and lens by applying gentle pressure with the forceps . dissected retina was placed in cooled pbs , freed from nonretinal tissue using the forceps , and immediately frozen in liquid nitrogen and stored at 80c for subsequent biochemical analysis . the other half of each eye was fixed by immersion in 10% neutral buffered formalin and paraffin - embedded for immunohistochemistry . seven days after the development of diabetes the mice were anesthetized by pentobarbital ( 45 mg / kg in saline ) . tropicamide ( 5% ) was instilled into the right eye of each animal , in order to induce dilatation of pupils , and tetracaine ( 1% ) was injected for local anaesthesia . physiological saline or mc receptor ligand preparations ( 5 l volume ) were administered via intravitreal injection into the right eye using sterile syringes fitted with a 30-gauge needle ( microfine ; becton dickinson ag , meylan , france ) , as previously described . the following mc receptor ligands were used , at the indicated dose as selected from the reported publications : bms-470539 , 33 mol ; mtii , 9.3 nmol ; shu 9119 , 9 nmol ; pg-901 , 7.32 nm ; pg20n , 130 nm ; agouti related protein or agrp , 1 g . fag was performed by using a topcon trc-50dx apparatus ( topcon , tokyo , japan ) following intraperitoneal injection of 10% fluorescein sterile solution ( 1 ml / kg body weight , ak - fluor ; akorn , inc . ) . fundus photographs were captured in order to display the retinal vasculature and to evaluate the early typical alterations of diabetic microangiopathy . a commercial kit ( phoenix pharmaceuticals , inc . , karlsruhe , germany ) was used following the manufacturer protocol in order to assess the levels of the protein within the retina of nondiabetic and diabetic mice with retinopathy . total rna was extracted using rneasy plus mini kit ( qiagen , west sussex , uk ) , according to the manufacturer 's instructions . contaminating dna was removed from rna preparations using the ambion turbo dna - free system ( life technologies , paisley , uk ) using manufacturer 's instructions . the concentration and purity of the rna were then analysed using the nandrop nd-1000 ( nanodrop technologies , wilmington , de ) . complementary dna ( cdna ) was obtained by reverse transcription ( rt ) of 1 g of total dnase - treated rna , using the superscript iii reverse transcriptase system ( invitrogen , carlsbad , ca , usa ) and oligo(dt ) primers following manufacturer 's protocol . conventional pcr was performed for detecting the expression of murine mc1r , mc3r , and mc5r genes using cdna ( 150 ng / reaction ) , specific primers ( quantitect primer assays , qiagen , west sussex , uk ) , and thermo scientific 1.1x reddymix pcr master mix ( life technologies , paisley , uk ) . the following amplification profile was applied : 95c for 2 min ; 35 cycles94c for 30 s , 55c for 35 s , and 72c for 65 s , followed by final elongation step at 72c for 5 min . melanocortin receptor expression was quantified using the predesigned quantitect primers ( abi prism 7900 sequence detection system ; applied biosystems inc . ) and 2x power sybr green mastermix ( applied biosystems , thermo fisher scientific inc . , paisley , uk ) . cycle threshold ( ct ) values were measured and calculated by the sequence detector software . relative amounts of mrna in diabetic retinas were normalized to endogenous control ( gapdh ) and to the healthy controls . western blot was performed on the retinal tissues 16 weeks after the onset of diabetes , monitoring the m2 marker , the mannose receptor cd206 , and the m1 marker , the integrin x / cd11c , according to di filippo et al . . retinal samples were homogenized on ice using ripa buffer ( santa cruz biotechnology , milan , italy ) , containing a protease inhibitor cocktail tablet ( roche diagnostics , mannheim , germany ) . protein concentrations were determined using the bio - rad protein assay ( bio - rad laboratories , milan , italy ) . a total of 100 g of proteins were separated on denaturing 8% sds - page and transferred to pvdf membrane . the following primary antibodies were used : anti - m2 mannose receptor ( cd206 ) ( 1 : 400 , abcam , cambridge , uk ) , anti - m1 integrin alpha x / cd11c ( 1 : 200 , bioss , usa ) . donkey anti - rabbit polyclonal igg ( abcam , cambridge , uk ) and goat anti - mouse polyclonal igg ( santa cruz biotechnology , usa ) secondary antibodies were used at concentration of 1 : 1000 and 1 : 2000 , respectively . a specific kit ( ary006 , r&d systems , abingdon , uk ) was used for the simultaneous measurement of the production of a number of pro- and anti - inflammatory cytokines and chemokines from mouse retinas . to assess the levels of occludin and vascular endothelial growth factor ( vegf ) in the retina of diabetic mice , the quantikine elisa kits ( r&d systems , abington , uk ) were used , according to the manufacturer 's protocol . ocular tissue sections ( 5 m ) were serially cut from paraffin - embedded tissue and labelled for the detection of ki-67 by immunohistochemistry , according to previous published protocol . briefly , sections were incubated with primary mouse monoclonal anti - ki67 ( pp-67 ) antibody ( dilution 1 : 250 , abcam , cambridge , uk ) for 30 min at room temperature . sections were then washed with pbs and incubated with biotin - conjugated goat anti - mouse igg secondary antibodies and avidin - biotin peroxidase complex ( dba , milan , italy ) . for the in vivo experiments , statistical analyses were assessed either by student 's t - test ( when only two groups were compared ) or one - way analyses of variance ( anova ) , followed by dunnett 's post hoc test ( more than two experimental groups ) . intraperitoneal injection of stz ( 65 mg / kg ) to c57bl/6 mice caused an elevation of the glycemia levels , which remained almost constant throughout the duration of the 16-week observation ( table 1 ) . these levels were not affected by drug treatment at any of the time points under investigation . in contrast , the endogenous levels of -msh within the retina were significantly reduced after 16 weeks of diabetes ( nondiabetic , 72 ng / ml 7 ; diabetic , 27 ng / ml 11 ) . fluorescein angiography ( fag ) analysis performed over the 16 weeks of diabetes showed structural changes in the retinal vessels with an increased vascular tortuosity and microvascular changes in 8 of the 10 mice analyzed ( data at week 12 ) . indeed , an irregular retinal vessel caliber and microaneurysms were seen at both time points ( figure 1 ) . no signs of deviation from the normal retina vascularization were seen after 8 weeks of diabetes ( figure 1 ) . next we determined the expression patterns of selected mc receptor expression in the retina of diabetic mice that had developed retinopathy , with a focus on mc1 , mc3 , and mc5 receptors since implicated in the process of inflammation and tissue protection . by conventional pcr we could detect the mc1 and mc5 signals , but not the mc3 ( figure 2 ) . using qpcr , mc1 and mc5 displayed a plastic response to diabetes , with elevated expression being quantified by week 16 ( figure 2 ) . intravitreal injections of the mc1 receptor agonist bms-470539 ( 33 mol ) or mc5 receptor agonist pg-901 ( 7.32 nm ) decreased retinal damage , as demonstrated by fag . indeed , regular course and caliber of retinal vessels without microvascular changes or vessel leakage were present at each time point considered after intravitreal injection of mc1 receptor agonist bms-470539 and mc5 receptors agonists pg-901 , as compared to the untreated diabetic mice with retinopathy ( figure 3 ) . to investigated a potential involvement of the mc pathway by endogenous peptides , we tested the effect of receptor antagonists . intravitreal injection of pg20n ( mc5 antagonist ) and agrp ( mc1 antagonist ) worsened the retinal injury with evident changes already after 8 weeks after induction of diabetes . due to the presence of a venous loop ( figure 4 ) and an extensive retinal vessel leakage with progressive dye diffusion ( figure 4 ) at 16 weeks after induction , hyperfluorescent areas were observed after treatment with either of the two compounds . of interest , intravitreal injection of molecules that activate mc3 , like the agonist mtii ( dual mc3-mc4 agonist ) or the antagonist shu9119 ( dual mc3-mc4 antagonist ) , did not produce changes of the microvascular bed into the retina of diabetic mice ( figure 5 ) . in diabetic mice suffering from retinopathy , cellular tight junctions were damaged as demonstrated by the low levels of occludin detected in retinal homogenates ( figure 6 ) . of interest , levels of this cell junction marker progressively decreased with the development of retinopathy , reaching the lowest level detected at week 16 after stz ( figure 6 ) . such a nadir was further reduced following intravitreal injection of the mc1 and mc5 receptor antagonist agrp and pg20n , respectively , over the different time points ( figure 6 ) . conversely , administration of bms-470539 or pg-901 increased the retinal occludin levels at 8 weeks , 12 weeks ( data not shown ) , and 16 weeks , compared to untreated diabetic mice ( p < 0.01 , figure 6 ) , demonstrating a protective effect downstream activation of mc1 or mc5 , respectively . modulation of mc3 signals was without effects ( figure 6 ) . as we observed profound alterations in the microcirculation , visual images complemented by the loss of tight junction proteins ( of which occludin was selected as faithful marker ) , we then measured expression of a fundamental angiogenic factor . retinal levels of vegf were increased ( + 66 3% ) in diabetic mice suffering from retinopathy at 16 weeks after stz ( p < 0.01 versus nondiabetic ; figure 6 ) . these levels were further increased following intravitreal injection of the mc5 antagonist pg20n ( + 35 2.2% on top of diabetic values , p < 0.01 versus diabetic ; figure 6 ) or mc1 antagonist agrp ( + 36.2 1.8% , p < 0.01 versus diabetic ; figure 6 ) . agonism at mc1 or mc5 decreased levels of vegf in the retina of diabetic mice back to levels detected in retinas of untreated diabetic mice ( p < 0.01 ; figure 6 ) . mc3-mc4 appear not be involved in this protective effect , as the compounds mtii and shu9119 failed to modulate vegf levels assayed during the development of retinopathy in diabetic mice ( p > 0.05 ; figure 6 ) . in line with this trend , immunohistochemistry for ki-67 showed a decrease in the percentages of positive stained area / total stained area following intravitreal injection of bms-470539 ( mc1 agonist , 64.2 6% versus diabetic ) or pg-901 ( mc5 agonist , 68.6 7% ) as calculated against the values quantified in week 16 diabetic mice ( figure 7 ) . western blotting of retina homogenates showed that the presence of the cd11c marker for m1 macrophages was increased in diabetic mice at all time points considered , compared with healthy nondiabetic mice ( figure 8) . there was a good correlation between the number of m1 macrophages and the levels of occludin in the retina with an r = 0.9732 . the number of m1 macrophages was further increased by intravitreal injection of the mc1 or mc5 antagonist ( figure 8) . in contrast , intravitreal injection of the mc1 or mc5 agonist in diabetic mice affected by retinopathy decreased the binding for cd11c ( figure 8) . to complement these analyses , intravitreal treatment with bms-470539 or pg-901 increased cd206 detection , when compared to diabetic mice ( figure 8) , in line with the improvement of the ocular signs recorded with the fag . the proinflammatory cytokine il-1 was increased by ~61% in response to the development of retinopathy ( p < 0.01 versus nondiabetic mice without retinopathy ; figure 9 ) . the melanocortin receptor antagonists agrp and pg20n further increased expression levels for il-1 , il-1 , il-6 , mip-1 , mip-2 , and mip-3 ( figure 9 ) . for example , il-1 was increased by 26 2% and 28 1.8% , respectively , for agrp and pg20n ( p < 0.01 versus diabetic ) . administration of bms-470539 or pg-901 significantly diminished il-1 , il-1 , il-6 , mip-1 , mip-2 , and mip-3 in the retina ( p < 0.01 versus diabetic ; figure 9 ) . mtii and shu9119 did not significantly affect the expression of these proinflammatory mediators ( figure 9 ) . a similar trend was observed for the proinflammatory chemokines mip-1 , mip-2 , and mip-3 , where the highest levels were reached in diabetic mice 16 weeks after diabetes induction ( figure 9 ) . modulation of mc1 and mc5 receptor signaling by means of agonists and antagonists significantly modified mip-1 , mip-2 , and mip-3 levels ( figure 9 ) . finally , and of further interest , while il-10 levels were low in the retina of diabetic mice with retinopathy , the expression of this anti - inflammatory cytokine was significantly increased after the injection of either mc1 or mc5 agonist , for example , + 64 7% and + 56 4.2% for bms-470539 and pg-901 , respectively ( p < 0.01 versus diabetic mice ) ( figure 9 ) . in this study we investigated the putative protective effect of intravitreal injection of melanocortin agonists in a model of streptozotocin- ( stz- ) induced diabetic retinopathy ( dr ) in mice . this is in light of the incomplete and still ongoing knowledge of the pathogenetic molecular mechanism underlying dr , the lack of structural , functional , and biochemical studies in human subjects , and the need of new local therapeutic options . the data presented here indicates that mice suffering from prolonged ( 16 weeks ) diabetes develop retinal alterations typical of nonproliferative diabetic retinopathy ( dr ) , such as microaneurysms with irregular vascular course and vessel leakage , appearing from 12 weeks after the onset of diabetes . these alterations were markedly reduced by the intravitreal injection of the mc1 and mc5 melanocortin receptor agonists bms-470539 and pg-901 , respectively . these compounds preserved a regular course and caliber of the vessel with no signs of leakage even 16 weeks after diabetes induction . in contrast , animals treated with the mc1 antagonist agrp , or the mc5 antagonist pg20n , showed worsening of dr clinical signs . as early as 8 weeks after induction of diabetes , approximately 80% of mice treated with mc1mc5 antagonists display appearance of venous loop with marked leakage , due to an increased vascular permeability , and vascular tortuosity . therefore , these antagonists enable identification of a protective melanocortin tone in the eye during dr . dr is clinically divided in two types : nonproliferative ( npdr ) and proliferative ( pdr ) . thus , npdr is characterized by microaneurysms , dot and blot hemorrhages , and , in severe cases , retinal microvascular damage and intraretinal microvascular abnormalities . pdr , on the other hand , is characterized by abnormal retinal neovascularization due to capillary nonperfusion , and retinal ischemia . vascular changes characteristics of diabetic retinopathy in humans have been widely documented in diabetic rats , dogs , and cats [ 3439 ] , including the breakdown of the blood - retinal barrier , damage in nonvascular retinal neurons and mller glial cells , thickening of the capillary basement membrane , reduction in the number of pericytes , and an increase in the number of acellular capillaries [ 3439 ] . the model we used here replicates the early signs of nonproliferative dr , such as loss of retinal pericytes and capillaries , thickening of the vascular basement membrane , and increased vascular permeability , signs that were significantly reduced by activation of retinal mc1 and mc5 receptors . these receptors are part of the 4 receptors that are differentially expressed in the neuroretina layers [ 12 , 16 ] ; as mc3 and mc4 receptors are localised in the layer of retinal ganglion cells , mc5 receptors are expressed in the neural outer plexiform layer , whilst the retinal pigment epithelial cells express mc1 and mc5 receptors [ 16 , 17 ] . they translate the actions of melanocortins in ocular immunity , development , and health , and in neurotrophism of eye tissues [ 1820 ] , together with biomolecular changes relating to cell protection . mc3 and mc5 do not appear to transduce the protective effects of melanocortin peptides , at least in these setting and with respect to the markers under analysis in our setting . dr is a pathology initiated by hyperglycemia which overall ( i ) increases polyol pathway flux ; ( ii ) increases advanced glycation end - product ( age ) formation ; ( iii ) activates protein kinase c ( pkc ) isoforms ; and ( iv ) increases hexosamine pathway flux . these events lead to upregulation of vegf , insulin - like growth factor ( igf ) , angiopoietins ( ang-2 ) , tumor necrosis factor- ( tnf- ) , and il-6 [ 4143 ] , responsible for a florid inflammatory response within the eye . modulation of melanocortin receptor activity did not affect systemic glycemia , suggesting therefore local activation of protective mechanisms . although we can not rule out potential off target activity of the melanocortin receptor agonists that may influence the results , we propose that specific activation / deactivation of inflammatory and oxidative signaling regulated by selective melanocortin receptor agonists may constitute an alternative approach for the treatment of dr , affording protection of retinal vessels , slowing down , and/or preventing the onset of early vascular changes typical of dr . congruent with these effects , and the notion of localised responses , functional actions of mc1mc5 evoked changes in the main mediators of the inflammatory response including cytokines ( such as il-1 , il-1 , il-6 , and il-10 ) and chemokines ( such as mip-1 , mip-2 , and mip-3 ) . these actions were married to changes of the main markers of vessel proliferation such as vegf and ki-67 . this evidence is supported by previous and almost extensive studies on melanocortin activities in a wide range of settings , including their protective effects in chondrocytes [ 44 , 45 ] and human primary cells and in vivo models of diseases like rheumatoid arthritis , colitis , allergic airway inflammation , or ischemia reperfusion injury [ 12 , 16 , 36 , 47 ] . these multiple properties are likely due to the ability of melanocortins to reduce production of proinflammatory cytokines by inhibiting nf-b translocation to the nucleus as demonstrated by manna and aggarwal or production of anti - inflammatory cytokines such as il-10 from monocytes as demonstrated by redondo et al . further support to the data presented and discussed here derives from the protective and anti - inflammatory properties of melanocortins in experimental models of exogenous uveitis [ 20 , 48 ] and of retinal degeneration . retinal mc1mc5 activation also modified the retinal macrophage population commonly associated with the released cytokines , and with the disruption of normal retina structure . in this context we observed a high level of m1 macrophages within the retina of diabetic mice that developed retinopathy , as evidenced by the western blotting performed with the specific m1 marker cd11c , with respect to the mice that developed diabetes only without retinopathy . these macrophages are notoriously characterized by a strong propensity to the production of cytokines and nitric oxide and reactive oxygen , which contribute to the retinal vascular damage . m2 macrophages were abundantly present into the retina after the stimulation of the mc1 or mc5 for 8 weeks . noteworthily , these macrophages are associated with resolution of the immune - inflammatory responses into tissues activated by previous insults [ 50 , 51 ] and into the consequent tissue regeneration . although clinically dr can be managed through the control of the metabolic glucose pathway , we conclude this study by proposing that stimulation of the endogenous melanocortin system in the eye through the local activation of mc1 and mc5 may reduce the retinal damage caused by diabetes . this may be the start of a melanocortin - based therapy for dr , especially when considering that several natural and synthetic melanocortin receptor agonists are under clinical experimentation [ 52 , 53 ] .

we hypothesize that melanocortin receptors ( mc ) could activate tissue protective circuit in a model of streptozotocin- ( stz- ) induced diabetic retinopathy ( dr ) in mice . at 1216 weeks after diabetes induction , fluorescein angiography ( fag ) revealed an approximate incidence of 80% microvascular changes , typical of dr , in the animals , without signs of vascular leakage . occludin progressively decreased in the retina of mice developing retinopathy . qpcr of murine retina revealed expression of two mc receptors , mc1r and mc5r . the intravitreal injection ( 5 l ) of the selective mc1 small molecule agonist bms-470539 ( 33 mol ) and the mc5 peptidomimetic agonist pg-901 ( 7.32 nm ) elicited significant protection with regular course and caliber of retinal vessels , as quantified at weeks 12 and 16 after diabetes induction . mouse retina homogenate settings indicated an augmented release of il-1 , il-1 , il-6 , mip-1 , mip-2 , mip-3 , and vegf from diabetic compared to nondiabetic mice . application of pg20n or agrp and mc5 and mc1 antagonist , respectively , augmented the release of cytokines , while the agonists bms-470539 and pg-901 almost restored normal pattern of these mediators back to nondiabetic values . similar changes were quantified with respect to ki-67 staining . finally , application of mc3-mc4 agonist / antagonists resulted to be inactive with respect to all parameters under assessment .

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

it affects more than 90% of people with diabetes , ultimately leading to retinal edema , neovascularization , and , in some patients , vision loss [ 1 , 2 ] . systemic control of blood glucose can slow down the progression of diabetic retinopathy but fails to stop or reverse clinical signs of it [ 3 , 4 ] . hence understanding the molecular pathways governing the pathophysiology of dr and targeting them is essential to the prevention of catastrophic visual loss arising from vision - threatening complications of diabetic retinopathy such as macular edema , vitreous hemorrhage , and tractional retinal detachment . melanocortins are endogenous peptides that possess a wide range of biological activities , including inhibition of leukocyte activation , promotion of inflammation resolution , and the ensuing tissue protection [ 512 ] . these effects on the immune response are brought about by five distinct melanocortin receptors , termed from mc1 to mc5 , which are ubiquitously expressed except for the mc2 which is localised to the adrenal glands . within the eye , mc3 , mc4 , and mc5 are expressed in the inner neural retinal layers [ 14 , 15 ] , with mc3 and mc4 expression being reported also in the layer of retinal ganglion cells [ 14 , 15 ] . mc5 alone has been detected in the neural outer plexiform layer , whilst mc1 and mc5 are detected in retinal pigment epithelial cells [ 16 , 17 ] . work is limited to the most common melanocortin peptide , -melanocyte stimulating hormone ( -msh ) , which activates all mc receptors ( except mc2 ) , controls the development and neurotrophism of the ocular tissues [ 1820 ] , and exerts protective effects on the retinal vascular endothelial cells [ 21 , 22 ] . the present study aimed at establishing the efficacy of melanocortin peptides in the prevention of dr and characterizing the mc subtypes engaged . we made use of a mouse model of stz - induced dr , an experimental system suitable for replicating the early signs of nonproliferative dr , such as loss of retinal pericytes and capillaries , thickening of the vascular basement membrane and increased vascular permeability . ( saffron walden , essex , uk ) , and shu9119 from phoenix pharmaceuticals ( karlsruhe , germany ) . this study was performed according to the guidelines of the ethic committee for animal experiments at the second university of naples . development of diabetes ( defined by blood glucose greater than 250 mg / dl ) was verified 1 week after the stz injection ( glucometer elite xl ; bayer corp . c57bl/6 mice were divided into 8 groups ( n = 10 animals per group ) , labelled consecutively from 1 to 10 to repeat the fluorescein angiography ( fag ) to the same animal at each time point considered . mice were randomised into the following experimental groups : ( 1 ) nondiabetic mice ; ( 2 ) diabetic mice ; ( 3 ) diabetic mice treated with intravitreal injection of the mc1 receptor agonist bms-470539 ; ( 4 ) diabetic mice treated with intravitreal injection of the mixed mc3-mc4 receptor agonist mtii ; ( 5 ) diabetic mice treated with mc1 receptor antagonist agouti related protein ( agrp ; ) ; ( 6 ) diabetic mice treated with intravitreal , mc5 agonist pg-901 ; ( 7 ) diabetic mice treated with intravitreal mc3-mc4 receptor antagonist shu9119 , ; ( 8) diabetic mice treated with intravitreal mc5 receptor antagonist pg20n , . in all cases , animals were monitored over a 16-week period for the development of diabetes , with specific analyses at weeks 8 , 12 , and 16 when fluorescein angiography was conducted . at the end of each time course the animals were sacrificed and the eye ball was displaced forward by placing curved forceps around the posterior part and cut in two halves . on one half of each eye the cornea was cut using a sharp blade or scalpel , and the retina was squeezed through the cut together with residual pigment epithelium and lens by applying gentle pressure with the forceps . dissected retina was placed in cooled pbs , freed from nonretinal tissue using the forceps , and immediately frozen in liquid nitrogen and stored at 80c for subsequent biochemical analysis . tropicamide ( 5% ) was instilled into the right eye of each animal , in order to induce dilatation of pupils , and tetracaine ( 1% ) was injected for local anaesthesia . physiological saline or mc receptor ligand preparations ( 5 l volume ) were administered via intravitreal injection into the right eye using sterile syringes fitted with a 30-gauge needle ( microfine ; becton dickinson ag , meylan , france ) , as previously described . the following mc receptor ligands were used , at the indicated dose as selected from the reported publications : bms-470539 , 33 mol ; mtii , 9.3 nmol ; shu 9119 , 9 nmol ; pg-901 , 7.32 nm ; pg20n , 130 nm ; agouti related protein or agrp , 1 g . , karlsruhe , germany ) was used following the manufacturer protocol in order to assess the levels of the protein within the retina of nondiabetic and diabetic mice with retinopathy . complementary dna ( cdna ) was obtained by reverse transcription ( rt ) of 1 g of total dnase - treated rna , using the superscript iii reverse transcriptase system ( invitrogen , carlsbad , ca , usa ) and oligo(dt ) primers following manufacturer 's protocol . conventional pcr was performed for detecting the expression of murine mc1r , mc3r , and mc5r genes using cdna ( 150 ng / reaction ) , specific primers ( quantitect primer assays , qiagen , west sussex , uk ) , and thermo scientific 1.1x reddymix pcr master mix ( life technologies , paisley , uk ) . the following amplification profile was applied : 95c for 2 min ; 35 cycles94c for 30 s , 55c for 35 s , and 72c for 65 s , followed by final elongation step at 72c for 5 min . relative amounts of mrna in diabetic retinas were normalized to endogenous control ( gapdh ) and to the healthy controls . western blot was performed on the retinal tissues 16 weeks after the onset of diabetes , monitoring the m2 marker , the mannose receptor cd206 , and the m1 marker , the integrin x / cd11c , according to di filippo et al . donkey anti - rabbit polyclonal igg ( abcam , cambridge , uk ) and goat anti - mouse polyclonal igg ( santa cruz biotechnology , usa ) secondary antibodies were used at concentration of 1 : 1000 and 1 : 2000 , respectively . to assess the levels of occludin and vascular endothelial growth factor ( vegf ) in the retina of diabetic mice , the quantikine elisa kits ( r&d systems , abington , uk ) were used , according to the manufacturer 's protocol . ocular tissue sections ( 5 m ) were serially cut from paraffin - embedded tissue and labelled for the detection of ki-67 by immunohistochemistry , according to previous published protocol . these levels were not affected by drug treatment at any of the time points under investigation . fluorescein angiography ( fag ) analysis performed over the 16 weeks of diabetes showed structural changes in the retinal vessels with an increased vascular tortuosity and microvascular changes in 8 of the 10 mice analyzed ( data at week 12 ) . next we determined the expression patterns of selected mc receptor expression in the retina of diabetic mice that had developed retinopathy , with a focus on mc1 , mc3 , and mc5 receptors since implicated in the process of inflammation and tissue protection . intravitreal injections of the mc1 receptor agonist bms-470539 ( 33 mol ) or mc5 receptor agonist pg-901 ( 7.32 nm ) decreased retinal damage , as demonstrated by fag . indeed , regular course and caliber of retinal vessels without microvascular changes or vessel leakage were present at each time point considered after intravitreal injection of mc1 receptor agonist bms-470539 and mc5 receptors agonists pg-901 , as compared to the untreated diabetic mice with retinopathy ( figure 3 ) . intravitreal injection of pg20n ( mc5 antagonist ) and agrp ( mc1 antagonist ) worsened the retinal injury with evident changes already after 8 weeks after induction of diabetes . due to the presence of a venous loop ( figure 4 ) and an extensive retinal vessel leakage with progressive dye diffusion ( figure 4 ) at 16 weeks after induction , hyperfluorescent areas were observed after treatment with either of the two compounds . of interest , intravitreal injection of molecules that activate mc3 , like the agonist mtii ( dual mc3-mc4 agonist ) or the antagonist shu9119 ( dual mc3-mc4 antagonist ) , did not produce changes of the microvascular bed into the retina of diabetic mice ( figure 5 ) . of interest , levels of this cell junction marker progressively decreased with the development of retinopathy , reaching the lowest level detected at week 16 after stz ( figure 6 ) . such a nadir was further reduced following intravitreal injection of the mc1 and mc5 receptor antagonist agrp and pg20n , respectively , over the different time points ( figure 6 ) . conversely , administration of bms-470539 or pg-901 increased the retinal occludin levels at 8 weeks , 12 weeks ( data not shown ) , and 16 weeks , compared to untreated diabetic mice ( p < 0.01 , figure 6 ) , demonstrating a protective effect downstream activation of mc1 or mc5 , respectively . as we observed profound alterations in the microcirculation , visual images complemented by the loss of tight junction proteins ( of which occludin was selected as faithful marker ) , we then measured expression of a fundamental angiogenic factor . retinal levels of vegf were increased ( + 66 3% ) in diabetic mice suffering from retinopathy at 16 weeks after stz ( p < 0.01 versus nondiabetic ; figure 6 ) . these levels were further increased following intravitreal injection of the mc5 antagonist pg20n ( + 35 2.2% on top of diabetic values , p < 0.01 versus diabetic ; figure 6 ) or mc1 antagonist agrp ( + 36.2 1.8% , p < 0.01 versus diabetic ; figure 6 ) . agonism at mc1 or mc5 decreased levels of vegf in the retina of diabetic mice back to levels detected in retinas of untreated diabetic mice ( p < 0.01 ; figure 6 ) . in line with this trend , immunohistochemistry for ki-67 showed a decrease in the percentages of positive stained area / total stained area following intravitreal injection of bms-470539 ( mc1 agonist , 64.2 6% versus diabetic ) or pg-901 ( mc5 agonist , 68.6 7% ) as calculated against the values quantified in week 16 diabetic mice ( figure 7 ) . western blotting of retina homogenates showed that the presence of the cd11c marker for m1 macrophages was increased in diabetic mice at all time points considered , compared with healthy nondiabetic mice ( figure 8) . there was a good correlation between the number of m1 macrophages and the levels of occludin in the retina with an r = 0.9732 . the number of m1 macrophages was further increased by intravitreal injection of the mc1 or mc5 antagonist ( figure 8) . in contrast , intravitreal injection of the mc1 or mc5 agonist in diabetic mice affected by retinopathy decreased the binding for cd11c ( figure 8) . to complement these analyses , intravitreal treatment with bms-470539 or pg-901 increased cd206 detection , when compared to diabetic mice ( figure 8) , in line with the improvement of the ocular signs recorded with the fag . the proinflammatory cytokine il-1 was increased by ~61% in response to the development of retinopathy ( p < 0.01 versus nondiabetic mice without retinopathy ; figure 9 ) . the melanocortin receptor antagonists agrp and pg20n further increased expression levels for il-1 , il-1 , il-6 , mip-1 , mip-2 , and mip-3 ( figure 9 ) . for example , il-1 was increased by 26 2% and 28 1.8% , respectively , for agrp and pg20n ( p < 0.01 versus diabetic ) . administration of bms-470539 or pg-901 significantly diminished il-1 , il-1 , il-6 , mip-1 , mip-2 , and mip-3 in the retina ( p < 0.01 versus diabetic ; figure 9 ) . mtii and shu9119 did not significantly affect the expression of these proinflammatory mediators ( figure 9 ) . a similar trend was observed for the proinflammatory chemokines mip-1 , mip-2 , and mip-3 , where the highest levels were reached in diabetic mice 16 weeks after diabetes induction ( figure 9 ) . modulation of mc1 and mc5 receptor signaling by means of agonists and antagonists significantly modified mip-1 , mip-2 , and mip-3 levels ( figure 9 ) . finally , and of further interest , while il-10 levels were low in the retina of diabetic mice with retinopathy , the expression of this anti - inflammatory cytokine was significantly increased after the injection of either mc1 or mc5 agonist , for example , + 64 7% and + 56 4.2% for bms-470539 and pg-901 , respectively ( p < 0.01 versus diabetic mice ) ( figure 9 ) . in this study we investigated the putative protective effect of intravitreal injection of melanocortin agonists in a model of streptozotocin- ( stz- ) induced diabetic retinopathy ( dr ) in mice . this is in light of the incomplete and still ongoing knowledge of the pathogenetic molecular mechanism underlying dr , the lack of structural , functional , and biochemical studies in human subjects , and the need of new local therapeutic options . the data presented here indicates that mice suffering from prolonged ( 16 weeks ) diabetes develop retinal alterations typical of nonproliferative diabetic retinopathy ( dr ) , such as microaneurysms with irregular vascular course and vessel leakage , appearing from 12 weeks after the onset of diabetes . these alterations were markedly reduced by the intravitreal injection of the mc1 and mc5 melanocortin receptor agonists bms-470539 and pg-901 , respectively . these compounds preserved a regular course and caliber of the vessel with no signs of leakage even 16 weeks after diabetes induction . in contrast , animals treated with the mc1 antagonist agrp , or the mc5 antagonist pg20n , showed worsening of dr clinical signs . as early as 8 weeks after induction of diabetes , approximately 80% of mice treated with mc1mc5 antagonists display appearance of venous loop with marked leakage , due to an increased vascular permeability , and vascular tortuosity . therefore , these antagonists enable identification of a protective melanocortin tone in the eye during dr . dr is clinically divided in two types : nonproliferative ( npdr ) and proliferative ( pdr ) . thus , npdr is characterized by microaneurysms , dot and blot hemorrhages , and , in severe cases , retinal microvascular damage and intraretinal microvascular abnormalities . vascular changes characteristics of diabetic retinopathy in humans have been widely documented in diabetic rats , dogs , and cats [ 3439 ] , including the breakdown of the blood - retinal barrier , damage in nonvascular retinal neurons and mller glial cells , thickening of the capillary basement membrane , reduction in the number of pericytes , and an increase in the number of acellular capillaries [ 3439 ] . the model we used here replicates the early signs of nonproliferative dr , such as loss of retinal pericytes and capillaries , thickening of the vascular basement membrane , and increased vascular permeability , signs that were significantly reduced by activation of retinal mc1 and mc5 receptors . these receptors are part of the 4 receptors that are differentially expressed in the neuroretina layers [ 12 , 16 ] ; as mc3 and mc4 receptors are localised in the layer of retinal ganglion cells , mc5 receptors are expressed in the neural outer plexiform layer , whilst the retinal pigment epithelial cells express mc1 and mc5 receptors [ 16 , 17 ] . mc3 and mc5 do not appear to transduce the protective effects of melanocortin peptides , at least in these setting and with respect to the markers under analysis in our setting . although we can not rule out potential off target activity of the melanocortin receptor agonists that may influence the results , we propose that specific activation / deactivation of inflammatory and oxidative signaling regulated by selective melanocortin receptor agonists may constitute an alternative approach for the treatment of dr , affording protection of retinal vessels , slowing down , and/or preventing the onset of early vascular changes typical of dr . congruent with these effects , and the notion of localised responses , functional actions of mc1mc5 evoked changes in the main mediators of the inflammatory response including cytokines ( such as il-1 , il-1 , il-6 , and il-10 ) and chemokines ( such as mip-1 , mip-2 , and mip-3 ) . further support to the data presented and discussed here derives from the protective and anti - inflammatory properties of melanocortins in experimental models of exogenous uveitis [ 20 , 48 ] and of retinal degeneration . retinal mc1mc5 activation also modified the retinal macrophage population commonly associated with the released cytokines , and with the disruption of normal retina structure . in this context we observed a high level of m1 macrophages within the retina of diabetic mice that developed retinopathy , as evidenced by the western blotting performed with the specific m1 marker cd11c , with respect to the mice that developed diabetes only without retinopathy . these macrophages are notoriously characterized by a strong propensity to the production of cytokines and nitric oxide and reactive oxygen , which contribute to the retinal vascular damage . m2 macrophages were abundantly present into the retina after the stimulation of the mc1 or mc5 for 8 weeks . although clinically dr can be managed through the control of the metabolic glucose pathway , we conclude this study by proposing that stimulation of the endogenous melanocortin system in the eye through the local activation of mc1 and mc5 may reduce the retinal damage caused by diabetes .

it encourages people to know their hiv status , thus helping to break the chain of transmission of the infection . vct is also a key entry point to care and support services for people living with hiv / aids . vct service utilization is low in ethiopia , only 20% of women and 21% of men have been tested for hiv and received the results in 2005 indicating that many people living with hiv / aids in ethiopia do not know their hiv status . however , to foster attitudes and behaviors that promote counseling and testing among community leaders and in communities , a community - based strategy called community conversation(cc ) was introduced in ethiopia in 2002 . cc is a facilitated interactive process which brings people together and engages them to explore the underlying causes fuelling the hiv / aids epidemics in their environment . it has explicit problem solving agenda , aiming to incite critical thinking that enables people to formulate solutions to local issues . cc is a process for dialogue and decision - making - for communities to investigate into the deep causes of the epidemic in their lives and generate their own solutions . its main purpose is empowerment of communities and individuals to identify and address issues that are important to them . the facilitators of cc in ethiopia are health extension workers ( hews ) who shared the ethnicity , home language , and had grown up in the same district of the cc participants . they conduct cc every 2 weeks for a cross section of 36 - 40 people from the community - men and women , old and young , people living with hiv / aids and those who are not infected , religious , community and traditional leaders , and members of the community at large . a cc participant graduates after completing 24 cc sessions . aided by skilled facilitators from their own communities , people openly talk to each other and explore the implications of hiv / aids , identify their own cultural norms and values that are fuelling the epidemic and the social capital within the community to overcome them . during cc session , the facilitator poses questions related to hiv including but not limited to driving factors for hiv transmission in the community , health , social , economical , and psychological impacts of hiv / aids and role of vct in hiv care and prevention . as problems and solutions are discussed , the facilitator guides the participants to develop concrete action plans . the facilitator convenes additional conversations on an on - going basis to discuss how implementing the action plans is going . though studies that related cc with hiv or vct are scarce , the role of cc in increasing hiv - related competence has been demonstrated in one qualitative study conducted in zimbabwe . also as a result of cc in ethiopia , it is reported that cc participants have travelled long distance to the nearest health facility to get tested for hiv and convinced their partner to get tested . moreover , cc is said to bring positive change with regard to receiving hiv test before marriage . it also is a powerful tool to reduce risk behavior toward hiv and promotes hiv prevention and stigma reduction . however , the role of cc on vct service uptake has not yet been evaluated using sound scientific methods in ethiopia . this study was designed to compare vct service utilization between rural communities with well cc performance and rural communities with poor cc performance in shebedino woreda , south ethiopia . the study was conducted in shebedino woreda , sidamo zone , southern nations nationalities and peoples region , ethiopia in november 2010 . the woreda comprises of 35 kebeles ( 3 urban kebeles and 32 rural kebeles ) . a cross - sectional comparative study was conducted among adult population who were in the age range ; 15 - 59 years in the selected six kebeles ( kebele is the smallest administrative unit ) of the woreda . the study protocol was approved by research ethical committee of the school of public health , addis ababa university . sample size was calculated using epi info sample size calculator ( epi info 3.3.2 , cdc ) for two population proportion by taking proportion of vct service utilization of 20.1% among communities with well cc performance . a 10% difference was assumed between well and poor cc kebeles so that the proportion of vct service utilization among poor cc communities was set at 10.1% . other assumptions considered were : 95% confidence level , 80% power , 5% level of significance , and the ratio of well to poor cc(r ) = 1 . the source population was 15 - 59 years old population residing in rural kebeles implementing cc in shebedino woreda . rural kebeles were classified into well and poor cc groups based on their major activities on cc performance . four major criteria ( cc methodology frame work , number of participants , number of cc groups , number of decisions undertaken , document handling , and reporting ) were considered to classify the study kebeles into well and poor cc groups . accordingly , all kebeles were evaluated out of 10 ( the sum of scores to each activity under the four criteria ) . a kebele was labeled as well performing cc group if its cc score was equal to or greater than the median value of 4.75 ; otherwise , it was labeled as a poor cc performing kebele . detail descriptions on the criteria used to score cc is annexed [ table 1 ] . list of criteria and weight used to classify kebeles into poor and well cc groups out of 35 rural kebeles in the woreda , 25 qualified to be sampled ( seven kebeles had stopped cc ) . the 25 rural kebeles were stratified by their cc status into well ( n = 13 ) and poor ( n = 12 ) . then , kebeles under the two strata ( well and poor ) were further stratified into proximal ( within 5 km ) , medium ( 5 - 10 km ) , and distal ( more than 10 km ) by their distance from the woreda capital . then households from the selected kebeles were selected using systematic random sampling technique through a door to door survey . from the selected households , data were collected from the selected adults in the age bracket of 15 - 59 years . when there were more than one eligible study participants , one was selected by lottery method . same gender interviewers were used to decrease discomfort as some of the questions were about personal sexual lifestyle . data were entered into epi info 3.3.2 as part of data quality management and analyzed using spss version 16.0 . upon checking completeness and consistency of responses , the magnitude of vct service uptake was determined by counting the number of study participants who underwent hiv test on voluntary basis . risky sexual behavior was documented if the study participant had at least one of the following : sex without condom , sex with multiple partners , inconsistent condom use , and sex after alcohol use . to measure knowledge , comprehensive set of knowledge questions a two sample proportion test was used to test the presence of statistical difference in the proportion of vct service utilization between the two cc groups . to determine the association between different predictors and vct service utilization ( dependent variable ) , a logistic regression model was employed . first , each predictor was entered into a separate binary logistic regression model to determine the crude effect of each variable on vct uptake . second , to account for the effect of confounders , a multiple logistic regression model was fitted with all predictors that have p 0.3 in the bivariate model . eventually , only those variables that remained significant at p 0.05 in the final model were retained as independent predictors of vct service uptake . sample size was calculated using epi info sample size calculator ( epi info 3.3.2 , cdc ) for two population proportion by taking proportion of vct service utilization of 20.1% among communities with well cc performance . a 10% difference was assumed between well and poor cc kebeles so that the proportion of vct service utilization among poor cc communities was set at 10.1% . other assumptions considered were : 95% confidence level , 80% power , 5% level of significance , and the ratio of well to poor cc(r ) = 1 . the source population was 15 - 59 years old population residing in rural kebeles implementing cc in shebedino woreda . rural kebeles were classified into well and poor cc groups based on their major activities on cc performance . four major criteria ( cc methodology frame work , number of participants , number of cc groups , number of decisions undertaken , document handling , and reporting ) were considered to classify the study kebeles into well and poor cc groups . accordingly , all kebeles were evaluated out of 10 ( the sum of scores to each activity under the four criteria ) . a kebele was labeled as well performing cc group if its cc score was equal to or greater than the median value of 4.75 ; otherwise , it was labeled as a poor cc performing kebele . detail descriptions on the criteria used to score cc is annexed [ table 1 ] . list of criteria and weight used to classify kebeles into poor and well cc groups out of 35 rural kebeles in the woreda , 25 qualified to be sampled ( seven kebeles had stopped cc ) . the 25 rural kebeles were stratified by their cc status into well ( n = 13 ) and poor ( n = 12 ) . then , kebeles under the two strata ( well and poor ) were further stratified into proximal ( within 5 km ) , medium ( 5 - 10 km ) , and distal ( more than 10 km ) by their distance from the woreda capital . then households from the selected kebeles were selected using systematic random sampling technique through a door to door survey . from the selected households , data were collected from the selected adults in the age bracket of 15 - 59 years . when there were more than one eligible study participants , one was selected by lottery method . same gender interviewers were used to decrease discomfort as some of the questions were about personal sexual lifestyle . data were entered into epi info 3.3.2 as part of data quality management and analyzed using spss version 16.0 . upon checking completeness and consistency of responses , the magnitude of vct service uptake was determined by counting the number of study participants who underwent hiv test on voluntary basis . risky sexual behavior was documented if the study participant had at least one of the following : sex without condom , sex with multiple partners , inconsistent condom use , and sex after alcohol use . to measure knowledge , comprehensive set of knowledge questions a two sample proportion test was used to test the presence of statistical difference in the proportion of vct service utilization between the two cc groups . to determine the association between different predictors and vct service utilization ( dependent variable ) , a logistic regression model was employed . first , each predictor was entered into a separate binary logistic regression model to determine the crude effect of each variable on vct uptake . second , to account for the effect of confounders , a multiple logistic regression model was fitted with all predictors that have p 0.3 in the bivariate model . eventually , only those variables that remained significant at p 0.05 in the final model were retained as independent predictors of vct service uptake . a total of 452 study participants completed the interview giving a response rate of 97.8% . of the 452 who participated in the study , 226 ( 50.0% ) were from well cc performing kebeles and 226 ( 50.0% ) from poor cc performing kebeles . majority of the study participants ; 119 ( 52.7% ) in well and 109 ( 48.2% ) in poor cc performing kebeles were in the age range of 15 - 24 years . with respect to educational status , near to half of the study participants in well ( 44.7% ) and in poor ( 46.9% ) cc groups had no formal education . regarding religion , about [ 183 ( 81.0% ) vs. 180 ( 79.6% ) ] of the study participants were protestant in poor and well cc groups . almost all of the study participants ; 218 ( 96.5% ) in well and 216 ( 95.6% ) in poor cc groups were sidama in ethnicity . married study participants were [ 141 ( 62.4% ) vs. 121 ( 53.5% ) ] followed by single [ 84 ( 37.4% ) vs. 102 ( 45.1% ) ] in poor and well cc groups , respectively . of the study participants ; ( 37.6% vs. 30.5% ) were students in occupation , followed by housewives ( 27.0% vs. 26.5% ) and farmers ( 23.0% vs. 23.7% ) in well and poor cc groups , respectively . majority of the study participants ; [ 146 ( 64.6% ) in well and 160 ( 70.6% ) ] in poor cc performing kebeles had started sexual intercourse by the time of data collection [ table 2 ] . the sociodemographic and sexual history of the study participants to determine the comparability of the study participants between well and poor cc performing kebeles , two sample test of proportion for major sociodemographic factors was computed . accordingly , the study participants in well and poor cc performing kebeles were not statistically different ( p > 0.05 ) by sex , age , religion , ethnicity , education , marital status , occupation , sexual practice , age at first sex , condom utilization , and risky behavior for hiv . therefore , the study participants in well and poor cc performing kebeles were comparable [ table 2 ] . significant number of study participants from well cc group compared with participants from poor cc group knew that vct is important to break the chain of hiv spread ( 97.8% vs.93.8% , p = 0.034 ) . more study participants from well cc performing kebeles utilized hiv voluntary counseling and testing service than from poor cc performing kebeles ( 73.0% vs. 54.9% , p < 0.001 ) . when the study was conducted , the operation period for cc was about 8 years . more than 97% of the study participants who utilized vct did so with in 4 years before the survey which is 4 years well after the initiation of cc . the association of different predictors with vct service uptake was assessed by fitting them into a logistic regression model . accordingly , the odds of hiv test uptake for participants from well cc performing kebeles was found to be 2.5 times that for participants from poor cc performing kebeles [ adjusted odds ratio ( aor ) ( 95% confidence interval ( ci ) = 2.51(1.63,3.86 ) ] . and also those found in the age bracket of 15 - 24 years had the higher odds of hiv test uptake than those between 35 and 59 years [ aor ( 95% ci ) = 3.14(1.37 , 7.20 ) ] . the odds of hiv test uptake was about three times higher in singles than in married [ aor ( 95% ci ) = 2.55 ( 1.09 , 5.99 ) ] . similarly , the odds of hiv test uptake was 3.36 times higher in traders than in farmers [ aor ( 95% ci ) = 3.36 ( 1.03 , 10.96 ) ] [ table 3 ] . also , the participants ' knowledge about the benefits of vct and the ways of hiv transmission were predictors of vct service uptake with their corresponding estimates of [ aor ( 95% ci ) = 3.51(1.15 , 10.67 ) ] and [ aor ( 95% ci ) = 2.03(1.05 , 3.91 ) ] , respectively [ table 3 ] . factors associated with voluntary counseling and testing uptake ( n = 452 ) however sex , religion , occupational status other than trader , alcohol consumption , the preferred ways to get vct result , attitude toward people living with hiv / aids ( plwhas ) and knowledge about hiv prevention did not show statistically significant association with vct uptake [ table 3 ] . a total of 452 study participants completed the interview giving a response rate of 97.8% . of the 452 who participated in the study , 226 ( 50.0% ) were from well cc performing kebeles and 226 ( 50.0% ) from poor cc performing kebeles . majority of the study participants ; 119 ( 52.7% ) in well and 109 ( 48.2% ) in poor cc performing kebeles were in the age range of 15 - 24 years . with respect to educational status , near to half of the study participants in well ( 44.7% ) and in poor ( 46.9% ) cc groups had no formal education . regarding religion , about [ 183 ( 81.0% ) vs. 180 ( 79.6% ) ] of the study participants were protestant in poor and well cc groups . almost all of the study participants ; 218 ( 96.5% ) in well and 216 ( 95.6% ) in poor cc groups were sidama in ethnicity . married study participants were [ 141 ( 62.4% ) vs. 121 ( 53.5% ) ] followed by single [ 84 ( 37.4% ) vs. 102 ( 45.1% ) ] in poor and well cc groups , respectively . of the study participants ; ( 37.6% vs. 30.5% ) were students in occupation , followed by housewives ( 27.0% vs. 26.5% ) and farmers ( 23.0% vs. 23.7% ) in well and poor cc groups , respectively . majority of the study participants ; [ 146 ( 64.6% ) in well and 160 ( 70.6% ) ] in poor cc performing kebeles had started sexual intercourse by the time of data collection [ table 2 ] . the sociodemographic and sexual history of the study participants to determine the comparability of the study participants between well and poor cc performing kebeles , two sample test of proportion for major sociodemographic factors was computed . accordingly , the study participants in well and poor cc performing kebeles were not statistically different ( p > 0.05 ) by sex , age , religion , ethnicity , education , marital status , occupation , sexual practice , age at first sex , condom utilization , and risky behavior for hiv . therefore , the study participants in well and poor cc performing kebeles were comparable [ table 2 ] . significant number of study participants from well cc group compared with participants from poor cc group knew that vct is important to break the chain of hiv spread ( 97.8% vs.93.8% , p = 0.034 ) . more study participants from well cc performing kebeles utilized hiv voluntary counseling and testing service than from poor cc performing kebeles ( 73.0% vs. 54.9% , p < 0.001 ) . when the study was conducted , the operation period for cc was about 8 years . more than 97% of the study participants who utilized vct did so with in 4 years before the survey which is 4 years well after the initiation of cc . the association of different predictors with vct service uptake was assessed by fitting them into a logistic regression model . accordingly , the odds of hiv test uptake for participants from well cc performing kebeles was found to be 2.5 times that for participants from poor cc performing kebeles [ adjusted odds ratio ( aor ) ( 95% confidence interval ( ci ) = 2.51(1.63,3.86 ) ] . and also those found in the age bracket of 15 - 24 years had the higher odds of hiv test uptake than those between 35 and 59 years [ aor ( 95% ci ) = 3.14(1.37 , 7.20 ) ] . the odds of hiv test uptake was about three times higher in singles than in married [ aor ( 95% ci ) = 2.55 ( 1.09 , 5.99 ) ] . similarly , the odds of hiv test uptake was 3.36 times higher in traders than in farmers [ aor ( 95% ci ) = 3.36 ( 1.03 , 10.96 ) ] [ table 3 ] . also , the participants ' knowledge about the benefits of vct and the ways of hiv transmission were predictors of vct service uptake with their corresponding estimates of [ aor ( 95% ci ) = 3.51(1.15 , 10.67 ) ] and [ aor ( 95% ci ) = 2.03(1.05 , 3.91 ) ] , respectively [ table 3 ] . factors associated with voluntary counseling and testing uptake ( n = 452 ) however sex , religion , occupational status other than trader , alcohol consumption , the preferred ways to get vct result , attitude toward people living with hiv / aids ( plwhas ) and knowledge about hiv prevention did not show statistically significant association with vct uptake [ table 3 ] . this study showed that significantly higher proportion of the study participants from well cc performing kebeles had utilized vct compared with participants from poor cc performing kebeles ( p < 0.001 ) . this could be due to change in the community understanding of the pandemic as a result of cc sessions . similarly , cc as a powerful tool in reducing risky sexual behavior embedded in the community and an early marriage has been documented in ethiopia . on the contrary , though could not reach statistical significance , representation of larger number of students in well cc group compared with poor cc group could have also positively affected the observed association . vct service uptake documented in the current study for well cc kebeles is higher than that reported in the recent ethiopia demographic and health survey ( edhs ) report ( 73% vs. 67% ) . our finding that cc is an independent predictor of vct service utilization is further supported by different reports from ethiopia , where after the cc program initiation , vct service uptake showed increment . moreover , being sexually active , having sex with a partner and having a colleague who utilized vct were the reported predictors of vct uptake among college youths in ethiopia . the proportion of the study participants who knew that vct service is important in reducing the spread of hiv infection was significantly higher in a well cc group ( p = 0.034 ) . the finding suggests that cc plays key role in improving knowledge of the community with regard to vct . positive attitude toward vct reported in this study ( 97.8% in well and 93.8% in poor cc groups ) was consistent with a harar and jijiga studies . consistent with a study done in north and south gonder , those who were in the age range of 15 - 24 years were found to significantly utilize vct service compared to those aged between 35 and 59 years . the finding that knowledge about hiv transmission is associated with hiv test uptake was also reported from blantyre , malawi . as with the current study , lack of association between sex and religion has been reported by a study conducted in ethiopia . this study is robust in that it has used a comparative cross - sectional design having a power of 80% to discriminate the difference . the study population were also homogenous on major sociodemographic characteristics , other than cc group , and hence were comparable . unlike the previous studies , this study determined the role of cc in vct service utilization . apart from its strengths , this study has short comings also : it was difficult to establish temporal relationship between cc and utilization of vct . moreover , the criteria used to classify kebeles into well and poor cc groups were quite general and not specific to hiv / vct topics . though cc is advocated as a powerful tool , its benefits are offset by its dependence on the facilitators ' skills . the criteria used to classify study kebeles into well or poor cc groups did not take facilitation skill into account . the bottom line assumption was if kebeles had generally well cc performance , they would address hiv / vct well . however , as there are lots of community felt problems to be discussed in cc , the share of hiv / vct might be low . consequently , even in a well cc kebele , hiv / vct topics might be addressed poorly . though it is known that vct uptake varies with vct modality as a community - based or facility - based , the study could not separate one modality from the other vct modality . another , noteworthy limitation is that the definition of risky sex in this study did not take into account the hiv status of the sex partner of the study participant .

background : voluntary counseling and testing ( vct ) is a cost - effective tool to prevent and control human immunodeficiency virus ( hiv)/acquired immodeficiency syndrome . community conversation ( cc ) is a community - based strategy meant to enhance the community utilization of vct . however , the role of cc in vct service uptake has not yet been evaluated.aims:this study was conducted to compare vct service utilization between rural communities with well cc performance and rural communities with poor cc performance in shebedino woreda.materials and methods : a cross - sectional comparative community - based study was conducted in 2010 among 462 selected adults in the age bracket of 15 - 59 years . vct service uptake was compared between well cc performing communities and poor cc performing communities using two sample test of proportion . predictors of vct service uptake were determined using logistic regression model.results:uptake of vct service and the related vct knowledge were statistically higher in well cc performing communities than poor cc performing communities ; [ 73.0% vs. 54.1% , p < 0.001 ) vs. 97.8% vs. 93.8% , p = 0.034 ] . cc , vct knowledge , and knowledge on hiv transmission were independent predictors of vct service utilization.conclusion:uptake of vct service is higher in well cc performing communities . emphasis should be given to strengthen cc performance .

Introduction Materials and Methods Sample size and sampling Variables Statistical analysis Results Description of the study population VCT Service utilization among the study participants Predictors of VCT service uptake Discussion Conclusion

vct service utilization is low in ethiopia , only 20% of women and 21% of men have been tested for hiv and received the results in 2005 indicating that many people living with hiv / aids in ethiopia do not know their hiv status . however , to foster attitudes and behaviors that promote counseling and testing among community leaders and in communities , a community - based strategy called community conversation(cc ) was introduced in ethiopia in 2002 . the facilitators of cc in ethiopia are health extension workers ( hews ) who shared the ethnicity , home language , and had grown up in the same district of the cc participants . they conduct cc every 2 weeks for a cross section of 36 - 40 people from the community - men and women , old and young , people living with hiv / aids and those who are not infected , religious , community and traditional leaders , and members of the community at large . aided by skilled facilitators from their own communities , people openly talk to each other and explore the implications of hiv / aids , identify their own cultural norms and values that are fuelling the epidemic and the social capital within the community to overcome them . during cc session , the facilitator poses questions related to hiv including but not limited to driving factors for hiv transmission in the community , health , social , economical , and psychological impacts of hiv / aids and role of vct in hiv care and prevention . though studies that related cc with hiv or vct are scarce , the role of cc in increasing hiv - related competence has been demonstrated in one qualitative study conducted in zimbabwe . also as a result of cc in ethiopia , it is reported that cc participants have travelled long distance to the nearest health facility to get tested for hiv and convinced their partner to get tested . it also is a powerful tool to reduce risk behavior toward hiv and promotes hiv prevention and stigma reduction . however , the role of cc on vct service uptake has not yet been evaluated using sound scientific methods in ethiopia . this study was designed to compare vct service utilization between rural communities with well cc performance and rural communities with poor cc performance in shebedino woreda , south ethiopia . the study was conducted in shebedino woreda , sidamo zone , southern nations nationalities and peoples region , ethiopia in november 2010 . a cross - sectional comparative study was conducted among adult population who were in the age range ; 15 - 59 years in the selected six kebeles ( kebele is the smallest administrative unit ) of the woreda . sample size was calculated using epi info sample size calculator ( epi info 3.3.2 , cdc ) for two population proportion by taking proportion of vct service utilization of 20.1% among communities with well cc performance . a 10% difference was assumed between well and poor cc kebeles so that the proportion of vct service utilization among poor cc communities was set at 10.1% . other assumptions considered were : 95% confidence level , 80% power , 5% level of significance , and the ratio of well to poor cc(r ) = 1 . the source population was 15 - 59 years old population residing in rural kebeles implementing cc in shebedino woreda . rural kebeles were classified into well and poor cc groups based on their major activities on cc performance . four major criteria ( cc methodology frame work , number of participants , number of cc groups , number of decisions undertaken , document handling , and reporting ) were considered to classify the study kebeles into well and poor cc groups . a kebele was labeled as well performing cc group if its cc score was equal to or greater than the median value of 4.75 ; otherwise , it was labeled as a poor cc performing kebele . list of criteria and weight used to classify kebeles into poor and well cc groups out of 35 rural kebeles in the woreda , 25 qualified to be sampled ( seven kebeles had stopped cc ) . then , kebeles under the two strata ( well and poor ) were further stratified into proximal ( within 5 km ) , medium ( 5 - 10 km ) , and distal ( more than 10 km ) by their distance from the woreda capital . from the selected households , data were collected from the selected adults in the age bracket of 15 - 59 years . upon checking completeness and consistency of responses , the magnitude of vct service uptake was determined by counting the number of study participants who underwent hiv test on voluntary basis . to measure knowledge , comprehensive set of knowledge questions a two sample proportion test was used to test the presence of statistical difference in the proportion of vct service utilization between the two cc groups . to determine the association between different predictors and vct service utilization ( dependent variable ) , a logistic regression model was employed . first , each predictor was entered into a separate binary logistic regression model to determine the crude effect of each variable on vct uptake . second , to account for the effect of confounders , a multiple logistic regression model was fitted with all predictors that have p 0.3 in the bivariate model . eventually , only those variables that remained significant at p 0.05 in the final model were retained as independent predictors of vct service uptake . sample size was calculated using epi info sample size calculator ( epi info 3.3.2 , cdc ) for two population proportion by taking proportion of vct service utilization of 20.1% among communities with well cc performance . a 10% difference was assumed between well and poor cc kebeles so that the proportion of vct service utilization among poor cc communities was set at 10.1% . other assumptions considered were : 95% confidence level , 80% power , 5% level of significance , and the ratio of well to poor cc(r ) = 1 . the source population was 15 - 59 years old population residing in rural kebeles implementing cc in shebedino woreda . rural kebeles were classified into well and poor cc groups based on their major activities on cc performance . four major criteria ( cc methodology frame work , number of participants , number of cc groups , number of decisions undertaken , document handling , and reporting ) were considered to classify the study kebeles into well and poor cc groups . a kebele was labeled as well performing cc group if its cc score was equal to or greater than the median value of 4.75 ; otherwise , it was labeled as a poor cc performing kebele . list of criteria and weight used to classify kebeles into poor and well cc groups out of 35 rural kebeles in the woreda , 25 qualified to be sampled ( seven kebeles had stopped cc ) . then , kebeles under the two strata ( well and poor ) were further stratified into proximal ( within 5 km ) , medium ( 5 - 10 km ) , and distal ( more than 10 km ) by their distance from the woreda capital . from the selected households , data were collected from the selected adults in the age bracket of 15 - 59 years . upon checking completeness and consistency of responses , the magnitude of vct service uptake was determined by counting the number of study participants who underwent hiv test on voluntary basis . to measure knowledge , comprehensive set of knowledge questions a two sample proportion test was used to test the presence of statistical difference in the proportion of vct service utilization between the two cc groups . to determine the association between different predictors and vct service utilization ( dependent variable ) , a logistic regression model was employed . first , each predictor was entered into a separate binary logistic regression model to determine the crude effect of each variable on vct uptake . second , to account for the effect of confounders , a multiple logistic regression model was fitted with all predictors that have p 0.3 in the bivariate model . eventually , only those variables that remained significant at p 0.05 in the final model were retained as independent predictors of vct service uptake . of the 452 who participated in the study , 226 ( 50.0% ) were from well cc performing kebeles and 226 ( 50.0% ) from poor cc performing kebeles . majority of the study participants ; 119 ( 52.7% ) in well and 109 ( 48.2% ) in poor cc performing kebeles were in the age range of 15 - 24 years . regarding religion , about [ 183 ( 81.0% ) vs. 180 ( 79.6% ) ] of the study participants were protestant in poor and well cc groups . almost all of the study participants ; 218 ( 96.5% ) in well and 216 ( 95.6% ) in poor cc groups were sidama in ethnicity . of the study participants ; ( 37.6% vs. 30.5% ) were students in occupation , followed by housewives ( 27.0% vs. 26.5% ) and farmers ( 23.0% vs. 23.7% ) in well and poor cc groups , respectively . majority of the study participants ; [ 146 ( 64.6% ) in well and 160 ( 70.6% ) ] in poor cc performing kebeles had started sexual intercourse by the time of data collection [ table 2 ] . the sociodemographic and sexual history of the study participants to determine the comparability of the study participants between well and poor cc performing kebeles , two sample test of proportion for major sociodemographic factors was computed . accordingly , the study participants in well and poor cc performing kebeles were not statistically different ( p > 0.05 ) by sex , age , religion , ethnicity , education , marital status , occupation , sexual practice , age at first sex , condom utilization , and risky behavior for hiv . therefore , the study participants in well and poor cc performing kebeles were comparable [ table 2 ] . significant number of study participants from well cc group compared with participants from poor cc group knew that vct is important to break the chain of hiv spread ( 97.8% vs.93.8% , p = 0.034 ) . more study participants from well cc performing kebeles utilized hiv voluntary counseling and testing service than from poor cc performing kebeles ( 73.0% vs. 54.9% , p < 0.001 ) . when the study was conducted , the operation period for cc was about 8 years . the association of different predictors with vct service uptake was assessed by fitting them into a logistic regression model . accordingly , the odds of hiv test uptake for participants from well cc performing kebeles was found to be 2.5 times that for participants from poor cc performing kebeles [ adjusted odds ratio ( aor ) ( 95% confidence interval ( ci ) = 2.51(1.63,3.86 ) ] . and also those found in the age bracket of 15 - 24 years had the higher odds of hiv test uptake than those between 35 and 59 years [ aor ( 95% ci ) = 3.14(1.37 , 7.20 ) ] . the odds of hiv test uptake was about three times higher in singles than in married [ aor ( 95% ci ) = 2.55 ( 1.09 , 5.99 ) ] . similarly , the odds of hiv test uptake was 3.36 times higher in traders than in farmers [ aor ( 95% ci ) = 3.36 ( 1.03 , 10.96 ) ] [ table 3 ] . also , the participants ' knowledge about the benefits of vct and the ways of hiv transmission were predictors of vct service uptake with their corresponding estimates of [ aor ( 95% ci ) = 3.51(1.15 , 10.67 ) ] and [ aor ( 95% ci ) = 2.03(1.05 , 3.91 ) ] , respectively [ table 3 ] . factors associated with voluntary counseling and testing uptake ( n = 452 ) however sex , religion , occupational status other than trader , alcohol consumption , the preferred ways to get vct result , attitude toward people living with hiv / aids ( plwhas ) and knowledge about hiv prevention did not show statistically significant association with vct uptake [ table 3 ] . of the 452 who participated in the study , 226 ( 50.0% ) were from well cc performing kebeles and 226 ( 50.0% ) from poor cc performing kebeles . majority of the study participants ; 119 ( 52.7% ) in well and 109 ( 48.2% ) in poor cc performing kebeles were in the age range of 15 - 24 years . regarding religion , about [ 183 ( 81.0% ) vs. 180 ( 79.6% ) ] of the study participants were protestant in poor and well cc groups . almost all of the study participants ; 218 ( 96.5% ) in well and 216 ( 95.6% ) in poor cc groups were sidama in ethnicity . married study participants were [ 141 ( 62.4% ) vs. 121 ( 53.5% ) ] followed by single [ 84 ( 37.4% ) vs. 102 ( 45.1% ) ] in poor and well cc groups , respectively . of the study participants ; ( 37.6% vs. 30.5% ) were students in occupation , followed by housewives ( 27.0% vs. 26.5% ) and farmers ( 23.0% vs. 23.7% ) in well and poor cc groups , respectively . majority of the study participants ; [ 146 ( 64.6% ) in well and 160 ( 70.6% ) ] in poor cc performing kebeles had started sexual intercourse by the time of data collection [ table 2 ] . the sociodemographic and sexual history of the study participants to determine the comparability of the study participants between well and poor cc performing kebeles , two sample test of proportion for major sociodemographic factors was computed . accordingly , the study participants in well and poor cc performing kebeles were not statistically different ( p > 0.05 ) by sex , age , religion , ethnicity , education , marital status , occupation , sexual practice , age at first sex , condom utilization , and risky behavior for hiv . therefore , the study participants in well and poor cc performing kebeles were comparable [ table 2 ] . significant number of study participants from well cc group compared with participants from poor cc group knew that vct is important to break the chain of hiv spread ( 97.8% vs.93.8% , p = 0.034 ) . more study participants from well cc performing kebeles utilized hiv voluntary counseling and testing service than from poor cc performing kebeles ( 73.0% vs. 54.9% , p < 0.001 ) . when the study was conducted , the operation period for cc was about 8 years . the association of different predictors with vct service uptake was assessed by fitting them into a logistic regression model . accordingly , the odds of hiv test uptake for participants from well cc performing kebeles was found to be 2.5 times that for participants from poor cc performing kebeles [ adjusted odds ratio ( aor ) ( 95% confidence interval ( ci ) = 2.51(1.63,3.86 ) ] . and also those found in the age bracket of 15 - 24 years had the higher odds of hiv test uptake than those between 35 and 59 years [ aor ( 95% ci ) = 3.14(1.37 , 7.20 ) ] . the odds of hiv test uptake was about three times higher in singles than in married [ aor ( 95% ci ) = 2.55 ( 1.09 , 5.99 ) ] . similarly , the odds of hiv test uptake was 3.36 times higher in traders than in farmers [ aor ( 95% ci ) = 3.36 ( 1.03 , 10.96 ) ] [ table 3 ] . also , the participants ' knowledge about the benefits of vct and the ways of hiv transmission were predictors of vct service uptake with their corresponding estimates of [ aor ( 95% ci ) = 3.51(1.15 , 10.67 ) ] and [ aor ( 95% ci ) = 2.03(1.05 , 3.91 ) ] , respectively [ table 3 ] . factors associated with voluntary counseling and testing uptake ( n = 452 ) however sex , religion , occupational status other than trader , alcohol consumption , the preferred ways to get vct result , attitude toward people living with hiv / aids ( plwhas ) and knowledge about hiv prevention did not show statistically significant association with vct uptake [ table 3 ] . this study showed that significantly higher proportion of the study participants from well cc performing kebeles had utilized vct compared with participants from poor cc performing kebeles ( p < 0.001 ) . this could be due to change in the community understanding of the pandemic as a result of cc sessions . similarly , cc as a powerful tool in reducing risky sexual behavior embedded in the community and an early marriage has been documented in ethiopia . on the contrary , though could not reach statistical significance , representation of larger number of students in well cc group compared with poor cc group could have also positively affected the observed association . vct service uptake documented in the current study for well cc kebeles is higher than that reported in the recent ethiopia demographic and health survey ( edhs ) report ( 73% vs. 67% ) . our finding that cc is an independent predictor of vct service utilization is further supported by different reports from ethiopia , where after the cc program initiation , vct service uptake showed increment . moreover , being sexually active , having sex with a partner and having a colleague who utilized vct were the reported predictors of vct uptake among college youths in ethiopia . the proportion of the study participants who knew that vct service is important in reducing the spread of hiv infection was significantly higher in a well cc group ( p = 0.034 ) . positive attitude toward vct reported in this study ( 97.8% in well and 93.8% in poor cc groups ) was consistent with a harar and jijiga studies . consistent with a study done in north and south gonder , those who were in the age range of 15 - 24 years were found to significantly utilize vct service compared to those aged between 35 and 59 years . the finding that knowledge about hiv transmission is associated with hiv test uptake was also reported from blantyre , malawi . this study is robust in that it has used a comparative cross - sectional design having a power of 80% to discriminate the difference . unlike the previous studies , this study determined the role of cc in vct service utilization . apart from its strengths , this study has short comings also : it was difficult to establish temporal relationship between cc and utilization of vct . moreover , the criteria used to classify kebeles into well and poor cc groups were quite general and not specific to hiv / vct topics . the bottom line assumption was if kebeles had generally well cc performance , they would address hiv / vct well . however , as there are lots of community felt problems to be discussed in cc , the share of hiv / vct might be low . though it is known that vct uptake varies with vct modality as a community - based or facility - based , the study could not separate one modality from the other vct modality .

histamine is a transmitter in the nervous system and a signaling molecule in the gut , the skin , and the immune system . histamine neurons in mammalian brain are located exclusively in the tuberomammillary nucleus of the posterior hypothalamus and their axons extend throughout the central nervous system ( cns ) . four known histamine receptors and histamine binding to glutamate nmda receptors serve multiple functions in the brain , particularly control of excitability and plasticity ( haas and panula , 2003 ; haas et al . , the h1 and h2 receptor - mediated actions are mostly excitatory , while h3 receptors act as inhibitory auto- and heteroreceptors . histamine neurons are proposed to have a dual effect on the cns , with both stimulatory and suppressive actions ( watanabe and yanai , 2001 ) . as a stimulator , neuronal histamine is one of the most important systems that stimulate and maintain attentive wakefulness . brain histamine also functions in bioprotection as a suppressor of various noxious and unfavorable stimuli of convulsion , drug sensitization , denervation supersensitivity , ischemic lesions , and stress susceptibility . we have examined the functions of histamine neurons using various approaches , such as histamine - related gene knockout mice and human positron emission tomography ( pet ) ( yanai and tashiro , 2007 ) . histamine neurons play an important role in the forebrain waking systems ( lin , 2000 ; eriksson et al . , 2001 ; their neuronal activity is specific for a high - vigilance waking state and histamine neurons might play a role not in the initiation of wakefulness , but in maintenance of the high level of vigilance necessary for cognitive processes ( takahashi et al . , 2008 ; an increase in histaminergic transmission promotes wakefulness , whereas its blockade by sedating antihistamines causes somnolence and impaired performance in humans ( yanai et al . , 2011 ) . several lines of evidence suggest that histamine modulates circadian rhythms ( tuomisto et al . , 2001 ; abe et al . , 2004 ) and it has even been suggested to play a pivotal role in circadian entrainment ( jacobs et al . , 2000 ) . accordingly , a clear circadian rhythm of histamine release was demonstrated in the anterior hypothalamus by in vivo microdialysis studies ( mochizuki et al . , 1992 ) . histamine release increased before the active phase and was maintained at an elevated level during the active phase . stress in our daily lives has been associated with various psychiatric disorders including depression , schizophrenia , cognitive disorders , and psychosomatic diseases such as anorexia nervosa and irritable bowel syndrome . to date , we have conducted several pet studies to elucidate the pathophysiological mechanism behind the above - mentioned disorders , focusing on alteration in neural transmission of the histaminergic neuron systems ( yanai and tashiro , 2007 ) . for pet studies , [ c]doxepin , a potent antagonist of histamine h1 receptors , has been utilized as an effective pet imaging tracer . using [ c]doxepin , increasing evidence has been accumulated regarding the role of the histaminergic neuron system in the pathophysiology of stress - related neuropsychiatric disorders . for example , histamine h1 receptor binding was measured using [ c]doxepin in patients with schizophrenia ( iwabuchi et al . , 2005 ) , major depression ( kano et al . , 2004 ) and anorexia nervosa ( yoshizawa et al . , 2009 ) . another application of [ c]doxepin - pet is the measurement of histamine h1 receptor occupancy by antihistamines . the inhibition of histaminergic activity at the brain h1 receptor level may have some favorable anxiolytic effects , however , more often this is accompanied by unfavorable effects like increased daytime somnolence , impaired memory and learning , decreased attention , and weight gain . the brain h1 receptor occupancy measured by [ c]doxepin - pet is one of the most reliable and objective methods to estimate the sedating properties of antihistamines ( yanai et al . , 2011 , 2012 ) . a critical step in validating [ c]doxepin to measure the brain h1 receptor occupancy is to evaluate the reproducibility of its binding potential ( bmax / kd ) in the different attentive conditions . 2011 ) , although [ c]doxepin binding to brain has not been examined on this aspect until now . previous studies have shown that imaging with pet radiotracers that are specific for brain receptors can be used to visualize changes in the release of neurotransmitters indirectly . most successful studies have focused on dopamine , since the dopamine neurons that project to the striatum have been shown to play a critical role in mediating motivational and addictive behaviors . these imaging studies successfully measured increased extracellular dopamine released by psychostimulants and physiological reward stimuli in humans with [ c]raclopride ( koepp et al . , 1998 ; rinne , 2003 ; hommer et al . , 2011 ) and however , some technical difficulties have been encountered for the imaging of changes in the release of other neurotransmitters . among these are low sensitivity , changes of neurotransmitter release pulse over time during pet imaging and the issue of affinity states , the contribution of carryover mass in the second pet scan , and internalization of receptors ( boecker et al . , 2008 ) . followed by the previous pet measurement of dopamine release , we examined whether neuronal histamine released as a result of mental stress and the circadian rhythm might change the levels of h1 receptors measured in vivo by pet and [ c]doxepin . therefore , we undertook the present study for the purpose of evaluating the test retest reliability of [ c]doxepin binding in the human brain during different attentive conditions and circadian rhythm in healthy human subjects . japanese male volunteers , who were physically and mentally healthy and had no history of allergy or long - term treatment with h1 antagonists , were recruited to participate in this study . concomitant medications , nicotine , caffeine , grapefruit or grapefruit juice , and alcohol were not allowed during the experimental period . the present study was approved by the committee on clinical investigation at tohoku university graduate school of medicine , japan , and was performed in accordance with the principles of the declaration of helsinki . all experiments were performed at the cyclotron and radioisotope center , tohoku university . in the first part of this study involving test retest measurements , six healthy male volunteers ( mean age sd : 24.6 2.1 years old ) were examined twice with [ c]doxepin - pet during a resting condition in the morning ( 11:00 a.m. ) and afternoon ( 3:00 p.m. ) of the same day in order to evaluate the circadian rhythm of h1 receptor binding . in the second part of this study involving investigation of ligand activation , 10 healthy men ( 22.3 1.0 years old ) they were scanned twice by pet ( set2400w ; shimadzu co. , kyoto , japan ) on the same day during attentive calculation tasks involving two - digit addition and during resting conditions with their eyes closed after administration of [ c]doxepin . we performed measurements of subjective feelings five times during pet scans before the scan ( pre ) , at interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of scan ( end ) . subjective feelings including alertness , tiredness , and sleepiness were measured during pet scans using line analog rating scale ( lars ) . in lars measurement , subjects mark a series of 100 mm linear analog scales ( + 50 to 50 mm ) , indicating their present feeling with regard to a midpoint , which represents their normal state of mind . the h1 receptors were examined with [ c]doxepin - pet during the resting and calculation conditions . the subjective feelings during pet scans were also measured during the 90 min before the scan ( pre ) , interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of the scan ( end ) . [ c]doxepin was synthesized by c - methylation of desmethyl - doxepin with [ c]methyl triflate as described previously . the radiochemical purity of [ c]doxepin was greater than 99% , and its specific radioactivity at the time of injection was 207.5 61.9 gbq/mol ( 5608 1673 mci/mol ) . the single injected dose and cold mass of [ c]doxepin were 119.8 10.5 mbq ( 3.23 0.283 mci ) and 0.577 0.051 nmol , respectively . for the measurement of h1 receptors , pet scans were carried out with an set2400w pet scanner ( shimadzu co. , kyoto , japan ) . pet data were acquired 30 s after the administration of [ c]doxepin with the subject 's eyes closed for 90 min . in order to calculate the binding potential ( bp ) of h1 receptors , brain pet images of each subject during resting and calculation conditions were subjected to inter - frame motion correction and then co - registered to an identical stereotaxic brain coordinate using a corresponding t1-weighted mri image . regions of interest ( roi ) were first placed on the following brain regions on the t1 images for which precise anatomical information was available : anterior and posterior cingulate gyrus , inferior prefrontal cortex , superior prefrontal cortex , temporal cortex , and cerebellum . roi was defined for each cortical region by 35 concentric circles with a diameter of 5.0 mm for each hemisphere in 45 consecutive brain transaxial slices . an averaged value from all rois was used as a representative value of each region . in addition , we produced a time - activity curve ( tac ) of each region from roi data . a standardized uptake value ( suv ) was calculated for the normalization of roi - tacs as follows : suv ( tac)=tac ( mbq / ml)/[injected tracer dose ( mbq)]/body weight ( g ) subsequently , logan graphical analysis with the reference tissue input ( lgar ) method was applied to calculate bp using pmod kinetic modeling tool ( pkin ) software ( pmod technologies ltd . , zurich , switzerland ) and tac ( suzuki et al . , 2005 ) , and we compared the bp between different conditions ( resting vs. calculation ; morning vs. afternoon ) . all data were analyzed by a repeated measure of anova followed by multiple comparisons ( tukey kramer test , scheffe 's f test , and bonferroni dunn test ) , and p < 0.05 was considered statistically significant . japanese male volunteers , who were physically and mentally healthy and had no history of allergy or long - term treatment with h1 antagonists , were recruited to participate in this study . concomitant medications , nicotine , caffeine , grapefruit or grapefruit juice , and alcohol were not allowed during the experimental period . the present study was approved by the committee on clinical investigation at tohoku university graduate school of medicine , japan , and was performed in accordance with the principles of the declaration of helsinki . all experiments were performed at the cyclotron and radioisotope center , tohoku university . in the first part of this study involving test retest measurements , six healthy male volunteers ( mean age sd : 24.6 2.1 years old ) were examined twice with [ c]doxepin - pet during a resting condition in the morning ( 11:00 a.m. ) and afternoon ( 3:00 p.m. ) of the same day in order to evaluate the circadian rhythm of h1 receptor binding . in the second part of this study involving investigation of ligand activation , 10 healthy men ( 22.3 1.0 years old ) they were scanned twice by pet ( set2400w ; shimadzu co. , kyoto , japan ) on the same day during attentive calculation tasks involving two - digit addition and during resting conditions with their eyes closed after administration of [ c]doxepin . we performed measurements of subjective feelings five times during pet scans before the scan ( pre ) , at interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of scan ( end ) . subjective feelings including alertness , tiredness , and sleepiness were measured during pet scans using line analog rating scale ( lars ) . in lars measurement , subjects mark a series of 100 mm linear analog scales ( + 50 to 50 mm ) , indicating their present feeling with regard to a midpoint , which represents their normal state of mind . the h1 receptors were examined with [ c]doxepin - pet during the resting and calculation conditions . the subjective feelings during pet scans were also measured during the 90 min before the scan ( pre ) , interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of the scan ( end ) . [ c]doxepin was synthesized by c - methylation of desmethyl - doxepin with [ c]methyl triflate as described previously . the radiochemical purity of [ c]doxepin was greater than 99% , and its specific radioactivity at the time of injection was 207.5 61.9 gbq/mol ( 5608 1673 mci/mol ) . the single injected dose and cold mass of [ c]doxepin were 119.8 10.5 mbq ( 3.23 0.283 mci ) and 0.577 0.051 nmol , respectively . for the measurement of h1 receptors , pet scans were carried out with an set2400w pet scanner ( shimadzu co. , kyoto , japan ) . pet data were acquired 30 s after the administration of [ c]doxepin with the subject 's eyes closed for 90 min . in order to calculate the binding potential ( bp ) of h1 receptors , brain pet images of each subject during resting and calculation conditions were subjected to inter - frame motion correction and then co - registered to an identical stereotaxic brain coordinate using a corresponding t1-weighted mri image . regions of interest ( roi ) were first placed on the following brain regions on the t1 images for which precise anatomical information was available : anterior and posterior cingulate gyrus , inferior prefrontal cortex , superior prefrontal cortex , temporal cortex , and cerebellum . roi was defined for each cortical region by 35 concentric circles with a diameter of 5.0 mm for each hemisphere in 45 consecutive brain transaxial slices . an averaged value from all rois was used as a representative value of each region . in addition , we produced a time - activity curve ( tac ) of each region from roi data . a standardized uptake value ( suv ) was calculated for the normalization of roi - tacs as follows : suv ( tac)=tac ( mbq / ml)/[injected tracer dose ( mbq)]/body weight ( g ) subsequently , logan graphical analysis with the reference tissue input ( lgar ) method was applied to calculate bp using pmod kinetic modeling tool ( pkin ) software ( pmod technologies ltd . , zurich , switzerland ) and tac ( suzuki et al . , 2005 ) , and we compared the bp between different conditions ( resting vs. calculation ; morning vs. afternoon ) . all data were analyzed by a repeated measure of anova followed by multiple comparisons ( tukey kramer test , scheffe 's f test , and bonferroni dunn test ) , and p < 0.05 was considered statistically significant . during the performance of the attentive task involving two - digit calculation , the percentage of correct answers was greater than 90% . subjects felt significantly more tired during the calculation task than in the resting condition ( figure 2a ) . subjects in the resting condition tended to have significantly higher sleepiness scores than those in the calculation condition ( figure 2b ) . these data suggest that there was a significant difference in the attention level between the resting and calculation conditions . subjective tiredness ( a ) and sleepiness ( b ) evaluated using lars during pet scans . data are presented as the means sem of lars ( mm ) from 10 healthy subjects in the resting and calculation conditions . all data were analyzed by a repeated measure of anova followed by multiple comparisons and p < 0.05 was considered statistically significant . six subjects were tested in the evaluation of the test retest reliability of [ c]doxepin pet . given concerns about the possibility that [ c]doxepin binding changes over days and week , the test and re - test trials were performed in the morning ( 11:00 a.m. ) and afternoon ( 3:00 p.m. ) of the same day . as shown in figure 3a , average suvs over time from test retest scans show that the tracer gradually entered the brain and the brain activity remained almost stable . the radioactivity in the anterior cingulate gyrus showed a slightly longer elimination phase in the test trial performed in the morning than that in the afternoon , but the difference was insignificant . the radioactivity in the cerebellum with negligible h1 receptor binding was essentially the same between the test and re - test trials . there was an apparent trend for decreased bp in the afternoon , but the bp values in the brain regions including anterior cingulate gyrus were not significantly higher in the morning test trial ( figure 3b ) , demonstrating that [ c]doxepin binding in the brain is essentially the same between in the morning and afternoon . test ( a ) tacs in terms of suvs for the regions of anterior cingulate gyrus ( ac ) and cerebellum ( cb ) of test retest scans . retest scans were performed in the morning and afternoon of the same day , demonstrating a gradual initial increase in suv followed by a longer elimination phase . abbreviations : ac , anterior cingulate gyrus ; ifrol , left inferior prefrontal cortex ; ifror , right inferior prefrontal cortex ; pc , posterior cingulate gyrus ; sfrol , left superior prefrontal cortex ; sfror , right superior prefrontal cortex ; templ , left temporal cortex ; tempr , right temporal cortex . in order to verify the effects of attentive waking on in vivo pet measurements of h1 receptor binding , [ c]doxepin binding was examined twice in the brains of 10 normal volunteers during resting and attentive waking conditions on the same day , as shown in figure 4 . the order of rest and calculation trials of pet studies was randomized to eliminate the effects of circadian rhythm on h1 receptor binding observed in previous experiments . the time courses in the regions of anterior cingulate gyrus ( h1 receptor rich region ) and cerebellum ( h1 receptor null region ) were not significantly different between the resting and attentive calculation conditions ( figure 4a ) . there was a trend for decreased bp during the calculation task compared with that in the resting condition , but the difference was not significant . these data suggest that [ c]doxepin binding in the brain is not significantly influenced by the performance of a calculation task as an example of an attentive waking condition ( a ) tacs in terms of suvs for the regions of anterior cingulate gyrus ( ac ) and cerebellum ( cb ) of resting and attentive waking conditions . ( b ) the binding potential ( bp ) in the resting and attentive conditions . abbreviations : ac , anterior cingulate gyrus ; ifrol , left inferior prefrontal cortex ; ifror , right inferior prefrontal cortex ; pc , posterior cingulate gyrus ; sfrol , left superior prefrontal cortex ; sfror , right superior prefrontal cortex ; templ , left temporal cortex ; tempr , right temporal cortex . the main objectives of this study were to analyze the specific brain uptake and kinetics of [ c]doxepin in normal volunteers under different conditions , and to assess the test retest reliability of quantitative pet measurements between morning and afternoon and between resting and attentive waking conditions . the test retest reliability for estimated bp was found to be sufficiently high to afford reasonable precision in the tracer binding determinations of h1 receptors . attentive waking and circadian rhythm might have some influences on the bp of h1 receptors , but only within the range of 10% over all regions . the low variability within 10% in the test retest studies can be compatible to other pet tracers such as [ c]dasb ( serotonin transporter ) , [ f]spa - rq ( nk-1 receptor ) , [ c]pib ( amyloid a ) , and [ c]flb457 ( d2/3 receptor ) ( kim et al . , 2006 ; yasuno et al . , 2007 ; edison et al . evidence from animal studies has implicated the histaminergic neuron system in the pathophysiology of stress - related disorders . although several antidepressants and atypical antipsychotics are potent h1r antagonists , the significance of their interaction with h1r in a clinical context of efficacy is still unclear . in previous pet studies , significant reduction in h1 it is suggested that prolonged massive histamine release due to repeated stress might lead to the down - regulation and/or internalization of h1r , which may result in decreased binding of [ c]doxepin in stress - related disorders ( endou et al . , 2001 ) . we previously demonstrated that normal female volunteers had significantly higher bp of [ c]doxepin to h1 receptors in the cerebral cortical areas than male volunteers ( yoshizawa et al . , 2009 ) . for example , there are gender differences in the size of the interstitial nucleus of anterior hypothalamus ( inah ) ( male > female ) . inah in homosexual men is only half the size of the nucleus in heterosexual men . the gender difference in human h1 receptors that we observed is reasonable because histamine neurons are exclusively located in the posterior hypothalamus . the density of histamine h1 receptors was higher in female rats than in male rats ( ghi et al . , 1999 ) , and hypothalamic histamine release was higher in male rats than in female rats ( ferretti et al . , 1998 ) . it is not ruled out that neuronal histamine release and in vivo h1 receptor binding are closely correlated . therefore , we should carefully consider unknown factors influencing in vivo [ c]doxepin binding in the brain . therefore , it is important to develop an objective and reliable method for measuring the strength of such sedative side effects , on which we have conducted numerous pet studies ( tagawa et al . [ c]doxepin - pet has been shown to be useful for evaluating their sedating side effects and the mechanisms involved . we succeeded in quantifying the strength of the sedating properties of antihistamines in terms of brain histamine h1 receptor occupancy . we previously reported an age - related decline in h1 receptor binding in normal human brain , especially in the prefrontal , temporal , cingulate , and parahippocampal regions ( yanai et al . , 1992 ) , which are known to be involved in attention and cognition . this study confirmed the reliability of values in h1 receptor occupancy by antihistamines because different pet studies were summarized to make the figures of occupancy ( yanai et al . this study demonstrates for the first time that subjects ' attentive conditions do not affect the reliability of h1 receptor binding measured by [ c]doxepin - pet . this study does not necessarily rule out the feasibility of measuring neuronal histamine release in the living human brain using pet , although pet tracers with better signal - to - noise properties should be developed in the future . for this purpose , h3 receptor binding would be more appropriate because h3 receptors are easily down - regulated by stress - related histamine release . the previous studies reported that histamine release was significantly increased during stressful conditions , and that the h3 receptor density rapidly decreased in response to stress ( ghi et al . 2002 ) . following the development of other histaminergic pet probes , non - invasive measurement of neuronal histamine release the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the 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 .

molecular imaging in neuroscience is a new research field that enables visualization of the impact of molecular events on brain structure and function in humans . while magnetic resonance - based imaging techniques can provide complex information at the level of system , positron emission tomography ( pet ) enables determination of the distribution and density of receptor and enzyme in the human brain . previous studies using [ 11c]raclopride and [ 11c]flb457 revealed that the release of neuronal dopamine was increased in human brain by psychostimulants or reward stimuli . following on from these previous [ 11c]raclopride studies , we examined whether the levels of neuronal release of histamine might change [ 11c]doxepin binding to the h1 receptors under the influence of physiological stimuli . the purpose of the present study was to evaluate the test retest reliability of quantitative measurement of [ 11c]doxepin binding between morning and afternoon and between resting and attentive waking conditions in healthy human subjects . there was a trend for a decrease in [ 11c]doxepin binding during attentive calculation tasks compared with that in resting conditions , but the difference ( less than 10% ) was not significant . similarly , the binding potential of [ 11c]doxepin in the cerebral cortex was slightly higher in the morning than that in the afternoon , but it was also insignificant . these data suggest that higher histamine release during wakefulness could not decrease the [ 11c]doxepin binding in the brain . this study confirmed the reproducibility and reliability of [ 11c]doxepin in the previous imaging studies to measure the h1 receptor .

Introduction Methods Subjects and study design PET image acquisition and data analysis Results Discussion Conflict of interest statement

histamine is a transmitter in the nervous system and a signaling molecule in the gut , the skin , and the immune system . histamine neurons in mammalian brain are located exclusively in the tuberomammillary nucleus of the posterior hypothalamus and their axons extend throughout the central nervous system ( cns ) . four known histamine receptors and histamine binding to glutamate nmda receptors serve multiple functions in the brain , particularly control of excitability and plasticity ( haas and panula , 2003 ; haas et al . , the h1 and h2 receptor - mediated actions are mostly excitatory , while h3 receptors act as inhibitory auto- and heteroreceptors . as a stimulator , neuronal histamine is one of the most important systems that stimulate and maintain attentive wakefulness . we have examined the functions of histamine neurons using various approaches , such as histamine - related gene knockout mice and human positron emission tomography ( pet ) ( yanai and tashiro , 2007 ) . histamine neurons play an important role in the forebrain waking systems ( lin , 2000 ; eriksson et al . , 2001 ; their neuronal activity is specific for a high - vigilance waking state and histamine neurons might play a role not in the initiation of wakefulness , but in maintenance of the high level of vigilance necessary for cognitive processes ( takahashi et al . , 2008 ; an increase in histaminergic transmission promotes wakefulness , whereas its blockade by sedating antihistamines causes somnolence and impaired performance in humans ( yanai et al . several lines of evidence suggest that histamine modulates circadian rhythms ( tuomisto et al . accordingly , a clear circadian rhythm of histamine release was demonstrated in the anterior hypothalamus by in vivo microdialysis studies ( mochizuki et al . histamine release increased before the active phase and was maintained at an elevated level during the active phase . to date , we have conducted several pet studies to elucidate the pathophysiological mechanism behind the above - mentioned disorders , focusing on alteration in neural transmission of the histaminergic neuron systems ( yanai and tashiro , 2007 ) . for pet studies , [ c]doxepin , a potent antagonist of histamine h1 receptors , has been utilized as an effective pet imaging tracer . using [ c]doxepin , increasing evidence has been accumulated regarding the role of the histaminergic neuron system in the pathophysiology of stress - related neuropsychiatric disorders . for example , histamine h1 receptor binding was measured using [ c]doxepin in patients with schizophrenia ( iwabuchi et al . another application of [ c]doxepin - pet is the measurement of histamine h1 receptor occupancy by antihistamines . the inhibition of histaminergic activity at the brain h1 receptor level may have some favorable anxiolytic effects , however , more often this is accompanied by unfavorable effects like increased daytime somnolence , impaired memory and learning , decreased attention , and weight gain . the brain h1 receptor occupancy measured by [ c]doxepin - pet is one of the most reliable and objective methods to estimate the sedating properties of antihistamines ( yanai et al . a critical step in validating [ c]doxepin to measure the brain h1 receptor occupancy is to evaluate the reproducibility of its binding potential ( bmax / kd ) in the different attentive conditions . 2011 ) , although [ c]doxepin binding to brain has not been examined on this aspect until now . previous studies have shown that imaging with pet radiotracers that are specific for brain receptors can be used to visualize changes in the release of neurotransmitters indirectly . most successful studies have focused on dopamine , since the dopamine neurons that project to the striatum have been shown to play a critical role in mediating motivational and addictive behaviors . these imaging studies successfully measured increased extracellular dopamine released by psychostimulants and physiological reward stimuli in humans with [ c]raclopride ( koepp et al . , 2011 ) and however , some technical difficulties have been encountered for the imaging of changes in the release of other neurotransmitters . among these are low sensitivity , changes of neurotransmitter release pulse over time during pet imaging and the issue of affinity states , the contribution of carryover mass in the second pet scan , and internalization of receptors ( boecker et al . followed by the previous pet measurement of dopamine release , we examined whether neuronal histamine released as a result of mental stress and the circadian rhythm might change the levels of h1 receptors measured in vivo by pet and [ c]doxepin . therefore , we undertook the present study for the purpose of evaluating the test retest reliability of [ c]doxepin binding in the human brain during different attentive conditions and circadian rhythm in healthy human subjects . japanese male volunteers , who were physically and mentally healthy and had no history of allergy or long - term treatment with h1 antagonists , were recruited to participate in this study . the present study was approved by the committee on clinical investigation at tohoku university graduate school of medicine , japan , and was performed in accordance with the principles of the declaration of helsinki . all experiments were performed at the cyclotron and radioisotope center , tohoku university . in the first part of this study involving test retest measurements , six healthy male volunteers ( mean age sd : 24.6 2.1 years old ) were examined twice with [ c]doxepin - pet during a resting condition in the morning ( 11:00 a.m. ) and afternoon ( 3:00 p.m. ) of the same day in order to evaluate the circadian rhythm of h1 receptor binding . in the second part of this study involving investigation of ligand activation , 10 healthy men ( 22.3 1.0 years old ) they were scanned twice by pet ( set2400w ; shimadzu co. , kyoto , japan ) on the same day during attentive calculation tasks involving two - digit addition and during resting conditions with their eyes closed after administration of [ c]doxepin . we performed measurements of subjective feelings five times during pet scans before the scan ( pre ) , at interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of scan ( end ) . the h1 receptors were examined with [ c]doxepin - pet during the resting and calculation conditions . the subjective feelings during pet scans were also measured during the 90 min before the scan ( pre ) , interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of the scan ( end ) . the radiochemical purity of [ c]doxepin was greater than 99% , and its specific radioactivity at the time of injection was 207.5 61.9 gbq/mol ( 5608 1673 mci/mol ) . the single injected dose and cold mass of [ c]doxepin were 119.8 10.5 mbq ( 3.23 0.283 mci ) and 0.577 0.051 nmol , respectively . for the measurement of h1 receptors , pet scans were carried out with an set2400w pet scanner ( shimadzu co. , kyoto , japan ) . pet data were acquired 30 s after the administration of [ c]doxepin with the subject 's eyes closed for 90 min . in order to calculate the binding potential ( bp ) of h1 receptors , brain pet images of each subject during resting and calculation conditions were subjected to inter - frame motion correction and then co - registered to an identical stereotaxic brain coordinate using a corresponding t1-weighted mri image . regions of interest ( roi ) were first placed on the following brain regions on the t1 images for which precise anatomical information was available : anterior and posterior cingulate gyrus , inferior prefrontal cortex , superior prefrontal cortex , temporal cortex , and cerebellum . in addition , we produced a time - activity curve ( tac ) of each region from roi data . a standardized uptake value ( suv ) was calculated for the normalization of roi - tacs as follows : suv ( tac)=tac ( mbq / ml)/[injected tracer dose ( mbq)]/body weight ( g ) subsequently , logan graphical analysis with the reference tissue input ( lgar ) method was applied to calculate bp using pmod kinetic modeling tool ( pkin ) software ( pmod technologies ltd . japanese male volunteers , who were physically and mentally healthy and had no history of allergy or long - term treatment with h1 antagonists , were recruited to participate in this study . the present study was approved by the committee on clinical investigation at tohoku university graduate school of medicine , japan , and was performed in accordance with the principles of the declaration of helsinki . all experiments were performed at the cyclotron and radioisotope center , tohoku university . in the first part of this study involving test retest measurements , six healthy male volunteers ( mean age sd : 24.6 2.1 years old ) were examined twice with [ c]doxepin - pet during a resting condition in the morning ( 11:00 a.m. ) and afternoon ( 3:00 p.m. ) of the same day in order to evaluate the circadian rhythm of h1 receptor binding . in the second part of this study involving investigation of ligand activation , 10 healthy men ( 22.3 1.0 years old ) they were scanned twice by pet ( set2400w ; shimadzu co. , kyoto , japan ) on the same day during attentive calculation tasks involving two - digit addition and during resting conditions with their eyes closed after administration of [ c]doxepin . we performed measurements of subjective feelings five times during pet scans before the scan ( pre ) , at interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of scan ( end ) . the h1 receptors were examined with [ c]doxepin - pet during the resting and calculation conditions . the subjective feelings during pet scans were also measured during the 90 min before the scan ( pre ) , interval 1 ( int1 ) , interval 2 ( int2 ) , interval 3 ( int3 ) , and at the end of the scan ( end ) . the radiochemical purity of [ c]doxepin was greater than 99% , and its specific radioactivity at the time of injection was 207.5 61.9 gbq/mol ( 5608 1673 mci/mol ) . the single injected dose and cold mass of [ c]doxepin were 119.8 10.5 mbq ( 3.23 0.283 mci ) and 0.577 0.051 nmol , respectively . for the measurement of h1 receptors , pet scans were carried out with an set2400w pet scanner ( shimadzu co. , kyoto , japan ) . pet data were acquired 30 s after the administration of [ c]doxepin with the subject 's eyes closed for 90 min . in order to calculate the binding potential ( bp ) of h1 receptors , brain pet images of each subject during resting and calculation conditions were subjected to inter - frame motion correction and then co - registered to an identical stereotaxic brain coordinate using a corresponding t1-weighted mri image . in addition , we produced a time - activity curve ( tac ) of each region from roi data . a standardized uptake value ( suv ) was calculated for the normalization of roi - tacs as follows : suv ( tac)=tac ( mbq / ml)/[injected tracer dose ( mbq)]/body weight ( g ) subsequently , logan graphical analysis with the reference tissue input ( lgar ) method was applied to calculate bp using pmod kinetic modeling tool ( pkin ) software ( pmod technologies ltd . during the performance of the attentive task involving two - digit calculation , the percentage of correct answers was greater than 90% . subjects felt significantly more tired during the calculation task than in the resting condition ( figure 2a ) . subjects in the resting condition tended to have significantly higher sleepiness scores than those in the calculation condition ( figure 2b ) . these data suggest that there was a significant difference in the attention level between the resting and calculation conditions . data are presented as the means sem of lars ( mm ) from 10 healthy subjects in the resting and calculation conditions . six subjects were tested in the evaluation of the test retest reliability of [ c]doxepin pet . given concerns about the possibility that [ c]doxepin binding changes over days and week , the test and re - test trials were performed in the morning ( 11:00 a.m. ) and afternoon ( 3:00 p.m. ) of the same day . as shown in figure 3a , average suvs over time from test retest scans show that the tracer gradually entered the brain and the brain activity remained almost stable . the radioactivity in the anterior cingulate gyrus showed a slightly longer elimination phase in the test trial performed in the morning than that in the afternoon , but the difference was insignificant . the radioactivity in the cerebellum with negligible h1 receptor binding was essentially the same between the test and re - test trials . there was an apparent trend for decreased bp in the afternoon , but the bp values in the brain regions including anterior cingulate gyrus were not significantly higher in the morning test trial ( figure 3b ) , demonstrating that [ c]doxepin binding in the brain is essentially the same between in the morning and afternoon . test ( a ) tacs in terms of suvs for the regions of anterior cingulate gyrus ( ac ) and cerebellum ( cb ) of test retest scans . retest scans were performed in the morning and afternoon of the same day , demonstrating a gradual initial increase in suv followed by a longer elimination phase . in order to verify the effects of attentive waking on in vivo pet measurements of h1 receptor binding , [ c]doxepin binding was examined twice in the brains of 10 normal volunteers during resting and attentive waking conditions on the same day , as shown in figure 4 . the order of rest and calculation trials of pet studies was randomized to eliminate the effects of circadian rhythm on h1 receptor binding observed in previous experiments . the time courses in the regions of anterior cingulate gyrus ( h1 receptor rich region ) and cerebellum ( h1 receptor null region ) were not significantly different between the resting and attentive calculation conditions ( figure 4a ) . there was a trend for decreased bp during the calculation task compared with that in the resting condition , but the difference was not significant . these data suggest that [ c]doxepin binding in the brain is not significantly influenced by the performance of a calculation task as an example of an attentive waking condition ( a ) tacs in terms of suvs for the regions of anterior cingulate gyrus ( ac ) and cerebellum ( cb ) of resting and attentive waking conditions . ( b ) the binding potential ( bp ) in the resting and attentive conditions . the main objectives of this study were to analyze the specific brain uptake and kinetics of [ c]doxepin in normal volunteers under different conditions , and to assess the test retest reliability of quantitative pet measurements between morning and afternoon and between resting and attentive waking conditions . the test retest reliability for estimated bp was found to be sufficiently high to afford reasonable precision in the tracer binding determinations of h1 receptors . attentive waking and circadian rhythm might have some influences on the bp of h1 receptors , but only within the range of 10% over all regions . the low variability within 10% in the test retest studies can be compatible to other pet tracers such as [ c]dasb ( serotonin transporter ) , [ f]spa - rq ( nk-1 receptor ) , [ c]pib ( amyloid a ) , and [ c]flb457 ( d2/3 receptor ) ( kim et al . evidence from animal studies has implicated the histaminergic neuron system in the pathophysiology of stress - related disorders . although several antidepressants and atypical antipsychotics are potent h1r antagonists , the significance of their interaction with h1r in a clinical context of efficacy is still unclear . in previous pet studies , significant reduction in h1 it is suggested that prolonged massive histamine release due to repeated stress might lead to the down - regulation and/or internalization of h1r , which may result in decreased binding of [ c]doxepin in stress - related disorders ( endou et al . we previously demonstrated that normal female volunteers had significantly higher bp of [ c]doxepin to h1 receptors in the cerebral cortical areas than male volunteers ( yoshizawa et al . for example , there are gender differences in the size of the interstitial nucleus of anterior hypothalamus ( inah ) ( male > female ) . inah in homosexual men is only half the size of the nucleus in heterosexual men . the gender difference in human h1 receptors that we observed is reasonable because histamine neurons are exclusively located in the posterior hypothalamus . the density of histamine h1 receptors was higher in female rats than in male rats ( ghi et al . , 1999 ) , and hypothalamic histamine release was higher in male rats than in female rats ( ferretti et al . it is not ruled out that neuronal histamine release and in vivo h1 receptor binding are closely correlated . therefore , we should carefully consider unknown factors influencing in vivo [ c]doxepin binding in the brain . we succeeded in quantifying the strength of the sedating properties of antihistamines in terms of brain histamine h1 receptor occupancy . we previously reported an age - related decline in h1 receptor binding in normal human brain , especially in the prefrontal , temporal , cingulate , and parahippocampal regions ( yanai et al . this study confirmed the reliability of values in h1 receptor occupancy by antihistamines because different pet studies were summarized to make the figures of occupancy ( yanai et al . this study demonstrates for the first time that subjects ' attentive conditions do not affect the reliability of h1 receptor binding measured by [ c]doxepin - pet . this study does not necessarily rule out the feasibility of measuring neuronal histamine release in the living human brain using pet , although pet tracers with better signal - to - noise properties should be developed in the future . for this purpose , h3 receptor binding would be more appropriate because h3 receptors are easily down - regulated by stress - related histamine release . the previous studies reported that histamine release was significantly increased during stressful conditions , and that the h3 receptor density rapidly decreased in response to stress ( ghi et al . following the development of other histaminergic pet probes , non - invasive measurement of neuronal histamine release the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the 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 .

individual needs , wishes , preferences , and ethics influence the meaning of value which , in turn , is influenced by different cultures or historical periods.1 the necessity of finding better ways of redirecting the incentives away from volume and toward value pushes patients , physicians , policy makers , and other stakeholders to turn their attention toward what value means and what are the main features of this concept.2 even if there is still no unanimous agreement on value s definition , it is commonly accepted that values in health care may be defined as normative guidelines helping us to evaluate actions or situations and influencing the decision - making process.35 different studies point out how the definition of value changes according to the reference sample : doctors values , most of the time , do not match the values of the patients , and vice versa.3,6,7 the presence of different opinions encourages some reputable organizations and associations to base their definitions of value on expert judgment or on empirical studies that correlate attributes of value with a measurable outcome . according to this , the american heart association ( aha ) underlines that , even though clinical efficacy and outcomes constitute the primary basis of good medical practice , value plays together with costs an important role , and it includes positive results in patient s outcome , safety , and satisfaction at a total cost that is reasonable and affordable.6 however , in 2008 , the institute of medicine7 ( iom ) held a 2-day workshop to explore key stakeholders perspectives on value in health care , seeking to understand the meaning of value . finding a mutually acceptable agreement among the different points of view , as expressed by patients , providers , economists , payers , and employers , is understandably complex . in fact , providers considered value on the basis of appropriateness of care and effective , evidence - based interventions ; economic representatives defined value as the clinical benefit achieved for the money spent . patients , however , place their attention on the ability of health care to satisfy their goals : a valuable intervention is a way of treating that also fulfills their needs.8 indeed , from a patient s perspective , the burden of illness is not limited to disease status , but it is also important to consider quality of life ( qol ) factors and , more precisely , health - related qol , referring to its clinical dimension . patient s needs are frequently measured taking into account different aspects of qol , such as pain , emotional and cognitive functioning , or functional impairment.9,10 moreover , a recent review11 on patient s perceptions of quality of care emphasizes how communication , health care access , and shared decision - making ( sdm ) are the key elements in a valuable health care environment . nowadays , even if we narrow our attention to the medical context , we are not able to identify the core features of a health care system based on value because every paradigm adopts different definitions of value . the absence of common and widely accepted meaning allows each movement in health care practice to take into account different components of value identifying different model , of care . the aim of this article is to provide an overview of the main approaches of the last 10 years that have considered value as the key theoretical concept . as we shall see , each movement adopts a particular definition of value leading to a different application of these paradigms in the health care system . two decades ago , the ebm was the first movement in health care that disregarded the paternalistic approach and revolutionized the idea of doing science . the ebm introduced a new way to make good clinical decisions : health care decisions should be based on the best available evidence mixed with the clinical expertise.12 external evidence and clinical expertise must be integrated with patients preferences in making medical decisions about their care ; only in this way , doctors will be able to identify the best interventions to maximize qol of patients and minimize the cost of their care.13 despite its success throughout the scientific world , some researchers have started asking whether this paradigm has been facing a crisis . among different reasons , greenhalgh et al14 suggested that , during the course of the years , ebm has forgotten the importance of individualism . evidence should be understandable by all patients , practitioners , and other stakeholders , and they should share discoveries and fears to take reasonable decisions.14 at the same time , preferences , wishes , thoughts , and all individual aspects of patients were included in the conceptual label of value , becoming like a constellation of principles with an important role in life . following the definition of sackett et al15 with patient values we mean the unique preferences , concerns and expectations each patient brings to a clinical encounter and which must be integrated into clinical decisions if they are to serve the patient . in the last 10 years , to re - emphasize the importance of patients preferences and qol , new paradigms were born , turning their attention toward individual aspects and focusing on patient s value . the term value - based was first introduced by brown et al who defines this new paradigm as the practice of medicine incorporating the highest level of evidence - based data with the patient perceived value conferred by health care interventions for the resources expended.16 ebm focuses its attention on clinical trial results and uses these data to provide the best care and , in the meanwhile , it usually ignores the importance of qol improvement . instead , the vbm leads to a higher level the discoveries of ebm calculating the value of operations in medical practice , based on pharmacoeconomic principles.1 the value is measured objectively by calculating the improvement in qol and life expectancy after surgery : the result is the benefit derived from an intervention for the costs expended.17,18 in other words , vbm takes ebm to a higher level , including the qol in the data analysis and interpreted the data in relation to the value and costs of an intervention . in so doing utility analysis where results could be interpreted in terms of $ /qaly ( quality - adjusted life year ) , considering the dollars spent for the improvement in length of life and/or qol on a continuum from 0.0 ( death ) to 1.0 ( perfect health).19 qaly is the arithmetic product of life expectancy combined with the measurement of the quality of remaining life years.20 the choice of the right methodology had been controversial , and they finally opted for a time trade - off cost utility analysis since it would be applicable in any specialty of medicine , allowing a comparison across different interventions.18,21 time trade - off methodology consists in asking a person how many additional years he or she expects to live and , at the same time , how much time he or she would be willing to trade in return for a treatment that would guarantee their conversion in a normal health state.21,22 in summary , vbm underlies the value aspect missing in ebm . in particular , ebm provides data that are converted to a measure of value through a cost utility analysis and later combined with costs.1 both vbm and ebm are paradigms involved in the growing complexity of decision - making process in health care because values and evidences affect all medical decisions.23,24 in fact , the increasing complication of evidences ( from which clinical decision - making depends ) and values ( on which clinical decision - making is based ) add up to a general growth of complexity in medicine , inspiring the necessity for an evidence and value - based practice ( vbp ) . today , a variety of disciplines play a significant role in clinical decision - making in day - to - day practice : medical humanities , social sciences , decision analysis , and health economics are only some of those that influence patient s choices.2528 consequently , the aim of vbp is to integrate complex and conflicting values in the process of medical decision - making through a skill - based approach where clinicians could make medical decisions on the basis of scientific evidence and social , ethical , and political values.2,29 another way to facilitate clinical decision - making and patient centeredness in health care is to organize the process of care around the concept of value : a health care system based on value has as a key component , the enhancement of patient doctor communication , and the use of sdm . in accordance with this , a study showed that 69% of patients with lung cancer and 81% of patients with colorectal cancer experienced some difficulties in understanding the goals of their treatments and , consequently , they were not able to make aware decisions.30 between 2006 and 2013 at the institute for strategy and competitiveness , based at the harvard business school , porter and teisberg developed and proposed a new health care system based on value , a breakthrough framework for redefining health care competition based on patient value.31 according to value - based principles , a health care system should co - create and measure outcomes that are meaningful for patients with similar needs along the whole care pathway.32 a value - based health system should refer to three important principles.33 first , its proper goal should be the value it provides to the patient . in health care , the meaning of value varies along a continuum where psychological and physical outcomes meet costs . in fact , we can refer to value either from a psychological or from an economic perspective . following porter s idea , value is expressed as the best health outcomes achieved per dollar spent.34 by health outcomes , it is meant the health results that matter for the patient s condition over the care cycle , and by costs , they refer to the total costs of care for patient s condition over the care cycle . second , treatment delivery should be based on medical conditions , and on the course of treatment , a patient has to undergo treatment and , finally , outcomes should be measurable and recorded.35 data must be collected along the entire patient cycle of care because the outcomes achieved are more effective measures than the number of services delivered that we could not previously know if they are properly and successfully used . moreover , outcomes should be interpreted on the basis of the true costs effectively delivered across the full care cycle because cost reduction without considering outcomes is dangerous and self - defeating . value - based approaches consider cost measurement in a similar way to outcomes measurement , that is , around individual patients for all their care , rather than the cost of each organization delivering care.33,36 a particular example where cost measurement is always integrated with other patient s outcomes measurement is represented by the area of rare diseases . in such context , treatment options are not so widely available , and they are very expensive ; the treatment care plan is usually highly personalized and centered on patient s health status and well - being taking into account the budget and patients rated outcomes.37,38 the right strategy for a high - value health care delivery system also includes the organization of care around customer s preferences , implementing an integrated practice unit where multidisciplinary personnel works together to serve groups of patients with similar primary and preventive care needs.32,35 the attention toward the health care system shifts from a volume of activity approach to a value - based system , focused on results concretely obtained:34 as porter and lee affirm , this proposal is an two decades ago , the ebm was the first movement in health care that disregarded the paternalistic approach and revolutionized the idea of doing science . the ebm introduced a new way to make good clinical decisions : health care decisions should be based on the best available evidence mixed with the clinical expertise.12 external evidence and clinical expertise must be integrated with patients preferences in making medical decisions about their care ; only in this way , doctors will be able to identify the best interventions to maximize qol of patients and minimize the cost of their care.13 despite its success throughout the scientific world , some researchers have started asking whether this paradigm has been facing a crisis . among different reasons , greenhalgh et al14 suggested that , during the course of the years , ebm has forgotten the importance of individualism . evidence should be understandable by all patients , practitioners , and other stakeholders , and they should share discoveries and fears to take reasonable decisions.14 at the same time , preferences , wishes , thoughts , and all individual aspects of patients were included in the conceptual label of value , becoming like a constellation of principles with an important role in life . following the definition of sackett et al15 with patient values we mean the unique preferences , concerns and expectations each patient brings to a clinical encounter and which must be integrated into clinical decisions if they are to serve the patient . in the last 10 years , to re - emphasize the importance of patients preferences and qol , new paradigms were born , turning their attention toward individual aspects and focusing on patient s value . the term value - based was first introduced by brown et al who defines this new paradigm as the practice of medicine incorporating the highest level of evidence - based data with the patient perceived value conferred by health care interventions for the resources expended.16 ebm focuses its attention on clinical trial results and uses these data to provide the best care and , in the meanwhile , it usually ignores the importance of qol improvement . instead , the vbm leads to a higher level the discoveries of ebm calculating the value of operations in medical practice , based on pharmacoeconomic principles.1 the value is measured objectively by calculating the improvement in qol and life expectancy after surgery : the result is the benefit derived from an intervention for the costs expended.17,18 in other words , vbm takes ebm to a higher level , including the qol in the data analysis and interpreted the data in relation to the value and costs of an intervention . in so doing , vbm utilizes a health care economic cost utility analysis where results could be interpreted in terms of $ /qaly ( quality - adjusted life year ) , considering the dollars spent for the improvement in length of life and/or qol on a continuum from 0.0 ( death ) to 1.0 ( perfect health).19 qaly is the arithmetic product of life expectancy combined with the measurement of the quality of remaining life years.20 the choice of the right methodology had been controversial , and they finally opted for a time trade - off cost utility analysis since it would be applicable in any specialty of medicine , allowing a comparison across different interventions.18,21 time trade - off methodology consists in asking a person how many additional years he or she expects to live and , at the same time , how much time he or she would be willing to trade in return for a treatment that would guarantee their conversion in a normal health state.21,22 in summary , vbm underlies the value aspect missing in ebm . in particular , ebm provides data that are converted to a measure of value through a cost utility analysis and later combined with costs.1 both vbm and ebm are paradigms involved in the growing complexity of decision - making process in health care because values and evidences affect all medical decisions.23,24 in fact , the increasing complication of evidences ( from which clinical decision - making depends ) and values ( on which clinical decision - making is based ) add up to a general growth of complexity in medicine , inspiring the necessity for an evidence and value - based practice ( vbp ) . today , a variety of disciplines play a significant role in clinical decision - making in day - to - day practice : medical humanities , social sciences , decision analysis , and health economics are only some of those that influence patient s choices.2528 consequently , the aim of vbp is to integrate complex and conflicting values in the process of medical decision - making through a skill - based approach where clinicians could make medical decisions on the basis of scientific evidence and social , ethical , and political values.2,29 another way to facilitate clinical decision - making and patient centeredness in health care is to organize the process of care around the concept of value : a health care system based on value has as a key component , the enhancement of patient doctor communication , and the use of sdm . in accordance with this , a study showed that 69% of patients with lung cancer and 81% of patients with colorectal cancer experienced some difficulties in understanding the goals of their treatments and , consequently , they were not able to make aware decisions.30 between 2006 and 2013 at the institute for strategy and competitiveness , based at the harvard business school , porter and teisberg developed and proposed a new health care system based on value , a breakthrough framework for redefining health care competition based on patient value.31 according to value - based principles , a health care system should co - create and measure outcomes that are meaningful for patients with similar needs along the whole care pathway.32 a value - based health system should refer to three important principles.33 first , its proper goal should be the value it provides to the patient . in health care , the meaning of value varies along a continuum where psychological and physical outcomes meet costs . in fact , we can refer to value either from a psychological or from an economic perspective . following porter s idea , value is expressed as the best health outcomes achieved per dollar spent.34 by health outcomes , it is meant the health results that matter for the patient s condition over the care cycle , and by costs , they refer to the total costs of care for patient s condition over the care cycle . second , treatment delivery should be based on medical conditions , and on the course of treatment , a patient has to undergo treatment and , finally , outcomes should be measurable and recorded.35 data must be collected along the entire patient cycle of care because the outcomes achieved are more effective measures than the number of services delivered that we could not previously know if they are properly and successfully used . moreover , outcomes should be interpreted on the basis of the true costs effectively delivered across the full care cycle because cost reduction without considering outcomes is dangerous and self - defeating . value - based approaches consider cost measurement in a similar way to outcomes measurement , that is , around individual patients for all their care , rather than the cost of each organization delivering care.33,36 a particular example where cost measurement is always integrated with other patient s outcomes measurement is represented by the area of rare diseases . in such context , treatment options are not so widely available , and they are very expensive ; the treatment care plan is usually highly personalized and centered on patient s health status and well - being taking into account the budget and patients rated outcomes.37,38 the right strategy for a high - value health care delivery system also includes the organization of care around customer s preferences , implementing an integrated practice unit where multidisciplinary personnel works together to serve groups of patients with similar primary and preventive care needs.32,35 the attention toward the health care system shifts from a volume of activity approach to a value - based system , focused on results concretely obtained:34 as porter and lee affirm , this proposal is an a value - based system goes hand in hand with patient centeredness : today s fragmented health care system should move toward what patients want , considering and respecting their feelings in clinical procedures . health care management is clearly moving toward a patient - centered system ( pcs ) , where outcomes achieved and units organized around the patient s needs are the main guidelines for the delivery of high - value care.32,40 in fact , the iom defines patient - centered care as care that is respectful of and respective to individual patient preference , needs and values , and ensure that patient values guide all clinical decisions.41,42 biological information must be integrated with beliefs and cognitive dispositions to empower patients and make them active participant in the treatment process.43 these features are also stressed by the p5 medicine approach to build a medicine that involves both medical and psycho - cognitive aspects . the health care system is moving toward a p5 medicine , which is a predictive , personalized , preventive , participatory , and psycho - cognitive medicine . the fifth p refers to an integration among needs , values , cognitive dispositions , and medical information , between psychological and biological aspects.41 the necessity of a psychological and cognitive profile , instead of a mere diagnostic patient s classification , leads to an assessment with psychometric tools that include cognitive , decision - making , and mental aspects , as well as clinical ones . the consideration of all these aspects is important to empower the patient , improve his / her qol , and move toward their becoming an active decision - maker.43 to obtain a complete evaluation of medical treatment and to determine its value , it is necessary both balancing the costs with the benefits and considering the range of patient preferences or costs offsets : only with a careful assessment of all these aspects , high - value care will be provided . if we do not keep in mind outcomes , safety , and patient satisfaction , we could run into unexpected costs and lower level of care . nowadays , different studies and updated clinical practice guidelines underline the importance of heeding both costs and values if we want a health care system effectively patient centered . new normative guidelines should include value and cost to achieve best performance measures and improve all health care system , as we can read on the report of american college of cardiology / aha statement on cost / value methodology.6 these guidelines should give some important recommendations for appropriate exercise testing in patients with cardiovascular disease.44 on the basis of these recommendations , a recent randomized trial45 regarding diagnostic testing in women with suspected coronary artery disease demonstrated how the only difference between two groups ( treadmill exercise electrocardiographic testing vs exercise myocardial perfusion imaging ) was costs and not outcomes : the first intervention was more cost - effective and less invasive than the second one . a good physician should be able to communicate in the right way with patients and to assess their health literacy and comprehension regarding different treatment options , possible benefits , and harm.46 these features are fundamental in the clinical decision - making process and are a cornerstone in a value - based health system . indeed , one of the main recommendations to implement a value - based health system is to actively involve the patient in his or her process of care.33 at the same time , medicine and the whole health care system are facing a shift toward the patient empowerment paradigm that is linked with different aspects of patient participation . although there is no unanimous agreement , current studies define this paradigm as a multidimensional concept where communication , decision , and health care system combined together and converge in the enhancement of the patient.47,48 every definition emphasizes a specific aspect of the empowerment process : awareness , responsibility , participation in the care process , sdm , and patient doctor communication . an empowered patient has developed specific abilities to interact with the health care system and to make better choices that include value for him or her . an empowerment process allows the patient to be an active and effective participant in their process of care and to look for valuable and useful information for their health . this is why empowerment should be considered in each step of the value chain and should influence the decision - making process in a pcs . these features are also prominent in the value health care model and attach importance to psychological aspects influencing the process of care because empowered patients become effective people , able to choose valuable paths of care and of life . the value - based system refers to a personalized care , where patient expectations and needs are included in a holistic approach of medicine that considers physical , mental , and spiritual well - being . physicians and patients should change their points of view , implementing a new process of care where they are actively and equally involved , each of them with their expertise : one with clinical knowledge and the other with his / her life . patient empowerment , value - based system , and p5 medicine seem to shed light on different aspects of a pcs , and this allows a better understanding of patients and their families in managing health and health care . therefore , the value - based health system and patient empowerment should be considered together , two sides of the same coin . patient s value may be fully expressed only when patients are the main actors of their care , and this means when they are fully empowered .

in the last 10 years , value has played a key role in the health care system . in this concept , innovations in medical practice and the increasing importance of patient centeredness have contributed to draw the attention of the medical community . nonetheless , a large consensus on the meaning of value is still lacking : patients , physicians , policy makers , and other health care professionals have different ideas on which component of value may play a prominent role . yet , shared clinical decision - making and patient empowerment have been recognized as fundamental features of the concept of value . different paradigms of health care system embrace different meanings of value , and the absence of common and widely accepted definition does not help to identify a unique model of care in health care system . our aim is to provide an overview of those paradigms that have considered value as a key theoretical concept and to investigate how the presence of value can influence the medical practice . this article may contribute to draw attention toward patients and propose a possible link between health care system based on value and new paradigms such as patient - centered system ( pcs ) , patient empowerment , and p5 medicine , in order to create a predictive , personalized , preventive , participatory , and psycho - cognitive model to treat patients . indeed , patient empowerment , value - based system , and p5 medicine seem to shed light on different aspects of a pcs , and this allows a better understanding of people under care .

Introduction From evidence-based medicine (EBM) toward value-based medicine (VBM) A health care system based on value Discussion Conclusion

individual needs , wishes , preferences , and ethics influence the meaning of value which , in turn , is influenced by different cultures or historical periods.1 the necessity of finding better ways of redirecting the incentives away from volume and toward value pushes patients , physicians , policy makers , and other stakeholders to turn their attention toward what value means and what are the main features of this concept.2 even if there is still no unanimous agreement on value s definition , it is commonly accepted that values in health care may be defined as normative guidelines helping us to evaluate actions or situations and influencing the decision - making process.35 different studies point out how the definition of value changes according to the reference sample : doctors values , most of the time , do not match the values of the patients , and vice versa.3,6,7 the presence of different opinions encourages some reputable organizations and associations to base their definitions of value on expert judgment or on empirical studies that correlate attributes of value with a measurable outcome . according to this , the american heart association ( aha ) underlines that , even though clinical efficacy and outcomes constitute the primary basis of good medical practice , value plays together with costs an important role , and it includes positive results in patient s outcome , safety , and satisfaction at a total cost that is reasonable and affordable.6 however , in 2008 , the institute of medicine7 ( iom ) held a 2-day workshop to explore key stakeholders perspectives on value in health care , seeking to understand the meaning of value . in fact , providers considered value on the basis of appropriateness of care and effective , evidence - based interventions ; economic representatives defined value as the clinical benefit achieved for the money spent . patients , however , place their attention on the ability of health care to satisfy their goals : a valuable intervention is a way of treating that also fulfills their needs.8 indeed , from a patient s perspective , the burden of illness is not limited to disease status , but it is also important to consider quality of life ( qol ) factors and , more precisely , health - related qol , referring to its clinical dimension . patient s needs are frequently measured taking into account different aspects of qol , such as pain , emotional and cognitive functioning , or functional impairment.9,10 moreover , a recent review11 on patient s perceptions of quality of care emphasizes how communication , health care access , and shared decision - making ( sdm ) are the key elements in a valuable health care environment . nowadays , even if we narrow our attention to the medical context , we are not able to identify the core features of a health care system based on value because every paradigm adopts different definitions of value . the absence of common and widely accepted meaning allows each movement in health care practice to take into account different components of value identifying different model , of care . the aim of this article is to provide an overview of the main approaches of the last 10 years that have considered value as the key theoretical concept . as we shall see , each movement adopts a particular definition of value leading to a different application of these paradigms in the health care system . the ebm introduced a new way to make good clinical decisions : health care decisions should be based on the best available evidence mixed with the clinical expertise.12 external evidence and clinical expertise must be integrated with patients preferences in making medical decisions about their care ; only in this way , doctors will be able to identify the best interventions to maximize qol of patients and minimize the cost of their care.13 despite its success throughout the scientific world , some researchers have started asking whether this paradigm has been facing a crisis . evidence should be understandable by all patients , practitioners , and other stakeholders , and they should share discoveries and fears to take reasonable decisions.14 at the same time , preferences , wishes , thoughts , and all individual aspects of patients were included in the conceptual label of value , becoming like a constellation of principles with an important role in life . in the last 10 years , to re - emphasize the importance of patients preferences and qol , new paradigms were born , turning their attention toward individual aspects and focusing on patient s value . the term value - based was first introduced by brown et al who defines this new paradigm as the practice of medicine incorporating the highest level of evidence - based data with the patient perceived value conferred by health care interventions for the resources expended.16 ebm focuses its attention on clinical trial results and uses these data to provide the best care and , in the meanwhile , it usually ignores the importance of qol improvement . instead , the vbm leads to a higher level the discoveries of ebm calculating the value of operations in medical practice , based on pharmacoeconomic principles.1 the value is measured objectively by calculating the improvement in qol and life expectancy after surgery : the result is the benefit derived from an intervention for the costs expended.17,18 in other words , vbm takes ebm to a higher level , including the qol in the data analysis and interpreted the data in relation to the value and costs of an intervention . in so doing utility analysis where results could be interpreted in terms of $ /qaly ( quality - adjusted life year ) , considering the dollars spent for the improvement in length of life and/or qol on a continuum from 0.0 ( death ) to 1.0 ( perfect health).19 qaly is the arithmetic product of life expectancy combined with the measurement of the quality of remaining life years.20 the choice of the right methodology had been controversial , and they finally opted for a time trade - off cost utility analysis since it would be applicable in any specialty of medicine , allowing a comparison across different interventions.18,21 time trade - off methodology consists in asking a person how many additional years he or she expects to live and , at the same time , how much time he or she would be willing to trade in return for a treatment that would guarantee their conversion in a normal health state.21,22 in summary , vbm underlies the value aspect missing in ebm . in particular , ebm provides data that are converted to a measure of value through a cost utility analysis and later combined with costs.1 both vbm and ebm are paradigms involved in the growing complexity of decision - making process in health care because values and evidences affect all medical decisions.23,24 in fact , the increasing complication of evidences ( from which clinical decision - making depends ) and values ( on which clinical decision - making is based ) add up to a general growth of complexity in medicine , inspiring the necessity for an evidence and value - based practice ( vbp ) . today , a variety of disciplines play a significant role in clinical decision - making in day - to - day practice : medical humanities , social sciences , decision analysis , and health economics are only some of those that influence patient s choices.2528 consequently , the aim of vbp is to integrate complex and conflicting values in the process of medical decision - making through a skill - based approach where clinicians could make medical decisions on the basis of scientific evidence and social , ethical , and political values.2,29 another way to facilitate clinical decision - making and patient centeredness in health care is to organize the process of care around the concept of value : a health care system based on value has as a key component , the enhancement of patient doctor communication , and the use of sdm . in accordance with this , a study showed that 69% of patients with lung cancer and 81% of patients with colorectal cancer experienced some difficulties in understanding the goals of their treatments and , consequently , they were not able to make aware decisions.30 between 2006 and 2013 at the institute for strategy and competitiveness , based at the harvard business school , porter and teisberg developed and proposed a new health care system based on value , a breakthrough framework for redefining health care competition based on patient value.31 according to value - based principles , a health care system should co - create and measure outcomes that are meaningful for patients with similar needs along the whole care pathway.32 a value - based health system should refer to three important principles.33 first , its proper goal should be the value it provides to the patient . in health care , the meaning of value varies along a continuum where psychological and physical outcomes meet costs . following porter s idea , value is expressed as the best health outcomes achieved per dollar spent.34 by health outcomes , it is meant the health results that matter for the patient s condition over the care cycle , and by costs , they refer to the total costs of care for patient s condition over the care cycle . second , treatment delivery should be based on medical conditions , and on the course of treatment , a patient has to undergo treatment and , finally , outcomes should be measurable and recorded.35 data must be collected along the entire patient cycle of care because the outcomes achieved are more effective measures than the number of services delivered that we could not previously know if they are properly and successfully used . in such context , treatment options are not so widely available , and they are very expensive ; the treatment care plan is usually highly personalized and centered on patient s health status and well - being taking into account the budget and patients rated outcomes.37,38 the right strategy for a high - value health care delivery system also includes the organization of care around customer s preferences , implementing an integrated practice unit where multidisciplinary personnel works together to serve groups of patients with similar primary and preventive care needs.32,35 the attention toward the health care system shifts from a volume of activity approach to a value - based system , focused on results concretely obtained:34 as porter and lee affirm , this proposal is an two decades ago , the ebm was the first movement in health care that disregarded the paternalistic approach and revolutionized the idea of doing science . the ebm introduced a new way to make good clinical decisions : health care decisions should be based on the best available evidence mixed with the clinical expertise.12 external evidence and clinical expertise must be integrated with patients preferences in making medical decisions about their care ; only in this way , doctors will be able to identify the best interventions to maximize qol of patients and minimize the cost of their care.13 despite its success throughout the scientific world , some researchers have started asking whether this paradigm has been facing a crisis . evidence should be understandable by all patients , practitioners , and other stakeholders , and they should share discoveries and fears to take reasonable decisions.14 at the same time , preferences , wishes , thoughts , and all individual aspects of patients were included in the conceptual label of value , becoming like a constellation of principles with an important role in life . in the last 10 years , to re - emphasize the importance of patients preferences and qol , new paradigms were born , turning their attention toward individual aspects and focusing on patient s value . the term value - based was first introduced by brown et al who defines this new paradigm as the practice of medicine incorporating the highest level of evidence - based data with the patient perceived value conferred by health care interventions for the resources expended.16 ebm focuses its attention on clinical trial results and uses these data to provide the best care and , in the meanwhile , it usually ignores the importance of qol improvement . instead , the vbm leads to a higher level the discoveries of ebm calculating the value of operations in medical practice , based on pharmacoeconomic principles.1 the value is measured objectively by calculating the improvement in qol and life expectancy after surgery : the result is the benefit derived from an intervention for the costs expended.17,18 in other words , vbm takes ebm to a higher level , including the qol in the data analysis and interpreted the data in relation to the value and costs of an intervention . in so doing , vbm utilizes a health care economic cost utility analysis where results could be interpreted in terms of $ /qaly ( quality - adjusted life year ) , considering the dollars spent for the improvement in length of life and/or qol on a continuum from 0.0 ( death ) to 1.0 ( perfect health).19 qaly is the arithmetic product of life expectancy combined with the measurement of the quality of remaining life years.20 the choice of the right methodology had been controversial , and they finally opted for a time trade - off cost utility analysis since it would be applicable in any specialty of medicine , allowing a comparison across different interventions.18,21 time trade - off methodology consists in asking a person how many additional years he or she expects to live and , at the same time , how much time he or she would be willing to trade in return for a treatment that would guarantee their conversion in a normal health state.21,22 in summary , vbm underlies the value aspect missing in ebm . in particular , ebm provides data that are converted to a measure of value through a cost utility analysis and later combined with costs.1 both vbm and ebm are paradigms involved in the growing complexity of decision - making process in health care because values and evidences affect all medical decisions.23,24 in fact , the increasing complication of evidences ( from which clinical decision - making depends ) and values ( on which clinical decision - making is based ) add up to a general growth of complexity in medicine , inspiring the necessity for an evidence and value - based practice ( vbp ) . today , a variety of disciplines play a significant role in clinical decision - making in day - to - day practice : medical humanities , social sciences , decision analysis , and health economics are only some of those that influence patient s choices.2528 consequently , the aim of vbp is to integrate complex and conflicting values in the process of medical decision - making through a skill - based approach where clinicians could make medical decisions on the basis of scientific evidence and social , ethical , and political values.2,29 another way to facilitate clinical decision - making and patient centeredness in health care is to organize the process of care around the concept of value : a health care system based on value has as a key component , the enhancement of patient doctor communication , and the use of sdm . in accordance with this , a study showed that 69% of patients with lung cancer and 81% of patients with colorectal cancer experienced some difficulties in understanding the goals of their treatments and , consequently , they were not able to make aware decisions.30 between 2006 and 2013 at the institute for strategy and competitiveness , based at the harvard business school , porter and teisberg developed and proposed a new health care system based on value , a breakthrough framework for redefining health care competition based on patient value.31 according to value - based principles , a health care system should co - create and measure outcomes that are meaningful for patients with similar needs along the whole care pathway.32 a value - based health system should refer to three important principles.33 first , its proper goal should be the value it provides to the patient . in health care , the meaning of value varies along a continuum where psychological and physical outcomes meet costs . following porter s idea , value is expressed as the best health outcomes achieved per dollar spent.34 by health outcomes , it is meant the health results that matter for the patient s condition over the care cycle , and by costs , they refer to the total costs of care for patient s condition over the care cycle . second , treatment delivery should be based on medical conditions , and on the course of treatment , a patient has to undergo treatment and , finally , outcomes should be measurable and recorded.35 data must be collected along the entire patient cycle of care because the outcomes achieved are more effective measures than the number of services delivered that we could not previously know if they are properly and successfully used . in such context , treatment options are not so widely available , and they are very expensive ; the treatment care plan is usually highly personalized and centered on patient s health status and well - being taking into account the budget and patients rated outcomes.37,38 the right strategy for a high - value health care delivery system also includes the organization of care around customer s preferences , implementing an integrated practice unit where multidisciplinary personnel works together to serve groups of patients with similar primary and preventive care needs.32,35 the attention toward the health care system shifts from a volume of activity approach to a value - based system , focused on results concretely obtained:34 as porter and lee affirm , this proposal is an a value - based system goes hand in hand with patient centeredness : today s fragmented health care system should move toward what patients want , considering and respecting their feelings in clinical procedures . health care management is clearly moving toward a patient - centered system ( pcs ) , where outcomes achieved and units organized around the patient s needs are the main guidelines for the delivery of high - value care.32,40 in fact , the iom defines patient - centered care as care that is respectful of and respective to individual patient preference , needs and values , and ensure that patient values guide all clinical decisions.41,42 biological information must be integrated with beliefs and cognitive dispositions to empower patients and make them active participant in the treatment process.43 these features are also stressed by the p5 medicine approach to build a medicine that involves both medical and psycho - cognitive aspects . the health care system is moving toward a p5 medicine , which is a predictive , personalized , preventive , participatory , and psycho - cognitive medicine . the fifth p refers to an integration among needs , values , cognitive dispositions , and medical information , between psychological and biological aspects.41 the necessity of a psychological and cognitive profile , instead of a mere diagnostic patient s classification , leads to an assessment with psychometric tools that include cognitive , decision - making , and mental aspects , as well as clinical ones . the consideration of all these aspects is important to empower the patient , improve his / her qol , and move toward their becoming an active decision - maker.43 to obtain a complete evaluation of medical treatment and to determine its value , it is necessary both balancing the costs with the benefits and considering the range of patient preferences or costs offsets : only with a careful assessment of all these aspects , high - value care will be provided . new normative guidelines should include value and cost to achieve best performance measures and improve all health care system , as we can read on the report of american college of cardiology / aha statement on cost / value methodology.6 these guidelines should give some important recommendations for appropriate exercise testing in patients with cardiovascular disease.44 on the basis of these recommendations , a recent randomized trial45 regarding diagnostic testing in women with suspected coronary artery disease demonstrated how the only difference between two groups ( treadmill exercise electrocardiographic testing vs exercise myocardial perfusion imaging ) was costs and not outcomes : the first intervention was more cost - effective and less invasive than the second one . a good physician should be able to communicate in the right way with patients and to assess their health literacy and comprehension regarding different treatment options , possible benefits , and harm.46 these features are fundamental in the clinical decision - making process and are a cornerstone in a value - based health system . indeed , one of the main recommendations to implement a value - based health system is to actively involve the patient in his or her process of care.33 at the same time , medicine and the whole health care system are facing a shift toward the patient empowerment paradigm that is linked with different aspects of patient participation . although there is no unanimous agreement , current studies define this paradigm as a multidimensional concept where communication , decision , and health care system combined together and converge in the enhancement of the patient.47,48 every definition emphasizes a specific aspect of the empowerment process : awareness , responsibility , participation in the care process , sdm , and patient doctor communication . this is why empowerment should be considered in each step of the value chain and should influence the decision - making process in a pcs . patient empowerment , value - based system , and p5 medicine seem to shed light on different aspects of a pcs , and this allows a better understanding of patients and their families in managing health and health care . therefore , the value - based health system and patient empowerment should be considered together , two sides of the same coin .

hospital - acquired infections ( hais ) are a significant problem in canadian health care , leading to higher mortality , longer hospitalization and increased use of health care resources [ 1 , 2 ] . we have recently published observations on the prevalence of hais in adult post - cardiac surgery patients at one canadian cardiac surgery center , showing an increased prevalence over an 18-year period from 8% in 1995 to 20% in 2013 . furthermore , this occurred despite advances in infection control practices that have been implemented to allow the institution to receive national accreditation . it is also important to highlight the fact that we demonstrated that our observed rates of increased infection occurred independent of patient risk factors such as age , urgency or important co - morbidities . while this trend in hais is alarming and of particular interest are previous studies that have shown that infections are commonly diagnosed with limited clinical criteria and often treated without lab confirmation . beyond this antimicrobial stewardship suggests that patients with unconfirmed infections in which antimicrobials are discontinued after 3 days have similar outcomes to those who receive full treatment . physician stewardship is believed to be a key component in reducing the prevalence of antimicrobial resistant bacteria , which involves the proper administration of antimicrobials . early initiation of treatment and minimization of its duration has been shown to reduce the probability of antimicrobial resistance epidemics in hospitals . diagnosing hais in postoperative cardiac patients can often be difficult due to several causes other than infection that lead to inflammation , such as tissue damage and systemic inflammatory response syndrome ( sirs ) . systemic antimicrobials administered before , during and after surgery may also contribute to lower blood culture yields , making confirmation of infection more difficult . while these factors may cause problems in positively identifying infection , verification of infection is still a very important aspect of patient care as it allows physicians to prescribe the optimal antimicrobial as well as cease treatment if lab results are negative and/or clinical improvement occurs . in addition , community and hospital - acquired antimicrobial resistant bacteria are becoming a more prevalent global problem and measures to mitigate infection by these bacteria species need to be undertaken to avoid further complications in patients . the practice of documenting , confirming or verifying hais has yet to be adequately studied or reviewed . in the present study , we extended our previously published findings to conduct a detailed chart review from a 2-month sample period of patients that underwent cardiac surgery at tertiary cardiac care center . patients acquiring at least one type of infection postoperatively were identified and further analyzed to better assess current physician practices . all consecutive patients who underwent cardiac surgical procedures during may and june 2013 at the queen elizabeth ii health sciences center ( qeii hsc ) in halifax , canada were identified . the period of time chosen for this audit represents 20% of the annual volume of cardiac surgery performed in halifax . the qeii hsc is the only cardiac surgical center in the province of nova scotia and serves a population of almost 1 million . the maritime heart center ( mhc ) registry is a prospective registry that has been used since 1995 and provides data on all cardiac surgical patients undergoing procedures at the qeii hsc . what is unique about this study compared to previous work by our group is that the registry data were supplemented using the horizon patient folder ( hpf ) database . hpf represents the pdf s version of all the actual medical records of each individual patient treated at the qeii hsc . institutional established standard operating procedures have been in place and continue to follow national guidelines at the qeii hsc . standard practice includes the use of prophylactic antimicrobials administration prior to skin incision ( first generation cephalosporin or vancomycin in penicillin - allergic patients ) . skin preparation has remained largely surgeon - dependent and included a combination of iodine and alcohol or a chlorhexidine - based solution . placement of central catheters prior to surgery was done under strict sterile conditions in the operating room by anesthesiologists . urinary catheters were also placed under sterile conditions in the operating room and were removed as soon as patients began to ambulate . standard pneumonia prevention protocols were in place during the study period and included mouth care , subglottic secretion drainage and semi - recumbent position . patients were classified as having a postoperative infection if at least one of the following three criteria were met : a wound opening that involved excision of tissue ; positive culture from respiratory secretions or pleural fluid , wound , blood , or urine ; or antibiotic treatment based on clinical suspicion . the society of thoracic surgeons ( sts ) data definitions were used to define how an infection was captured by the mhc registry and represent a well - known benchmark in cardiac surgical literature ( sts.org ) . we selected six types of infections based on recently published work by our group and due to their high relevance for our study population . these included : 1 ) urinary tract infection ( uti ) , 2 ) pneumonia , 3 ) harvest site infections , 4 ) superficial sternal site infection ( sssi ) , 5 ) sepsis , and 6 ) deep sternal site infection ( dssi ) . clinical characteristics of patients that were identified as having an infection or not were examined univariately . data captured also included : type of infection , verification through laboratory microbiology cultures , date of bacteria detection , type of antimicrobials used , duration of antimicrobial treatment , whether antimicrobials were discontinued after suspected infection was confirmed negative , and the type of bacteria identified . continuous variables were compared using student s t - test and categorical variables were analyzed using fischer s exact test . the prism 6 ( graph pad prism6 ) statistical software package was used to complete all statistical analyses . the study was completed with full approval from the institutional ( capital district health authority ) research ethics board ( reb ) . given the retrospective nature of the work , all consecutive patients who underwent cardiac surgical procedures during may and june 2013 at the queen elizabeth ii health sciences center ( qeii hsc ) in halifax , canada were identified . the period of time chosen for this audit represents 20% of the annual volume of cardiac surgery performed in halifax . the qeii hsc is the only cardiac surgical center in the province of nova scotia and serves a population of almost 1 million . the maritime heart center ( mhc ) registry is a prospective registry that has been used since 1995 and provides data on all cardiac surgical patients undergoing procedures at the qeii hsc . what is unique about this study compared to previous work by our group is that the registry data were supplemented using the horizon patient folder ( hpf ) database . hpf represents the pdf s version of all the actual medical records of each individual patient treated at the qeii hsc . institutional established standard operating procedures have been in place and continue to follow national guidelines at the qeii hsc . standard practice includes the use of prophylactic antimicrobials administration prior to skin incision ( first generation cephalosporin or vancomycin in penicillin - allergic patients ) . skin preparation has remained largely surgeon - dependent and included a combination of iodine and alcohol or a chlorhexidine - based solution . placement of central catheters prior to surgery was done under strict sterile conditions in the operating room by anesthesiologists . urinary catheters were also placed under sterile conditions in the operating room and were removed as soon as patients began to ambulate . standard pneumonia prevention protocols were in place during the study period and included mouth care , subglottic secretion drainage and semi - recumbent position . patients were classified as having a postoperative infection if at least one of the following three criteria were met : a wound opening that involved excision of tissue ; positive culture from respiratory secretions or pleural fluid , wound , blood , or urine ; or antibiotic treatment based on clinical suspicion . the society of thoracic surgeons ( sts ) data definitions were used to define how an infection was captured by the mhc registry and represent a well - known benchmark in cardiac surgical literature ( sts.org ) . we selected six types of infections based on recently published work by our group and due to their high relevance for our study population . these included : 1 ) urinary tract infection ( uti ) , 2 ) pneumonia , 3 ) harvest site infections , 4 ) superficial sternal site infection ( sssi ) , 5 ) sepsis , and 6 ) deep sternal site infection ( dssi ) . clinical characteristics of patients that were identified as having an infection or not were examined univariately . data captured also included : type of infection , verification through laboratory microbiology cultures , date of bacteria detection , type of antimicrobials used , duration of antimicrobial treatment , whether antimicrobials were discontinued after suspected infection was confirmed negative , and the type of bacteria identified . continuous variables were compared using student s t - test and categorical variables were analyzed using fischer s exact test . the prism 6 ( graph pad prism6 ) statistical software package was used to complete all statistical analyses . the study was completed with full approval from the institutional ( capital district health authority ) research ethics board ( reb ) . given the retrospective nature of the work , a waiver of consent was granted by the reb . during the time period of may and june 2013 , 185 consecutive patients underwent cardiac surgical procedures requiring a heart - lung machine at the qeii hsc . most patients were male ( 82% ) , had an average age of 64.4 12.6 years , underwent isolated coronary artery bypass graft ( cabg ) procedures ( 52% ) , and were hospitalized prior to intervention ( 51% ) . preoperatively , 28% had diabetes mellitus , 11% had a low ejection fraction ( 40% ) , and 8% had renal insufficiency ( creatinine > 176 mmol / l ) . a total of 39 patients ( 21% ) developed at least one hai during their indexed cardiac surgical admission , defined from the time of the operation until discharge from hospital ( table 1 ) . all infection definitions were based on established sts definitions and represent the standard of reporting in the cardiac surgical literature . unadjusted findings suggest that infection occurs more often in patients presenting with the following clinical conditions : older age , male sex , low ejection fraction , peripheral vascular disease or cerebral vascular disease , renal insufficiency , diabetes mellitus , congestive heart failure , chronic obstructive pulmonary disease ( copd ) , and complex procedures other than isolated cabg . the most common infection was utis ( 8% ) , followed by pneumonia ( 7% ) , leg harvest site infection ( 5% ) , and sssi ( 4% ) . sepsis was the least common infection and was only seen in 2% of patients ( table 2 ) . a total of six patients developed more than one type of infection during their hospital admission . cabg : coronary artery bypass graft ; chf : congestive heart failure ; copd : chronic obstructive pulmonary disease ; cr : creatinine ; cvd : cerebral vascular disease ; ef : ejection fraction ; isol : isolated ; nyha : new york heart association ; pvd : peripheral vascular disease ; n / s : not significant . los : length of stay ; sssi : superficial surgical site infection ; uti : urinary tract infection . however , the in - house mortality of patients with an hai was 10% , being much higher than patients without an infection , which was 2% ( p = 0.0369 ) . similarly the median los for patients with an hai was 14 days compared to 8 days for those who did not contract an infection ( p 0.0001 ) . this study used a standard definition of los exceeding 9 days as prolonged los , consistent with our previous study . in patients that contracted an hai , all individual patient records were reviewed to determine the circumstances surrounding the diagnosis of an infection . specifically , confirmation with bacteriology differed significantly across infection types : 100% in patients diagnosed with sepsis , 67% in patients with utis and pneumonia , 25% in sssi , and only 10% in patients with a leg infection ( fig . the average time for the diagnosis of infection postoperatively was 6.3 4.0 days ( fig . 2 ) . there was no significant difference in the detection times between infection types analyzed , ranging from 5.8 days for leg infections to 7.2 days for pneumonia . a large diversity of bacterial species was detected in the period studied and varied based on infection type . specific bacteria associated with each type of infection were identified ( table 3 ) . to the best of our knowledge , there was no case of methicillin - resistant staphylococcus aureus ( mrsa ) or vancomycin - resistant enterococcus ( vre ) during the study period . uti : urinary tract infection ; leginf : leg infection ; infstsup : superficial surgical site infection . infstsup : superficial surgical site infection ; uti : urinary tract infection ; leginf : leg infection . the range of treatment duration was from 4 to 16 days . however , overall antimicrobial treatment duration was consistent across infection types with the exception of superficial surgical site infections , which had median treatment duration that was 3 days greater than all other infections being investigated ( fig . this was in spite of 16/39 ( 41% ) infections not confirmed by a positive culture . all patients with unverified hais did not have antimicrobials discontinued after negative cultures were obtained . infstsup : superficial surgical site infection ; leginf : leg infection ; uti : urinary tract infection . during the time period of may and june 2013 , 185 consecutive patients underwent cardiac surgical procedures requiring a heart - lung machine at the qeii hsc . most patients were male ( 82% ) , had an average age of 64.4 12.6 years , underwent isolated coronary artery bypass graft ( cabg ) procedures ( 52% ) , and were hospitalized prior to intervention ( 51% ) . preoperatively , 28% had diabetes mellitus , 11% had a low ejection fraction ( 40% ) , and 8% had renal insufficiency ( creatinine > 176 mmol / l ) . a total of 39 patients ( 21% ) developed at least one hai during their indexed cardiac surgical admission , defined from the time of the operation until discharge from hospital ( table 1 ) . all infection definitions were based on established sts definitions and represent the standard of reporting in the cardiac surgical literature . unadjusted findings suggest that infection occurs more often in patients presenting with the following clinical conditions : older age , male sex , low ejection fraction , peripheral vascular disease or cerebral vascular disease , renal insufficiency , diabetes mellitus , congestive heart failure , chronic obstructive pulmonary disease ( copd ) , and complex procedures other than isolated cabg . the most common infection was utis ( 8% ) , followed by pneumonia ( 7% ) , leg harvest site infection ( 5% ) , and sssi ( 4% ) . sepsis was the least common infection and was only seen in 2% of patients ( table 2 ) . a total of six patients developed more than one type of infection during their hospital admission . cabg : coronary artery bypass graft ; chf : congestive heart failure ; copd : chronic obstructive pulmonary disease ; cr : creatinine ; cvd : cerebral vascular disease ; ef : ejection fraction ; isol : isolated ; nyha : new york heart association ; pvd : peripheral vascular disease ; n / s : not significant . los : length of stay ; sssi : superficial surgical site infection ; uti : urinary tract infection . however , the in - house mortality of patients with an hai was 10% , being much higher than patients without an infection , which was 2% ( p = 0.0369 ) . similarly the median los for patients with an hai was 14 days compared to 8 days for those who did not contract an infection ( p 0.0001 ) . this study used a standard definition of los exceeding 9 days as prolonged los , consistent with our previous study . in patients that contracted an hai , all individual patient records were reviewed to determine the circumstances surrounding the diagnosis of an infection . specifically , confirmation with bacteriology differed significantly across infection types : 100% in patients diagnosed with sepsis , 67% in patients with utis and pneumonia , 25% in sssi , and only 10% in patients with a leg infection ( fig . the average time for the diagnosis of infection postoperatively was 6.3 4.0 days ( fig . 2 ) . there was no significant difference in the detection times between infection types analyzed , ranging from 5.8 days for leg infections to 7.2 days for pneumonia . a large diversity of bacterial species was detected in the period studied and varied based on infection type . specific bacteria associated with each type of infection were identified ( table 3 ) . to the best of our knowledge , there was no case of methicillin - resistant staphylococcus aureus ( mrsa ) or vancomycin - resistant enterococcus ( vre ) during the study period . uti : urinary tract infection ; leginf : leg infection ; infstsup : superficial surgical site infection . infstsup : superficial surgical site infection ; uti : urinary tract infection ; leginf : leg infection . the range of treatment duration was from 4 to 16 days . however , overall antimicrobial treatment duration was consistent across infection types with the exception of superficial surgical site infections , which had median treatment duration that was 3 days greater than all other infections being investigated ( fig . this was in spite of 16/39 ( 41% ) infections not confirmed by a positive culture . all patients with unverified hais did not have antimicrobials discontinued after negative cultures were obtained . infstsup : superficial surgical site infection ; leginf : leg infection ; uti : urinary tract infection . the present study represents a 2-month detailed review of all reported infection occurring in all cardiac surgery performed at the only cardiac center for the province of nova scotia , canada . in our study period , the overall rate of hais was 21% ( 39/185 ) , similar to recently published data suggesting that hai prevalence in cardiac surgery patients was 20% in the last 5 years and comparable to similar patient populations [ 3 , 11 , 12 ] . the present study was not designed to focus on risk factors for hai as previously published , but instead prompted the present review of current clinical practice in the setting of suspected infection . we specifically looked at how commonly suspected hais were treated , verified through microbiology tests and in the case of unconfirmed hais , if antimicrobial treatment was discontinued . we found that a significant number ( 38% ) of suspected infections were not verified or confirmed by positive microbiology cultures . furthermore , our findings demonstrate that in all patients in which negative cultures were obtained ( 16 out of 39 ) , none of these patients had antimicrobial treatment discontinued . this is not surprising for infections such as sssi where bacterial confirmation may not be of much clinical value . however , our findings do show that clinicians appear to generally continue a course of antimicrobials even in the context of clinical improvement and lack of correlating evidence such as bacteriology . evidence supporting antimicrobial stewardship by treating clinicians has been limited in cardiac surgery but shown in other areas to be safe and appropriate [ 5 , 10 , 13 ] . effective antimicrobial stewardship relies on selecting the best drug at an optimal dosage and duration to effectively treat an infection while simultaneously reducing the selection of antimicrobial resistant bacteria . numerous studies have shown that antimicrobial use can lead to selective pressure for resistance and other side effects [ 14 - 16 ] . one should note , however , that there were no cases of antimicrobial resistant bacteria in the period studied , such as mrsa or vre and these low numbers are consistent with what we have published before . our findings highlight physician s tendency to continue treatment with antimicrobials in times where it has been demonstrated in select cases to safely discontinue these drugs and limit antimicrobial use [ 15 , 17 ] . our findings are in keeping with similar reports looking at nosocomial pneumonia where it was shown that once antimicrobials were prescribed , it was continued in all patients , including those with negative cultures . although the effects of improperly prescribing antimicrobials are commonly known among physicians , translating knowledge into policies that are adequate and effectively implemented still remains a significant problem . there are many published guidelines on the implementation of effective antimicrobial stewardship , which involves streamlining or de - escalating therapy once cultures are received . it is believed that by doing so , the causative pathogen can be more appropriately treated , selective pressure for antimicrobial resistant bacteria is decreased , and cost savings can be achieved . we show here that while these guidelines are well known , their translation into clinical practice may not be optimal and require more concerted effort . we have shown with this study the prevalence of different types of infections in postoperative cardiac surgery patients . consistent with previous studies , utis and pneumonia are the most common types of infections postoperatively while sepsis occurs in a much smaller segment of patients [ 1 , 3 ] . the overall rate of infection was within the range of other studies investigating hais , which ranged from 7% to 20% [ 11 , 12 ] . the time until detection of infection was consistent across the different types identified , averaging 6.3 days . we believe the narrow range of detection time across infections may be due to similarities in the patient population such as comorbidities and types of surgical procedures performed and reflect a clean type of procedure in which infection should be rare in the first few days after surgery . treatment duration was also consistent across infections allowing a 7 - 10 day course of antibiotics with the exception of surgical site infections , which had a median duration of 3 days greater than all other infections . we speculate this may be due to the significant concern physicians have regarding surgical site infections in patients requiring a sternotomy to avoid a deep sternal wound complication which is known to be associated with significant morbidity and mortality . while the sample size of the present study was relatively small , consisting of 185 patients , our findings were consistent with a recently published study from our group that looked at nearly 20,000 patients undergoing cardiac surgery . what is unique about our study is that it contains very detailed patient information obtained from direct chart review rather than registry data only . this included information on pathogens , antimicrobials administered , duration of treatment , and diagnostic criteria used . we acknowledge that examining infection at a single institution can not address infections specific to all other regions , limiting the generalizability of our findings . however , the present study is from a large regional center responsible for all advanced cardiac care for a province in canada . furthermore , the qeii hsc has received full national accreditation including implementation of national standards regarding hai prevention , making the conclusions relevant for canadian health care providers . despite these limitations , furthermore , we have shown that common types of infection are generally approached in a similar fashion by the clinician . this means that when suspected infections are treated clinicians prescribe a full course of antimicrobials without discontinuation even after negative lab results are received . while this study was not designed to review the application of published guidelines for antimicrobial stewardship , it does suggest some difficulty in translating this knowledge to clinical practice . furthermore , we have shown that common types of infection are generally approached in a similar fashion by the clinician . this means that when suspected infections are treated clinicians prescribe a full course of antimicrobials without discontinuation even after negative lab results are received . while this study was not designed to review the application of published guidelines for antimicrobial stewardship , it does suggest some difficulty in translating this knowledge to clinical practice . hospital acquired infection systemic inflammatory response syndrome queen elizabeth ii health sciences center maritime heart center horizon patient folder society of thoracic surgeons urinary tract infection superficial sternal site infection deep sternal site infection coronary artery bypass graft chronic obstructive pulmonary disease methicillin - resistant staphylococcus aureus vancomycin - resistant enterococcus

backgroundthe management of hospital - acquired infections ( hais ) with respect to physician practices remains largely unexplored despite increasing efforts to standardize care . in the present study , we report findings from a 2-month audit of all patients that have undergone cardiac surgery at a large referral center in atlantic canada.methodsall patients who underwent cardiac surgical procedures during may and june 2013 at the queen elizabeth ii health sciences center in halifax , nova scotia were identified . the prevalence of urinary tract infections ( utis ) , pneumonia , leg harvest site infections , superficial sternal wound infections , deep sternal wound infections , and sepsis was examined to determine physician approaches in terms of verification rates ( microbiology ) , time of diagnosis and duration of treatment . continuous variables were compared using student s t - test and categorical variables were analyzed using fischer s exact test.resultsa total of 185 consecutive patients underwent cardiac surgical procedures , of which 39 ( 21% ) developed at least one postoperative infection . the overall prevalence of infection types , from highest to lowest , was uti ( 8% ) , pneumonia ( 7% ) , leg harvest site infection ( 5% ) , superficial surgical site infection ( 4% ) , and sepsis ( 2% ) . there were no deep sternal wound infections . the overall in - hospital mortality rate was 3.8% with a median length of stay ( los ) of 8 days . the overall infection verification rate was 50% ( ranged from 100% in sepsis to 10% in leg harvest site infections ) . in all cases , a full course of antibiotics was administered despite negative microbiology cultures or limited evidence of an actual infection.conclusionshais are commonly treated without being verified and treatment is often not discontinued after negative cultures are received . our findings highlight the fact that antibiotic treatment is not always supported by evidence , and the effect of this could contribute to increased selective pressure for antimicrobial resistant bacteria .

Introduction Methods Setting and patients Standard procedures related to HAIs Study design Ethics approval and consent to participate Results Patient population Variation in clinical practice with regard to HAIs Discussion Conclusions Abbreviations

hospital - acquired infections ( hais ) are a significant problem in canadian health care , leading to higher mortality , longer hospitalization and increased use of health care resources [ 1 , 2 ] . we have recently published observations on the prevalence of hais in adult post - cardiac surgery patients at one canadian cardiac surgery center , showing an increased prevalence over an 18-year period from 8% in 1995 to 20% in 2013 . furthermore , this occurred despite advances in infection control practices that have been implemented to allow the institution to receive national accreditation . it is also important to highlight the fact that we demonstrated that our observed rates of increased infection occurred independent of patient risk factors such as age , urgency or important co - morbidities . while this trend in hais is alarming and of particular interest are previous studies that have shown that infections are commonly diagnosed with limited clinical criteria and often treated without lab confirmation . beyond this antimicrobial stewardship suggests that patients with unconfirmed infections in which antimicrobials are discontinued after 3 days have similar outcomes to those who receive full treatment . physician stewardship is believed to be a key component in reducing the prevalence of antimicrobial resistant bacteria , which involves the proper administration of antimicrobials . early initiation of treatment and minimization of its duration has been shown to reduce the probability of antimicrobial resistance epidemics in hospitals . systemic antimicrobials administered before , during and after surgery may also contribute to lower blood culture yields , making confirmation of infection more difficult . while these factors may cause problems in positively identifying infection , verification of infection is still a very important aspect of patient care as it allows physicians to prescribe the optimal antimicrobial as well as cease treatment if lab results are negative and/or clinical improvement occurs . in addition , community and hospital - acquired antimicrobial resistant bacteria are becoming a more prevalent global problem and measures to mitigate infection by these bacteria species need to be undertaken to avoid further complications in patients . the practice of documenting , confirming or verifying hais has yet to be adequately studied or reviewed . in the present study , we extended our previously published findings to conduct a detailed chart review from a 2-month sample period of patients that underwent cardiac surgery at tertiary cardiac care center . patients acquiring at least one type of infection postoperatively were identified and further analyzed to better assess current physician practices . all consecutive patients who underwent cardiac surgical procedures during may and june 2013 at the queen elizabeth ii health sciences center ( qeii hsc ) in halifax , canada were identified . the period of time chosen for this audit represents 20% of the annual volume of cardiac surgery performed in halifax . the qeii hsc is the only cardiac surgical center in the province of nova scotia and serves a population of almost 1 million . the maritime heart center ( mhc ) registry is a prospective registry that has been used since 1995 and provides data on all cardiac surgical patients undergoing procedures at the qeii hsc . hpf represents the pdf s version of all the actual medical records of each individual patient treated at the qeii hsc . institutional established standard operating procedures have been in place and continue to follow national guidelines at the qeii hsc . placement of central catheters prior to surgery was done under strict sterile conditions in the operating room by anesthesiologists . urinary catheters were also placed under sterile conditions in the operating room and were removed as soon as patients began to ambulate . patients were classified as having a postoperative infection if at least one of the following three criteria were met : a wound opening that involved excision of tissue ; positive culture from respiratory secretions or pleural fluid , wound , blood , or urine ; or antibiotic treatment based on clinical suspicion . the society of thoracic surgeons ( sts ) data definitions were used to define how an infection was captured by the mhc registry and represent a well - known benchmark in cardiac surgical literature ( sts.org ) . these included : 1 ) urinary tract infection ( uti ) , 2 ) pneumonia , 3 ) harvest site infections , 4 ) superficial sternal site infection ( sssi ) , 5 ) sepsis , and 6 ) deep sternal site infection ( dssi ) . clinical characteristics of patients that were identified as having an infection or not were examined univariately . data captured also included : type of infection , verification through laboratory microbiology cultures , date of bacteria detection , type of antimicrobials used , duration of antimicrobial treatment , whether antimicrobials were discontinued after suspected infection was confirmed negative , and the type of bacteria identified . continuous variables were compared using student s t - test and categorical variables were analyzed using fischer s exact test . given the retrospective nature of the work , all consecutive patients who underwent cardiac surgical procedures during may and june 2013 at the queen elizabeth ii health sciences center ( qeii hsc ) in halifax , canada were identified . the period of time chosen for this audit represents 20% of the annual volume of cardiac surgery performed in halifax . the qeii hsc is the only cardiac surgical center in the province of nova scotia and serves a population of almost 1 million . the maritime heart center ( mhc ) registry is a prospective registry that has been used since 1995 and provides data on all cardiac surgical patients undergoing procedures at the qeii hsc . hpf represents the pdf s version of all the actual medical records of each individual patient treated at the qeii hsc . institutional established standard operating procedures have been in place and continue to follow national guidelines at the qeii hsc . placement of central catheters prior to surgery was done under strict sterile conditions in the operating room by anesthesiologists . urinary catheters were also placed under sterile conditions in the operating room and were removed as soon as patients began to ambulate . patients were classified as having a postoperative infection if at least one of the following three criteria were met : a wound opening that involved excision of tissue ; positive culture from respiratory secretions or pleural fluid , wound , blood , or urine ; or antibiotic treatment based on clinical suspicion . the society of thoracic surgeons ( sts ) data definitions were used to define how an infection was captured by the mhc registry and represent a well - known benchmark in cardiac surgical literature ( sts.org ) . these included : 1 ) urinary tract infection ( uti ) , 2 ) pneumonia , 3 ) harvest site infections , 4 ) superficial sternal site infection ( sssi ) , 5 ) sepsis , and 6 ) deep sternal site infection ( dssi ) . clinical characteristics of patients that were identified as having an infection or not were examined univariately . data captured also included : type of infection , verification through laboratory microbiology cultures , date of bacteria detection , type of antimicrobials used , duration of antimicrobial treatment , whether antimicrobials were discontinued after suspected infection was confirmed negative , and the type of bacteria identified . continuous variables were compared using student s t - test and categorical variables were analyzed using fischer s exact test . given the retrospective nature of the work , a waiver of consent was granted by the reb . during the time period of may and june 2013 , 185 consecutive patients underwent cardiac surgical procedures requiring a heart - lung machine at the qeii hsc . most patients were male ( 82% ) , had an average age of 64.4 12.6 years , underwent isolated coronary artery bypass graft ( cabg ) procedures ( 52% ) , and were hospitalized prior to intervention ( 51% ) . preoperatively , 28% had diabetes mellitus , 11% had a low ejection fraction ( 40% ) , and 8% had renal insufficiency ( creatinine > 176 mmol / l ) . a total of 39 patients ( 21% ) developed at least one hai during their indexed cardiac surgical admission , defined from the time of the operation until discharge from hospital ( table 1 ) . all infection definitions were based on established sts definitions and represent the standard of reporting in the cardiac surgical literature . unadjusted findings suggest that infection occurs more often in patients presenting with the following clinical conditions : older age , male sex , low ejection fraction , peripheral vascular disease or cerebral vascular disease , renal insufficiency , diabetes mellitus , congestive heart failure , chronic obstructive pulmonary disease ( copd ) , and complex procedures other than isolated cabg . the most common infection was utis ( 8% ) , followed by pneumonia ( 7% ) , leg harvest site infection ( 5% ) , and sssi ( 4% ) . sepsis was the least common infection and was only seen in 2% of patients ( table 2 ) . a total of six patients developed more than one type of infection during their hospital admission . los : length of stay ; sssi : superficial surgical site infection ; uti : urinary tract infection . however , the in - house mortality of patients with an hai was 10% , being much higher than patients without an infection , which was 2% ( p = 0.0369 ) . similarly the median los for patients with an hai was 14 days compared to 8 days for those who did not contract an infection ( p 0.0001 ) . in patients that contracted an hai , all individual patient records were reviewed to determine the circumstances surrounding the diagnosis of an infection . specifically , confirmation with bacteriology differed significantly across infection types : 100% in patients diagnosed with sepsis , 67% in patients with utis and pneumonia , 25% in sssi , and only 10% in patients with a leg infection ( fig . the average time for the diagnosis of infection postoperatively was 6.3 4.0 days ( fig . there was no significant difference in the detection times between infection types analyzed , ranging from 5.8 days for leg infections to 7.2 days for pneumonia . a large diversity of bacterial species was detected in the period studied and varied based on infection type . specific bacteria associated with each type of infection were identified ( table 3 ) . uti : urinary tract infection ; leginf : leg infection ; infstsup : superficial surgical site infection . infstsup : superficial surgical site infection ; uti : urinary tract infection ; leginf : leg infection . the range of treatment duration was from 4 to 16 days . however , overall antimicrobial treatment duration was consistent across infection types with the exception of superficial surgical site infections , which had median treatment duration that was 3 days greater than all other infections being investigated ( fig . all patients with unverified hais did not have antimicrobials discontinued after negative cultures were obtained . infstsup : superficial surgical site infection ; leginf : leg infection ; uti : urinary tract infection . during the time period of may and june 2013 , 185 consecutive patients underwent cardiac surgical procedures requiring a heart - lung machine at the qeii hsc . most patients were male ( 82% ) , had an average age of 64.4 12.6 years , underwent isolated coronary artery bypass graft ( cabg ) procedures ( 52% ) , and were hospitalized prior to intervention ( 51% ) . preoperatively , 28% had diabetes mellitus , 11% had a low ejection fraction ( 40% ) , and 8% had renal insufficiency ( creatinine > 176 mmol / l ) . a total of 39 patients ( 21% ) developed at least one hai during their indexed cardiac surgical admission , defined from the time of the operation until discharge from hospital ( table 1 ) . all infection definitions were based on established sts definitions and represent the standard of reporting in the cardiac surgical literature . unadjusted findings suggest that infection occurs more often in patients presenting with the following clinical conditions : older age , male sex , low ejection fraction , peripheral vascular disease or cerebral vascular disease , renal insufficiency , diabetes mellitus , congestive heart failure , chronic obstructive pulmonary disease ( copd ) , and complex procedures other than isolated cabg . the most common infection was utis ( 8% ) , followed by pneumonia ( 7% ) , leg harvest site infection ( 5% ) , and sssi ( 4% ) . sepsis was the least common infection and was only seen in 2% of patients ( table 2 ) . a total of six patients developed more than one type of infection during their hospital admission . los : length of stay ; sssi : superficial surgical site infection ; uti : urinary tract infection . however , the in - house mortality of patients with an hai was 10% , being much higher than patients without an infection , which was 2% ( p = 0.0369 ) . similarly the median los for patients with an hai was 14 days compared to 8 days for those who did not contract an infection ( p 0.0001 ) . in patients that contracted an hai , all individual patient records were reviewed to determine the circumstances surrounding the diagnosis of an infection . specifically , confirmation with bacteriology differed significantly across infection types : 100% in patients diagnosed with sepsis , 67% in patients with utis and pneumonia , 25% in sssi , and only 10% in patients with a leg infection ( fig . the average time for the diagnosis of infection postoperatively was 6.3 4.0 days ( fig . there was no significant difference in the detection times between infection types analyzed , ranging from 5.8 days for leg infections to 7.2 days for pneumonia . a large diversity of bacterial species was detected in the period studied and varied based on infection type . specific bacteria associated with each type of infection were identified ( table 3 ) . uti : urinary tract infection ; leginf : leg infection ; infstsup : superficial surgical site infection . infstsup : superficial surgical site infection ; uti : urinary tract infection ; leginf : leg infection . the range of treatment duration was from 4 to 16 days . however , overall antimicrobial treatment duration was consistent across infection types with the exception of superficial surgical site infections , which had median treatment duration that was 3 days greater than all other infections being investigated ( fig . all patients with unverified hais did not have antimicrobials discontinued after negative cultures were obtained . infstsup : superficial surgical site infection ; leginf : leg infection ; uti : urinary tract infection . the present study represents a 2-month detailed review of all reported infection occurring in all cardiac surgery performed at the only cardiac center for the province of nova scotia , canada . in our study period , the overall rate of hais was 21% ( 39/185 ) , similar to recently published data suggesting that hai prevalence in cardiac surgery patients was 20% in the last 5 years and comparable to similar patient populations [ 3 , 11 , 12 ] . the present study was not designed to focus on risk factors for hai as previously published , but instead prompted the present review of current clinical practice in the setting of suspected infection . we specifically looked at how commonly suspected hais were treated , verified through microbiology tests and in the case of unconfirmed hais , if antimicrobial treatment was discontinued . we found that a significant number ( 38% ) of suspected infections were not verified or confirmed by positive microbiology cultures . furthermore , our findings demonstrate that in all patients in which negative cultures were obtained ( 16 out of 39 ) , none of these patients had antimicrobial treatment discontinued . this is not surprising for infections such as sssi where bacterial confirmation may not be of much clinical value . however , our findings do show that clinicians appear to generally continue a course of antimicrobials even in the context of clinical improvement and lack of correlating evidence such as bacteriology . evidence supporting antimicrobial stewardship by treating clinicians has been limited in cardiac surgery but shown in other areas to be safe and appropriate [ 5 , 10 , 13 ] . effective antimicrobial stewardship relies on selecting the best drug at an optimal dosage and duration to effectively treat an infection while simultaneously reducing the selection of antimicrobial resistant bacteria . numerous studies have shown that antimicrobial use can lead to selective pressure for resistance and other side effects [ 14 - 16 ] . one should note , however , that there were no cases of antimicrobial resistant bacteria in the period studied , such as mrsa or vre and these low numbers are consistent with what we have published before . our findings highlight physician s tendency to continue treatment with antimicrobials in times where it has been demonstrated in select cases to safely discontinue these drugs and limit antimicrobial use [ 15 , 17 ] . our findings are in keeping with similar reports looking at nosocomial pneumonia where it was shown that once antimicrobials were prescribed , it was continued in all patients , including those with negative cultures . although the effects of improperly prescribing antimicrobials are commonly known among physicians , translating knowledge into policies that are adequate and effectively implemented still remains a significant problem . there are many published guidelines on the implementation of effective antimicrobial stewardship , which involves streamlining or de - escalating therapy once cultures are received . it is believed that by doing so , the causative pathogen can be more appropriately treated , selective pressure for antimicrobial resistant bacteria is decreased , and cost savings can be achieved . we have shown with this study the prevalence of different types of infections in postoperative cardiac surgery patients . the overall rate of infection was within the range of other studies investigating hais , which ranged from 7% to 20% [ 11 , 12 ] . the time until detection of infection was consistent across the different types identified , averaging 6.3 days . we believe the narrow range of detection time across infections may be due to similarities in the patient population such as comorbidities and types of surgical procedures performed and reflect a clean type of procedure in which infection should be rare in the first few days after surgery . treatment duration was also consistent across infections allowing a 7 - 10 day course of antibiotics with the exception of surgical site infections , which had a median duration of 3 days greater than all other infections . we speculate this may be due to the significant concern physicians have regarding surgical site infections in patients requiring a sternotomy to avoid a deep sternal wound complication which is known to be associated with significant morbidity and mortality . while the sample size of the present study was relatively small , consisting of 185 patients , our findings were consistent with a recently published study from our group that looked at nearly 20,000 patients undergoing cardiac surgery . this included information on pathogens , antimicrobials administered , duration of treatment , and diagnostic criteria used . we acknowledge that examining infection at a single institution can not address infections specific to all other regions , limiting the generalizability of our findings . however , the present study is from a large regional center responsible for all advanced cardiac care for a province in canada . despite these limitations , furthermore , we have shown that common types of infection are generally approached in a similar fashion by the clinician . this means that when suspected infections are treated clinicians prescribe a full course of antimicrobials without discontinuation even after negative lab results are received . while this study was not designed to review the application of published guidelines for antimicrobial stewardship , it does suggest some difficulty in translating this knowledge to clinical practice . furthermore , we have shown that common types of infection are generally approached in a similar fashion by the clinician . this means that when suspected infections are treated clinicians prescribe a full course of antimicrobials without discontinuation even after negative lab results are received . while this study was not designed to review the application of published guidelines for antimicrobial stewardship , it does suggest some difficulty in translating this knowledge to clinical practice . hospital acquired infection systemic inflammatory response syndrome queen elizabeth ii health sciences center maritime heart center horizon patient folder society of thoracic surgeons urinary tract infection superficial sternal site infection deep sternal site infection coronary artery bypass graft chronic obstructive pulmonary disease methicillin - resistant staphylococcus aureus vancomycin - resistant enterococcus

it has been generally held that about half of the human genome is derived from mobile genomic elements , but according to a recent estimate over two - thirds of our genome may result from the presence or ancient activity of ' jumping genes ' . this massive amount of dna includes domesticated elements with evolutionarily spectacular functions , such as the rag ( recombination activating ) genes that form the basis of v(d)j recombination in our immune system , and the ervwe1 ( endogenous retrovirus group w , member 1 ) gene that plays a role in placental development . contrary to their evolutionary gifts , mobile elements are most notorious for being junk or for causing life - threatening human disorders by insertional mutagenesis and homologous recombination . as we usher in the era of genome - scale studies , it is clear that these elements have the potential to cause intra - individual and inter - individual variation and probably common disease through structural variation , deregulated transcriptional activity or epigenetic effects . furthermore , large - scale studies have expanded the pool of human disorders resulting from retrotransposon - mediated insertional mutagenesis . recent reviews have discussed the technical aspects of these new methods [ 5 - 8 ] . we focus here on the known , as well as inferred , potential health impact of their novel findings . dna transposons move by a cut - and - paste mechanism , while retrotransposons mobilize by a copy - and - paste mechanism via an rna intermediate , a process called retrotransposition . retrotransposons can be further subdivided into long terminal repeat ( ltr ) and non - ltr elements . ltr retrotransposons are human endogenous retroviruses ( hervs ) that have an intracellular existence as a result of a non - functional envelope gene . non - ltr elements are classified as long interspersed elements ( lines ; the prototype of which is the rna polymerase ii transcribed line-1 ( l1 ) ) , and short interspersed elements ( sines ) , the latter consisting essentially of rna polymerase iii transcribed alus . svas ( sine - r / vntr ( variable number of tandem repeat)/alu ) are also active non - ltr retrotransposable elements that are intermediate in size relative to alus and l1s , and are likely to be transcribed by rna polymerase ii . l1s are the only known autonomously active human retrotransposons , as only they encode proteins ( open reading frame 1 ( orf1 ) and orf2 ) with which they can be mobilized . l1s are also responsible for the mobilization of the non - autonomous alus , svas [ 10 - 13 ] and processed pseudogenes , which are cellular mrnas that are reverse transcribed and inserted into the genome . accordingly , no human disease is known to arise as a result of their activity . also , hervs are thought to be retrotransposition defective in humans , but some may have retained their ability to move . for example , herv - k113 has intact orfs and has insertional polymorphisms in the human population , implying recent evolutionary activity . although no insertional mutagenesis by hervs has been described , oncogenic etv1 ( ets variant 1)-herv - k fusions generated by chromosomal translocation have been observed in prostate cancer , and herv expression has been suggested as a potential contributor to autoimmune diseases . furthermore , syncytin , an endogenous retroviral envelope protein playing a role in placental trophoblast cell fusion , is involved in breast cancer - endothelial cell and endometrial carcinoma cell fusions . also , a ltr of a malr human endogenous retrovirus has been shown to aberrantly activate a proto - oncogene , thereby causing lymphoma . the predominant mechanism by which l1s cause disease is insertional mutagenesis into or near genes . l1 insertions are often accompanied by 3 ' transduction , the co - mobilization of dna sequences downstream of an l1 as a consequence of transcriptional read - through resulting from a weak l1 poly(a ) signal [ 24 - 27 ] . alu sequences predominantly cause disease by homologous recombination between two alu sequences , but insertion into or near exons , and aberrant alu splicing from introns , also frequently result in pathological conditions [ 28 - 30 ] . furthermore , alu rna toxicity , a new disease - causing phenomenon , has been recently proposed to result in macular degeneration by dicer1 deficit . regarding the role of alu elements in eye disorders , it is interesting that an alu insertion polymorphism in the ace ( angiotensin i converting enzyme ) gene has been associated with protection from the dry / atrophic form of age - related macular degeneration . sva elements also have the ability to interrupt genes through insertional mutagenesis that can be coupled with 3 ' transduction , genomic deletion or aberrant splicing [ 11,33 - 35 ] . the wide spectrum of disease cases caused by retrotransposons ranges from hemophilia to muscular dystrophy and cancer , and has been thoroughly reviewed [ 36 - 38 ] . there have been 96 known retrotransposon insertions in disease cases , of which 25 are caused by l1s , while the other 71 are also l1-mediated . among the latter , 60 cases are attributable to alus , 7 to svas , and 4 to truncated inserts with only poly(a ) sequence remaining . overall , retrotransposon insertions account for about 1 in 250 ( 0.4% ) of disease - causing mutations . processed pseudogenes have not yet been found to cause human disease by de novo insertional mutagenesis , but facioscapulohumeral dystrophy has been demonstrated to arise as a result of the contraction of macrosatellite repeats leading to aberrant expression of an array of dux4 retrogenes residing within the repeats . , mutations in utp14c have been associated with male infertility , mutations in tacstd2/m1s1 result in gelatinous drop - like corneal dystrophy , and ptenp1 is selectively lost in human cancers . the main characteristics of mobile elements capable of causing human disease are summarized in figure 1 . env , envelope ; gag , group specific antigen ; herv , human endogenous retrovirus ; kb , kilobase ; ltr , long terminal repeat ; orf , open reading frame ; pol , polymerase ; sine , short interspersed element ; sva , sine - r / vntr / alu ; tprt , target primed reverse transcription ; utr , untranslated region ; vntr , variable number of tandem repeats . . we discuss large - scale genome , transcriptome and methylation profiling studies of mobile elements in human diseases . we also discuss some non - genome - scale studies that support or contradict the implications of these novel findings . high - throughput sequencing has increased our capacity to generate large datasets at an unprecedented resolution . it is now possible to characterize genome sequences of scarce samples or even single cells . a next - generation sequencing technique with a high coverage of germline polymorphic human - specific l1 ( l1hs ) retrotransposition events has been developed by ewing and kazazian , comprising hemi - specific pcr coupled to illumina sequencing . using this approach , it has been demonstrated that many l1hs elements are population - specific , and recapitulate genetic ancestry similar to alu insertion polymorphisms . retrotransposons are not only excellent markers for exploring population history , but can also give rise to population - specific diseases . for example , a homozygous alu insertion in an exon of the mak ( male germ - cell - associated kinase ) gene has been identified in 21 patients of jewish ancestry who were diagnosed with retinitis pigmentosa . oddly , the discovery of this mutation using agilent exome capture and subsequent illumina and abi sequencing was paradoxical , as attempts to remove repetitive sequences from the analysis led to the identification of the insertion . another population - specific disease caused by retrotransposon mutagenesis is fukuyama - type congenital muscular dystrophy . it is one of the most common autosomal recessive disorders in japan and was the first human disease found to result from ancestral insertion of an sva element . a similar example of an apparently ethnic - specific retrotransposon allele - mediated disease is an l1-mediated orphan 3 ' transduction into the dystrophin gene leading to duchenne muscular dystrophy in a japanese boy . an unexpected finding of state - of - the - art , large - scale approaches to study retrotransposon insertions has been that highly active ( or ' hot ' ) l1s are much more abundant in humans than previously appreciated . the outcome of a fosmid - based paired - end dna sequencing strategy , coupled with a cell culture assay for retrotransposition , was that over half of newly identified l1s are hot , expanding the number of known hot l1s from 6 to 43 . these l1s are not only expected to be a major source of inter - individual genetic variation , but hot l1s account for most examples of disease - causing insertions . another unusual finding of genome - scale approaches in retrotransposon biology has been that retrotransposition occurs at a very high frequency in somatic cells . specifically , the brain was announced to be a bona fide territory for retrotransposition . among three somatic tissues tested , this organ supported the highest level of endogenous l1 copy number , as assessed by quantitative pcr ( qpcr ) . in another study that awaits further validation , over 7,700 l1s , 13,600 alus and 1,300 sva putative somatic insertions were found in the hippocampus of three individuals using retrotransposon capture sequencing , which is based on transposon array capture followed by illumina paired - end sequencing . surprisingly , in this study , l1 and alu insertions were over - represented in protein - coding genes and targeted genes , such as hdac1 ( histone deacetylase 1 ) and rai1 ( retinoic acid induced 1 ) , which are known to be mutated in neurological disorders . these findings suggest that if a retrotransposon inserts into a gene that functions in neurological development or psychological functioning early in development , it might affect a large enough area of the brain to lead to disease . one might further speculate that retrotransposition in a single brain cell could have some physiological consequences or impact memory formation through altered extracellular signaling to neighboring neurons . if such neuronal plasticity exists , it could affect behavioral phenotypes , and could be modulated by environmental factors . conversely , knockdown of an l1 regulating cellular factor has demonstrated an effect on l1 retrotransposition in the neurodevelopmental disorder rett syndrome . mecp2 ( methyl cpg binding protein 2 ) has been shown to repress l1 expression and retrotransposition , and increased l1 retrotransposition has been observed in induced pluripotent stem cells of patients with rett syndrome who carry mecp2 mutations . ten brain tumors were examined for somatic l1 insertions by 454 pyrosequencing , but interestingly no retrotransposon insertions were discovered . however , nine somatic l1 insertions were found in 6 out of 20 lung tumors with the same technique . it was not determined though whether the normal tissues also contained some number of l1 insertions relative to the tumor tissue , and thus whether insertions in the cancer represented an elevated level of retrotransposon mobilization . furthermore , it is not known if these insertions are transcribed or affect gene expression , and whether they were drivers or merely passengers of the tumorigenic process . the genome - wide methylation status of the lung tumors and adjacent normal tissue was also examined using an illumina platform . all 6 patient dna samples exhibiting tumor - specific l1 insertions were clustered together as hypomethylated , compared with 13 out of the remaining 14 samples that lacked somatic insertions . these data imply that a methylation signature distinguishes l1-permissive tumors from non - permissive tumors . another genome - scale method to genotype common retrotransposon insertion polymorphisms ( rips ) to identify genotype - phenotype associations uses array - based technology . commonly , single nucleotide polymorphisms ( snps ) and copy - number variants have been used as markers in genome - wide association studies ( gwass ) to map loci involved in human disease . rips are a valuable resource to investigate the role of these elements in phenotypic variation and disease . also , generally a rip is much more likely to be the causal variant than a snp , because a large insertion is more likely to be disruptive of gene function than a single nucleotide alteration , and retrotransposons have many features that can interfere with gene expression ( reviewed by goodier and kazazian ) . on the other hand , strong selection exists against retroelement insertions into coding regions , where they are under - represented compared with snps . currently , one array - based approach has been conducted to detect retrotransposon insertions in human disease . using transposon insertion profiling by microarray ( tip - chip ) , several novel l1 insertions on the x chromosome were discovered in male probands with presumptively x - linked intellectual disability . interestingly , one of the insertions occurred in the nhs gene , which is mutated in nance - horan syndrome , a condition associated with intellectual disability . another promising insertion occurred in the dach2 ( dachshund homolog 2 ) gene that regulates neuronal differentiation . however , confirmation studies are needed to demonstrate whether these insertions are the underlying cause of intellectual disability in these patients . except for the baillie et al . study , which analyzed l1 , alu and sva somatic insertions , the studies mentioned above concentrated on genome - wide detection of new l1 insertions . a notable study by witherspoon et al . developed a robust technique , termed mobile element scanning , to find new insertions of young alu elements using pcr methods coupled with high - throughput sequencing . their technique is applicable to all mobile elements , and is amenable to significant multiplexing of a number of dna samples in one sequencing run . it is speculated that one of the main roles of dna methylation , in addition to epigenetic reprogramming , is to silence transposable elements . most methylation studies of human transposons have investigated malignancies and showed consistent hypomethylation ( for example , ) , the extent of which , however , was variable in different tissues . as the malignant phenotype is inherently associated with global as well as tumor - type - specific methylation changes , and transposable elements comprise the majority of the human genome , it is difficult to establish the role of transposon demethylation per se in tumorigenesis , especially without accompanying functional studies . it is possible that pathogenic cellular stress responses could result in local or global transposon deregulation - for example , via demethylation or chromatin modification . once out of control , such an epigenetic deregulation might result in single or multiple retrotransposition events . they might also express non - coding rnas , and retrotransposons in the 3 ' utr ( untranslated region ) of genes show strong evidence of reducing the expression of the respective gene , as assessed by cap analysis gene expression and pyrosequencing . thus , an altered retrotransposon methylation state is expected to affect either the transcription of the retrotransposon itself or that of nearby genes . accordingly , it has been shown that hypomethylation of l1s can cause altered gene expression . specifically , an l1 is located in the met ( hepatocyte growth factor receptor ) oncogene , and hypomethylation of a promoter in this l1 induced an alternative met transcript within the urothelium of tumor - bearing bladders . at the same time , in the bladder epithelium of cancer - free donors the methylation level of this l1 promoter was high and expression of the alternative met transcript was low . there are few studies that correlate human retrotransposon methylation with their transcription level on a genome - wide scale . according to one study , expression of l1 5 ' and 3 ' utr sequences in prostate cancer different herv - k families showed opposite trends in expression levels , and the expression of evolutionarily young alu families was restricted to individual prostate tumors as assessed by rt - qpcr and pyrosequencing . in agreement with that study , transcriptional activation of l1s was not observed in globally hypomethylated hepatocellular carcinoma compared with matched normal tissue , as assessed by rt - qpcr . of note , the quantification of some types of expressed retroelements by using classical methods may prove ambiguous . since alu sequences are abundant in rna polymerase ii transcripts , quantification of the relatively rare rna polymerase iii transcribed alu transcripts by rt - qpcr is fraught with potential error . transcribed l1 sequences embedded in genes may similarly confound the results of such quantitative measurements of l1 expression . using a custom genechip microarray for transcriptome analysis of several herv families , it was shown that numerous herv - w loci were overexpressed in testicular cancer . interestingly , one of these was an ervwe1 transcript whose expression is usually restricted to the placenta . methylation was severely or completely diminished at herv - w sequences in the tumor dna , suggesting that dna methylation and herv - w expression is interrelated in this tumor context . with a genome - wide technique termed selective differential display of rnas containing interspersed repeats and with its modified version , termed l1 chimera display , it has been also demonstrated that the levels of many herv - k ltr transcripts differ between normal and testicular germ cell tumor tissues , and that the l1 antisense promoter gives rise to novel chimeric transcripts that are unique in tumor samples . furthermore , the cancer - specific chimeric l1 transcripts could be induced in non - malignant cells by using the demethylating drug 5-azacytidine . it will be interesting to learn if tumor - specific retrotransposon profiles reveal enhanced retroelement mobility . for example , l1 retrotransposition is associated with genetic instability , a hallmark of cancer . thus , local or global overactivation of l1s could have the potential to contribute to tumorigenesis . in particular , germ cell tumors are good candidates to examine cancer - specific retroelement activity , because the genome of germ cells goes through epigenetic reprogramming through methylation at cpg sites . thus , deregulation of this process might easily lead to the derepression of transposable elements , and potentially to germ cell tumors . in support of this hypothesis , the l1 orf1 protein was overexpressed in all 62 cases of investigated childhood malignant germ cell tumors relative to adjacent normal tissue and was associated with poor differentiation . testicular germ cell tumors should also be examined for l1-conferred hereditary disease , as no high penetrance susceptibility genes have been identified in this condition . with pyrosequencing of bisulfite - treated dna using l1-specific primers , transgenerational l1 methylation inheritance was implicated to be associated with testicular cancer risk . thus , l1s are attractive candidates for both somatic drivers and hereditary predisposition factors in germ cell tumors and possibly in other cancer types . however , currently their functional impact in malignancy is poorly understood . genome - scale technologies now provide us with the opportunity to investigate retrotransposon biology in unprecedented detail . ultimately , it will be important to test the functional consequences of these results , such as the effect of rips on gene function , and their role in cancer and neurological disorders . this outcome might be accomplished by classical functional studies , or by combining the results of several genome - scale experiments . for instance , if comprehensive rip profiles were coupled with next - generation rna sequencing data , it would allow testing of hypotheses pertaining to retrotransposons and their effects upon gene expression . such platforms would also be useful to explore whether there is a role for common rips in common disease and if these rips convey the disease phenotype through expression . in a similar manner , one could incorporate chromatin / methyl - seq / rna / chip - seq profiles for dna - binding or rna - binding proteins with the respective rip profiles . it would also be advantageous to carry out studies to explore whether any overlap between a gwas hit and a known rip exists , as the rip might indeed be the causal variant . as an alternative genome - scale approach to understand the impact of human transposons on disease , for instance , haploid cell lines and blm ( bloom syndrome , recq helicase - like)-deficient cells that can be converted to generate a genome - wide library of homozygous mutant cells [ 85 - 87 ] are available to be mutagenized and screened for any desirable phenotype , such as altered retrotransposition activity , using suitable read - out systems . one such system could be a retrotransposition assay , where an l1 reporter construct has been designed so that translation of the reporter ( drug - resistance gene or enhanced green fluorescent protein ) occurs only after l1 reverse transcription and insertion of its cdna copy into the genome . also , genome - wide mutagenesis might be accomplished with mobile elements themselves , such as retroviruses or dna transposons [ 85 - 87 ] . similarly , large - scale small interfering rna ( sirna ) and cdna functional genetics screening strategies could be designed to identify host cell factors modulating l1 activity . one should also investigate whether some host factors elicit a disease phenotype through deregulated retrotransposon activity . for example , the remarkable finding of the role of alu rna toxicity due to dicer1 deficiency in macular degeneration needs to be replicated by alternative methods . those methods should exclude the possibility that what is really being detected is amplification of the closely related 7sl rna or alu sequences contained in rna polymerase ii transcripts , which vastly outnumber the rna polymerase iii - transcribed alu elements . also , functional genetic follow - up studies should circumvent - if at all feasible - non - specific toxicity arising as a result of ectopic alu overexpression or antisense oligo - mediated downregulation of essential rna polymerase ii transcripts with embedded alu sequences . dicer1 may also have a role in tumorigenesis through retrotransposon overexpression , as germline mutations in this gene have been found in familial pleuropulmonary blastoma and in familial multinodular goiter with ovarian sertoli - leydig cell tumors . sense and antisense transcripts derived from l1 promoters could be processed to sirnas that might suppress retrotransposition by rna interference , and dicer1 has been implicated in this process . these data raise the possibility that genomic instability in some malignancies could arise - at least partly - from retrotransposon overdose as a consequence of a mutated small non - coding rna pathway . this could lead eventually to retrotransposon rna toxicity , genotoxic stress through dna nicking by orf2 , or elevated insertional mutagenesis . for the future of personalized medicine it will be vital not to exclude the transposon profile of patients , as exemplified by the case of an alu insertion in a retinitis pigmentosa proband . another aspect of personalized medicine is gene therapy . in one form of gene therapy , antisense oligonucleotides that block aberrant splicing into an intronic sva that causes fukuyama muscular dystrophy has been suggested . another aspect of gene therapy is the use of dna transposons that hold the promise of lower immunogenicity , enhanced safety profile and reduced manufacturing costs compared with viral vectors . two dna transposons from non - mammalian species have emerged as gene therapy tools based on their efficient transposition in humans : the reconstructed tc1/mariner element sleeping beauty from salmonid fish and piggybac from the baculovirus genome . the first ex vivo gene therapy clinical trial using sleeping beauty has been approved , and induced pluripotent stem cells are now being generated after targeted gene correction using piggybac technology . once the potential side - effects of these therapies - such as secondary mutagenesis resulting from transposon hopping or activation of nearby genes - are overcome , the roles of mobile elements can be redefined from being just ' junk ' or ' enemy ' to ' life - guards ' of our genomes . : human - specific l1 ; line-1/l1 : long interspersed element ; ltr : long terminal repeat ; orf : open reading frame ; qpcr : quantitative pcr ; rip : retrotransposon insertion polymorphism ; sine : short interspersed element ; sirna : small interfering rna ; snp : single nucleotide polymorphism ; sva : sine - r / vntr / alu ; tprt : target primed reverse transcription ; utr : untranslated region ; vntr : variable number of tandem repeat . we thank dustin c hancks , john l goodier , adam d ewing and tara doucet for their comments on the manuscript . research in the kazazian laboratory is funded by grants from the national institutes of health awarded to hhk .

perhaps as much as two - thirds of the mammalian genome is composed of mobile genetic elements ( ' jumping genes ' ) , a fraction of which is still active or can be reactivated . by their sheer number and mobility , retrotransposons , dna transposons and endogenous retroviruses have shaped our genotype and phenotype both on an evolutionary scale and on an individual level . notably , at least the non - long terminal repeat retrotransposons are still able to cause disease by insertional mutagenesis , recombination , providing enzymatic activities for other mobile dna , and perhaps by transcriptional overactivation and epigenetic effects . currently , there are nearly 100 examples of known retroelement insertions that cause disease . in this review , we highlight those genome - scale technologies that have expanded our knowledge of the diseases that these mobile elements can elicit , and we discuss the potential impact of these findings for medicine . it is now likely that at least some types of cancer and neurological disorders arise as a result of retrotransposon mutagenesis .

The emerging importance of mobile DNA elements in disease Types of mobile elements in the human genome and the main disease mechanisms Genome-scale approaches to identify new retrotransposon insertions Genome-wide methylation studies and transcriptome analysis of retrotransposons Concluding remarks and future directions Abbreviations Competing interests Acknowledgements

it has been generally held that about half of the human genome is derived from mobile genomic elements , but according to a recent estimate over two - thirds of our genome may result from the presence or ancient activity of ' jumping genes ' . this massive amount of dna includes domesticated elements with evolutionarily spectacular functions , such as the rag ( recombination activating ) genes that form the basis of v(d)j recombination in our immune system , and the ervwe1 ( endogenous retrovirus group w , member 1 ) gene that plays a role in placental development . contrary to their evolutionary gifts , mobile elements are most notorious for being junk or for causing life - threatening human disorders by insertional mutagenesis and homologous recombination . as we usher in the era of genome - scale studies , it is clear that these elements have the potential to cause intra - individual and inter - individual variation and probably common disease through structural variation , deregulated transcriptional activity or epigenetic effects . furthermore , large - scale studies have expanded the pool of human disorders resulting from retrotransposon - mediated insertional mutagenesis . we focus here on the known , as well as inferred , potential health impact of their novel findings . dna transposons move by a cut - and - paste mechanism , while retrotransposons mobilize by a copy - and - paste mechanism via an rna intermediate , a process called retrotransposition . retrotransposons can be further subdivided into long terminal repeat ( ltr ) and non - ltr elements . ltr retrotransposons are human endogenous retroviruses ( hervs ) that have an intracellular existence as a result of a non - functional envelope gene . non - ltr elements are classified as long interspersed elements ( lines ; the prototype of which is the rna polymerase ii transcribed line-1 ( l1 ) ) , and short interspersed elements ( sines ) , the latter consisting essentially of rna polymerase iii transcribed alus . svas ( sine - r / vntr ( variable number of tandem repeat)/alu ) are also active non - ltr retrotransposable elements that are intermediate in size relative to alus and l1s , and are likely to be transcribed by rna polymerase ii . l1s are the only known autonomously active human retrotransposons , as only they encode proteins ( open reading frame 1 ( orf1 ) and orf2 ) with which they can be mobilized . l1s are also responsible for the mobilization of the non - autonomous alus , svas [ 10 - 13 ] and processed pseudogenes , which are cellular mrnas that are reverse transcribed and inserted into the genome . accordingly , no human disease is known to arise as a result of their activity . although no insertional mutagenesis by hervs has been described , oncogenic etv1 ( ets variant 1)-herv - k fusions generated by chromosomal translocation have been observed in prostate cancer , and herv expression has been suggested as a potential contributor to autoimmune diseases . also , a ltr of a malr human endogenous retrovirus has been shown to aberrantly activate a proto - oncogene , thereby causing lymphoma . the predominant mechanism by which l1s cause disease is insertional mutagenesis into or near genes . l1 insertions are often accompanied by 3 ' transduction , the co - mobilization of dna sequences downstream of an l1 as a consequence of transcriptional read - through resulting from a weak l1 poly(a ) signal [ 24 - 27 ] . alu sequences predominantly cause disease by homologous recombination between two alu sequences , but insertion into or near exons , and aberrant alu splicing from introns , also frequently result in pathological conditions [ 28 - 30 ] . furthermore , alu rna toxicity , a new disease - causing phenomenon , has been recently proposed to result in macular degeneration by dicer1 deficit . regarding the role of alu elements in eye disorders , it is interesting that an alu insertion polymorphism in the ace ( angiotensin i converting enzyme ) gene has been associated with protection from the dry / atrophic form of age - related macular degeneration . sva elements also have the ability to interrupt genes through insertional mutagenesis that can be coupled with 3 ' transduction , genomic deletion or aberrant splicing [ 11,33 - 35 ] . the wide spectrum of disease cases caused by retrotransposons ranges from hemophilia to muscular dystrophy and cancer , and has been thoroughly reviewed [ 36 - 38 ] . there have been 96 known retrotransposon insertions in disease cases , of which 25 are caused by l1s , while the other 71 are also l1-mediated . among the latter , 60 cases are attributable to alus , 7 to svas , and 4 to truncated inserts with only poly(a ) sequence remaining . processed pseudogenes have not yet been found to cause human disease by de novo insertional mutagenesis , but facioscapulohumeral dystrophy has been demonstrated to arise as a result of the contraction of macrosatellite repeats leading to aberrant expression of an array of dux4 retrogenes residing within the repeats . , mutations in utp14c have been associated with male infertility , mutations in tacstd2/m1s1 result in gelatinous drop - like corneal dystrophy , and ptenp1 is selectively lost in human cancers . the main characteristics of mobile elements capable of causing human disease are summarized in figure 1 . env , envelope ; gag , group specific antigen ; herv , human endogenous retrovirus ; kb , kilobase ; ltr , long terminal repeat ; orf , open reading frame ; pol , polymerase ; sine , short interspersed element ; sva , sine - r / vntr / alu ; tprt , target primed reverse transcription ; utr , untranslated region ; vntr , variable number of tandem repeats . we discuss large - scale genome , transcriptome and methylation profiling studies of mobile elements in human diseases . we also discuss some non - genome - scale studies that support or contradict the implications of these novel findings . it is now possible to characterize genome sequences of scarce samples or even single cells . using this approach , it has been demonstrated that many l1hs elements are population - specific , and recapitulate genetic ancestry similar to alu insertion polymorphisms . retrotransposons are not only excellent markers for exploring population history , but can also give rise to population - specific diseases . for example , a homozygous alu insertion in an exon of the mak ( male germ - cell - associated kinase ) gene has been identified in 21 patients of jewish ancestry who were diagnosed with retinitis pigmentosa . oddly , the discovery of this mutation using agilent exome capture and subsequent illumina and abi sequencing was paradoxical , as attempts to remove repetitive sequences from the analysis led to the identification of the insertion . another population - specific disease caused by retrotransposon mutagenesis is fukuyama - type congenital muscular dystrophy . it is one of the most common autosomal recessive disorders in japan and was the first human disease found to result from ancestral insertion of an sva element . an unexpected finding of state - of - the - art , large - scale approaches to study retrotransposon insertions has been that highly active ( or ' hot ' ) l1s are much more abundant in humans than previously appreciated . the outcome of a fosmid - based paired - end dna sequencing strategy , coupled with a cell culture assay for retrotransposition , was that over half of newly identified l1s are hot , expanding the number of known hot l1s from 6 to 43 . these l1s are not only expected to be a major source of inter - individual genetic variation , but hot l1s account for most examples of disease - causing insertions . another unusual finding of genome - scale approaches in retrotransposon biology has been that retrotransposition occurs at a very high frequency in somatic cells . in another study that awaits further validation , over 7,700 l1s , 13,600 alus and 1,300 sva putative somatic insertions were found in the hippocampus of three individuals using retrotransposon capture sequencing , which is based on transposon array capture followed by illumina paired - end sequencing . surprisingly , in this study , l1 and alu insertions were over - represented in protein - coding genes and targeted genes , such as hdac1 ( histone deacetylase 1 ) and rai1 ( retinoic acid induced 1 ) , which are known to be mutated in neurological disorders . these findings suggest that if a retrotransposon inserts into a gene that functions in neurological development or psychological functioning early in development , it might affect a large enough area of the brain to lead to disease . if such neuronal plasticity exists , it could affect behavioral phenotypes , and could be modulated by environmental factors . mecp2 ( methyl cpg binding protein 2 ) has been shown to repress l1 expression and retrotransposition , and increased l1 retrotransposition has been observed in induced pluripotent stem cells of patients with rett syndrome who carry mecp2 mutations . it was not determined though whether the normal tissues also contained some number of l1 insertions relative to the tumor tissue , and thus whether insertions in the cancer represented an elevated level of retrotransposon mobilization . furthermore , it is not known if these insertions are transcribed or affect gene expression , and whether they were drivers or merely passengers of the tumorigenic process . the genome - wide methylation status of the lung tumors and adjacent normal tissue was also examined using an illumina platform . all 6 patient dna samples exhibiting tumor - specific l1 insertions were clustered together as hypomethylated , compared with 13 out of the remaining 14 samples that lacked somatic insertions . these data imply that a methylation signature distinguishes l1-permissive tumors from non - permissive tumors . another genome - scale method to genotype common retrotransposon insertion polymorphisms ( rips ) to identify genotype - phenotype associations uses array - based technology . rips are a valuable resource to investigate the role of these elements in phenotypic variation and disease . also , generally a rip is much more likely to be the causal variant than a snp , because a large insertion is more likely to be disruptive of gene function than a single nucleotide alteration , and retrotransposons have many features that can interfere with gene expression ( reviewed by goodier and kazazian ) . on the other hand , strong selection exists against retroelement insertions into coding regions , where they are under - represented compared with snps . currently , one array - based approach has been conducted to detect retrotransposon insertions in human disease . using transposon insertion profiling by microarray ( tip - chip ) , several novel l1 insertions on the x chromosome were discovered in male probands with presumptively x - linked intellectual disability . interestingly , one of the insertions occurred in the nhs gene , which is mutated in nance - horan syndrome , a condition associated with intellectual disability . study , which analyzed l1 , alu and sva somatic insertions , the studies mentioned above concentrated on genome - wide detection of new l1 insertions . their technique is applicable to all mobile elements , and is amenable to significant multiplexing of a number of dna samples in one sequencing run . it is speculated that one of the main roles of dna methylation , in addition to epigenetic reprogramming , is to silence transposable elements . most methylation studies of human transposons have investigated malignancies and showed consistent hypomethylation ( for example , ) , the extent of which , however , was variable in different tissues . as the malignant phenotype is inherently associated with global as well as tumor - type - specific methylation changes , and transposable elements comprise the majority of the human genome , it is difficult to establish the role of transposon demethylation per se in tumorigenesis , especially without accompanying functional studies . it is possible that pathogenic cellular stress responses could result in local or global transposon deregulation - for example , via demethylation or chromatin modification . they might also express non - coding rnas , and retrotransposons in the 3 ' utr ( untranslated region ) of genes show strong evidence of reducing the expression of the respective gene , as assessed by cap analysis gene expression and pyrosequencing . thus , an altered retrotransposon methylation state is expected to affect either the transcription of the retrotransposon itself or that of nearby genes . specifically , an l1 is located in the met ( hepatocyte growth factor receptor ) oncogene , and hypomethylation of a promoter in this l1 induced an alternative met transcript within the urothelium of tumor - bearing bladders . at the same time , in the bladder epithelium of cancer - free donors the methylation level of this l1 promoter was high and expression of the alternative met transcript was low . there are few studies that correlate human retrotransposon methylation with their transcription level on a genome - wide scale . according to one study , expression of l1 5 ' and 3 ' utr sequences in prostate cancer different herv - k families showed opposite trends in expression levels , and the expression of evolutionarily young alu families was restricted to individual prostate tumors as assessed by rt - qpcr and pyrosequencing . of note , the quantification of some types of expressed retroelements by using classical methods may prove ambiguous . interestingly , one of these was an ervwe1 transcript whose expression is usually restricted to the placenta . methylation was severely or completely diminished at herv - w sequences in the tumor dna , suggesting that dna methylation and herv - w expression is interrelated in this tumor context . with a genome - wide technique termed selective differential display of rnas containing interspersed repeats and with its modified version , termed l1 chimera display , it has been also demonstrated that the levels of many herv - k ltr transcripts differ between normal and testicular germ cell tumor tissues , and that the l1 antisense promoter gives rise to novel chimeric transcripts that are unique in tumor samples . furthermore , the cancer - specific chimeric l1 transcripts could be induced in non - malignant cells by using the demethylating drug 5-azacytidine . for example , l1 retrotransposition is associated with genetic instability , a hallmark of cancer . thus , deregulation of this process might easily lead to the derepression of transposable elements , and potentially to germ cell tumors . testicular germ cell tumors should also be examined for l1-conferred hereditary disease , as no high penetrance susceptibility genes have been identified in this condition . genome - scale technologies now provide us with the opportunity to investigate retrotransposon biology in unprecedented detail . ultimately , it will be important to test the functional consequences of these results , such as the effect of rips on gene function , and their role in cancer and neurological disorders . this outcome might be accomplished by classical functional studies , or by combining the results of several genome - scale experiments . as an alternative genome - scale approach to understand the impact of human transposons on disease , for instance , haploid cell lines and blm ( bloom syndrome , recq helicase - like)-deficient cells that can be converted to generate a genome - wide library of homozygous mutant cells [ 85 - 87 ] are available to be mutagenized and screened for any desirable phenotype , such as altered retrotransposition activity , using suitable read - out systems . one such system could be a retrotransposition assay , where an l1 reporter construct has been designed so that translation of the reporter ( drug - resistance gene or enhanced green fluorescent protein ) occurs only after l1 reverse transcription and insertion of its cdna copy into the genome . also , genome - wide mutagenesis might be accomplished with mobile elements themselves , such as retroviruses or dna transposons [ 85 - 87 ] . similarly , large - scale small interfering rna ( sirna ) and cdna functional genetics screening strategies could be designed to identify host cell factors modulating l1 activity . for example , the remarkable finding of the role of alu rna toxicity due to dicer1 deficiency in macular degeneration needs to be replicated by alternative methods . those methods should exclude the possibility that what is really being detected is amplification of the closely related 7sl rna or alu sequences contained in rna polymerase ii transcripts , which vastly outnumber the rna polymerase iii - transcribed alu elements . also , functional genetic follow - up studies should circumvent - if at all feasible - non - specific toxicity arising as a result of ectopic alu overexpression or antisense oligo - mediated downregulation of essential rna polymerase ii transcripts with embedded alu sequences . dicer1 may also have a role in tumorigenesis through retrotransposon overexpression , as germline mutations in this gene have been found in familial pleuropulmonary blastoma and in familial multinodular goiter with ovarian sertoli - leydig cell tumors . sense and antisense transcripts derived from l1 promoters could be processed to sirnas that might suppress retrotransposition by rna interference , and dicer1 has been implicated in this process . these data raise the possibility that genomic instability in some malignancies could arise - at least partly - from retrotransposon overdose as a consequence of a mutated small non - coding rna pathway . this could lead eventually to retrotransposon rna toxicity , genotoxic stress through dna nicking by orf2 , or elevated insertional mutagenesis . for the future of personalized medicine it will be vital not to exclude the transposon profile of patients , as exemplified by the case of an alu insertion in a retinitis pigmentosa proband . another aspect of gene therapy is the use of dna transposons that hold the promise of lower immunogenicity , enhanced safety profile and reduced manufacturing costs compared with viral vectors . two dna transposons from non - mammalian species have emerged as gene therapy tools based on their efficient transposition in humans : the reconstructed tc1/mariner element sleeping beauty from salmonid fish and piggybac from the baculovirus genome . the first ex vivo gene therapy clinical trial using sleeping beauty has been approved , and induced pluripotent stem cells are now being generated after targeted gene correction using piggybac technology . once the potential side - effects of these therapies - such as secondary mutagenesis resulting from transposon hopping or activation of nearby genes - are overcome , the roles of mobile elements can be redefined from being just ' junk ' or ' enemy ' to ' life - guards ' of our genomes . : human - specific l1 ; line-1/l1 : long interspersed element ; ltr : long terminal repeat ; orf : open reading frame ; qpcr : quantitative pcr ; rip : retrotransposon insertion polymorphism ; sine : short interspersed element ; sirna : small interfering rna ; snp : single nucleotide polymorphism ; sva : sine - r / vntr / alu ; tprt : target primed reverse transcription ; utr : untranslated region ; vntr : variable number of tandem repeat .

crohn 's disease ( cd ) is a chronic , progressive , and potentially disabling disease , characterised by relapsing and remitting episodes of transmural inflammation of the gastrointestinal tract which might be associated with arthritic manifestations . both cd and ulcerative colitis ( uc ) can be categorized under the name of inflammatory bowel disease ( ibd ) . the differentiation between uc and cd is mainly based on clinical manifestations and the scale of bowel involvements . patients with either cd or uc , however , are likely to have associated extraintestinal manifestations . ibd is classified as one of the constituents of a group of diseases collectively termed spondyloarthropathies ( spas ) . the other entities of this group include ankylosing spondylitis ( as ) , reactive arthritis , psoriatic arthropathy , and undifferentiated spa . there are certain features frequently associated with this group of diseases and these include spinal / sacroiliac arthritis , oligoarthritis , enthesitis , uveitis , negativity for rheumatoid factors , and a positive family history . a positive family history and a high degree of association of spas with hla - b27 genetic markers have placed these conditions under the umbrella of what is called b27 diseases . furthermore , patients with cd seem to share more genetic , clinical , laboratory , immunological , and pathological features with as patients [ 57 ] . it has a high impact on the psychological condition , as well as the social status and work abilities in patients with this disease . based on these negative effects on the general health and welfare of patients with cd and the drawbacks of the currently used medical treatments as well as the increased likelihood of surgical complications in these patients , a search for the causative factor(s ) together with a proposal for the use of an alternative therapeutic strategy involving eradication of the causative factors could be of crucial importance in the management of this disease . the incidence of cd has been increasing substantially in the last few decades , where more than 40,000 new cases in europe and north america have been estimated to be diagnosed each year . in a more recent analytical review study , it was shown that the incidence and prevalence of cd are increasing with time and in different regions around the world , particularly among the caucasians . the same increase in the incidence of cd these rises in the occurrence of cd could largely be explained by altered lifestyles and environmental exposures and/or changes in the dietary habits among the patients with this disease . at least three interacting elements including genetic factors , intestinal microbes , and immune - mediated inflammation are involved in the pathogenesis of cd . the most popular theory , however , is that the disease usually results from an abnormal immune response to certain enterobacterial pathogens present in the gut lumen . evidence from twin and familial aggregation as well as genome - wide association studies indicates that genetic factors play an important role in the aetiopathogenesis of cd ( as shown in the lists below ) . although many of the non - hla genes such as nod2/card15 , il23r , and ctla4 have been suggested to play a role in the susceptibility to develop cd until now , however , scientists have not been able to discover a gene which might solely contribute to the aetiology of this disease . aetiopathogenetic evidence for the roles of genetic / microbial ( especially klebsiella ) factors in cd evidence of the genetic involvement positive family history.a considerable increase in the risk of developing the disease among monozygotic twin of patients with cd.higher risk of developing the disease among relatives of patients with cd or other entities of spas.presence of hla - b27 genetic markers in more than 50% of patients with cd having spondylitis.significant association with nod2/card15 and some other genetic mutations.transgenic rats carrying human hla - b27 genes are more prone to develop spontaneous bowel lesions . positive family history . a considerable increase in the risk of developing the disease among monozygotic twin of patients with cd . higher risk of developing the disease among relatives of patients with cd or other entities of spas . presence of hla - b27 genetic markers in more than 50% of patients with cd having spondylitis . transgenic rats carrying human hla - b27 genes are more prone to develop spontaneous bowel lesions . evidence of the environmental / microbial factors more than 80% of dizygotic twins of patients with cd fail to develop the disease.increased incidence of cd among friends and clustering of unrelated people as well as immigrants moving from low to high risk areas.associated bouts of remissions and exacerbations with spatial and temporal variations in the incidence and the clinical course of the disease in cd.increased gut mucosal leukocyte trafficking and enhanced microbial innate immune responses , as well as increased risk of developing the disease after appendectomy in patients with cd . more than 80% of dizygotic twins of patients with cd fail to develop the disease . increased incidence of cd among friends and clustering of unrelated people as well as immigrants moving from low to high risk areas . associated bouts of remissions and exacerbations with spatial and temporal variations in the incidence and the clinical course of the disease in cd . increased gut mucosal leukocyte trafficking and enhanced microbial innate immune responses , as well as increased risk of developing the disease after appendectomy in patients with cd . evidence of the role of klebsiella and crossreactive self antigens increased load of klebsiella culture from fecal matter in the large bowel.occurrence of intercurrent klebsiella intestinal colitis in patients with cd.significantly enhanced antibody responses to klebsiella microbes were found on many occasions in patients with cd when compared to healthy subjects . increased antibody levels against collagens i , iii , and iv , all crossreactive with klebsiella pullulanase enzymes , were found in patients with cd when compared to healthy controls.molecular similarities were found between different antigens present in klebsiella nitrogenase and pullulanase puld enzymes and hla - b27 self - antigens . enzymes were found to possess a molecular structure similar to the trihelical shape of collagen i , iii , and iv fibres . increased load of klebsiella culture from fecal matter in the large bowel . occurrence of intercurrent klebsiella intestinal colitis in patients with cd . significantly enhanced antibody responses to klebsiella microbes were found on many occasions in patients with cd when compared to healthy subjects . increased antibody levels against collagens i , iii , and iv , all crossreactive with klebsiella pullulanase enzymes , were found in patients with cd when compared to healthy controls . molecular similarities were found between different antigens present in klebsiella nitrogenase and pullulanase puld enzymes and hla - b27 self - antigens . enzymes were found to possess a molecular structure similar to the trihelical shape of collagen i , iii , and iv fibres . the link between hla - b27 and cd on the other hand is relatively more obvious especially in patients with associated spondylitis . for example , more than forty percent of patients with cd , especially those having spondylitis / sacroiliitis , were found to be positive for the hla - b27 markers , but the prevalence of this gene in patients with cd without spondylitis / sacroiliitis merely parallels its frequency in the caucasian general population . furthermore , hla - b27 positive caucasians are 20 times more likely to develop one or other entities of the spas , including ibd . there is also evidence of high risk and cross - risk ratios among the first- , second- , or third - degree relatives to develop ibd or as . moreover , in an experimental study by a group from dallas , transgenic rats with high level of human hla - b27 expression were found to be more prone to develop bowel inflammation with arthritis , and the severity of intestinal features was correlating with concentration of the large bowel microbial flora . extensive evidence supports the role of certain environmental and/or microbial factors in the aetiopathogenesis of ibd and particularly in the causation of cd ( as shown in the first three lists).a negative family history in more than 95% and a low concordance rate among twins of patients with cd support the role of environmental factors in the development of this disease.there is a report for an increased clustering of cd among unrelated individuals and closely living friends , as well as amid second - generation immigrants moving from low to high risk areas . in a previous study on a large moroccan family and one parent with cd emigrated to belgium , 4 out of the 8 children were found to have subsequently developed the disease . failure to find significant genetic variants with high incidence of cd among this family suggests the direct role of a major environmental factor which probably has been encountered in the new residing area and is not related to the sanitation in childhood . patients with cd show intermittent variations in the clinical onset and activity during the disease course as well as during different seasons of the year with associated concurrent intestinal bacterial infections . increased incidence of cd after appendectomy , together with involvement of neutrophils and lymphocytes in the gut inflammation as well as enhanced general immune response to enterobacterial antigens in these patients , supports the role of gut microbes in the aetiopathogenesis of this disease.transgenic rats do not develop colitis under germ - free conditions , but when these rats are transferred into conventional environments , they develop signs of intestinal inflammation and colitis . these results indicate that colonic microflora plays a key role in the development or exacerbation of the intestinal inflammation resembling that occurring in patients with cd . a negative family history in more than 95% and a low concordance rate among twins of patients with cd support the role of environmental factors in the development of this disease . there is a report for an increased clustering of cd among unrelated individuals and closely living friends , as well as amid second - generation immigrants moving from low to high risk areas . in a previous study on a large moroccan family and one parent with cd emigrated to belgium , failure to find significant genetic variants with high incidence of cd among this family suggests the direct role of a major environmental factor which probably has been encountered in the new residing area and is not related to the sanitation in childhood . patients with cd show intermittent variations in the clinical onset and activity during the disease course as well as during different seasons of the year with associated concurrent intestinal bacterial infections . increased incidence of cd after appendectomy , together with involvement of neutrophils and lymphocytes in the gut inflammation as well as enhanced general immune response to enterobacterial antigens in these patients , supports the role of gut microbes in the aetiopathogenesis of this disease . transgenic rats do not develop colitis under germ - free conditions , but when these rats are transferred into conventional environments , they develop signs of intestinal inflammation and colitis . these results indicate that colonic microflora plays a key role in the development or exacerbation of the intestinal inflammation resembling that occurring in patients with cd . during the last few decades , various microbial agents , including also klebsiella pneumonia , have been implicated to have a role in the aetiopathogenesis of cd . a considerable amount of microbiological , molecular , and immunological evidence exists which supports the role of klebsiella microbes in the aetiology of cd ( figure 1 ) ( as shown in the first three lists ) . it was reported that , in more than 20% of patients with cd , klebsiella microorganisms have been isolated from the large bowel specimens ( hring et al . , 1991 ) . the disease relapses in patients with cd were found also to be associated with attacks of klebsiella oxytoca colitis . in a study on the biopsy specimens taken from 12 patients with cd , it has been shown that invasion of the bowel mucosa by microbial agents including also enterobacteriaceae spp . was evident in 55.6% of the ileal and in 25% of the colonic specimens from the cd patients , but no bacteria were detected in the tissues of the non - cd controls . in another study using immunohistochemistry , however , the majority of biopsy specimens taken from the bowel mucosa of patients with ibd have shown negative staining for escherichia , listeria , and even klebsiella microbes . this latter finding could indicate that it is the microbial bulk in intestinal lumen rather than at the sites of the pathological lesions , which is important in evoking both local mucosal and general antibacterial immune responses . for example , nitrogenase reductase enzyme possesses a hexameric amino acid sequence , qtdred , which has been found to be homologous to another sequence present in hla - b27 molecules . in a later study , puld secretion protein from klebsiella pullulanase enzyme , which carries tetramer amino acid residues , drde , was found to be homologous to dred sequence present in hla - b27 molecules , whilst repeating gly - x - pro amino acid sequences present in the pula secretion components of klebsiella pullulanase enzyme were found to be similar to those present in the trihelical structures of collagen types i , iii , and iv . significant cytotoxic reactions shown as percentage hemolysis for sheep red blood cells coated with hla - b2705 peptide were detected in the serum samples containing high anti - klebsiella antibodies concentrations taken from as patients when compared to control sera . it was reported that , in more than 20% of patients with cd , klebsiella microorganisms have been isolated from the large bowel specimens ( hring et al . , 1991 ) . the disease relapses in patients with cd were found also to be associated with attacks of klebsiella oxytoca colitis . in a study on the biopsy specimens taken from 12 patients with cd , it has been shown that invasion of the bowel mucosa by microbial agents including also enterobacteriaceae spp . was evident in 55.6% of the ileal and in 25% of the colonic specimens from the cd patients , but no bacteria were detected in the tissues of the non - cd controls . in another study using immunohistochemistry , however , the majority of biopsy specimens taken from the bowel mucosa of patients with ibd have shown negative staining for escherichia , listeria , and even klebsiella microbes . this latter finding could indicate that it is the microbial bulk in intestinal lumen rather than at the sites of the pathological lesions , which is important in evoking both local mucosal and general antibacterial immune responses . for example , nitrogenase reductase enzyme possesses a hexameric amino acid sequence , qtdred , which has been found to be homologous to another sequence present in hla - b27 molecules . in a later study , puld secretion protein from klebsiella pullulanase enzyme , which carries tetramer amino acid residues , drde , was found to be homologous to dred sequence present in hla - b27 molecules , whilst repeating gly - x - pro amino acid sequences present in the pula secretion components of klebsiella pullulanase enzyme were found to be similar to those present in the trihelical structures of collagen types i , iii , and iv . significant cytotoxic reactions shown as percentage hemolysis for sheep red blood cells coated with hla - b2705 peptide were detected in the serum samples containing high anti - klebsiella antibodies concentrations taken from as patients when compared to control sera . collagen - induced enterocolitis as well as arthritis has been observed in experimental animals when immunized with homologous colonic extracts and collagen type ii together with klebsiella lipopolysaccharides . elevated levels of humoral immune responses to klebsiella microbes in patients with cd as well as uc have been shown in several studies carried out at six different gastroenterology centres in the united kingdom.significantly increased levels of antibodies against klebsiella and yersinia enterobacterial agents were observed in patients with cd and uc from birmingham when compared to corresponding healthy controls .iga antibody levels against klebsiella microbes were found to be significantly increased in patients with ibd and as from glasgow when compared to corresponding control subjects .anti - klebsiella antibody levels were found to be significantly higher in patients with cd and as from edinburgh when compared to corresponding controls .in three consecutive studies carried out at different gastroenterology centres in london and winchester , similar results have been reported . in one study , klebsiella antibody levels were found to be significantly higher in patients with ibd and as when compared to healthy or other disease controls , whilst no such elevations were observed in the antibody levels against e. coli or other anaerobic intestinal microbes . in a second study , the levels of class - specific isotypic antibodies against many klebsiella antigens were found to be significantly elevated in patients with cd and as when compared to patients with coeliac disease or to healthy controls . in a third study , the levels of class - specific antibodies against klebsiella microbes and crossreactive collagens i , iii , iv , and v were found to be significantly increased in patients with early and late cd as well as in patients with as when compared to healthy controls . moreover , a positive correlation was demonstrated between antibody levels to collagen types i , iii , and iv and klebsiella in both groups of patients with cd and as . significantly increased levels of antibodies against klebsiella and yersinia enterobacterial agents were observed in patients with cd and uc from birmingham when compared to corresponding healthy controls . iga antibody levels against klebsiella microbes were found to be significantly increased in patients with ibd and as from glasgow when compared to corresponding control subjects . anti - klebsiella antibody levels were found to be significantly higher in patients with cd and as from edinburgh when compared to corresponding controls . in three consecutive studies carried out at different gastroenterology centres in london and winchester , klebsiella antibody levels were found to be significantly higher in patients with ibd and as when compared to healthy or other disease controls , whilst no such elevations were observed in the antibody levels against e. coli or other anaerobic intestinal microbes . in a second study , the levels of class - specific isotypic antibodies against many klebsiella antigens were found to be significantly elevated in patients with cd and as when compared to patients with coeliac disease or to healthy controls . in a third study , the levels of class - specific antibodies against klebsiella microbes and crossreactive collagens i , iii , iv , and v were found to be significantly increased in patients with early and late cd as well as in patients with as when compared to healthy controls . moreover , a positive correlation was demonstrated between antibody levels to collagen types i , iii , and iv and klebsiella in both groups of patients with cd and as . it is , therefore , plausible that cd disease could result from repeated subclinical infections by klebsiella microbes in the large bowel with the consequent inflammations and tissue damage in the bowel and joints resulting from the binding of anti - klebsiella and anti - self tissue antibodies to the crossreactive targeted antigens . dietary macronutrients especially carbohydrates are considered as a major factor driving the composition and metabolism of the colonic microbiota . recent evidence from molecular ecology has shown that the amount and type of nondigestible carbohydrates including starch influence the species composition of the gut microbiota both in short - term dietary intervention and in response to habitual long - term dietary intake [ 50 , 51 ] . in a recent study , a measure of 120 g / l of glucose was found to be one of the optimal requirements for the cell growth and lipid synthesis of lipomyces starkeyi yeast . it is composed of two types of structurally distinct -glucan polymer , amylase , and amylopectin . amylose , which constitutes around 2030% of the starch particle , consists of long chains of several thousand -glucosyl units joined by (14)-linkages , whilst amylopectin , which constitutes the other 7080% part of starch , is a branched molecule comprising short chains of (14)-linked -glucosyl units , joined by (16 ) branch linkages . degradation of the starch molecules is carried out by various catalytic enzymes , which are produced by plants , microbes , and human body . among these starch - debranching enzymes , however , pullulanases and -amylases are known to be among the potent groups . klebsiella microbes can survive in harsh environments exploiting some of their enzymatic products , which are required for the protection , maintenance , and survival of these microbes . apart from nitrogenase reductases these microbes can utilize starch as the sole carbon and energy source via two metabolic routes . the first one involves the extracellular degradation into linear maltodextrins by hydrolysis of the glycosidic bonds via the cell - surface - associated pullulanase and then the subsequent cleavage of the glycosidic linkages by the action of the extracellular glycosyltransferase . the large bowel in humans is described as a complex ecosystem , containing a large number of microbial species . although the composition of the colonic microbiota in adults is relatively stable , its concentration , however , can be manipulated by dietary means . for example , it has been reported that a high intake of carbohydrate , particularly oligosaccharides , can stimulate the growth of bifidobacterium spp . a considerable part of the consumed starchy food escapes digestion in the human small intestine and directly enters the large bowel . these starches that are not degraded in the small intestine and referred to as resistant starch can be used as substrate for bacterial fermentation . it has been shown that up to 20% of the total starch materials consumed by normal individuals fail the absorption process when assessed by oral hydrogen excretion studies . these starch materials are basically present in potatoes and wheat flour products such as pasta and bread ( as shown in the lists below ) . recommended diet for crohn 's disease and ankylosing spondylitis patients decreased intake of high - starch - containing foods flour and related products : breads , biscuits , cakes , puddings , pies , and popcorn . rice varieties : brown or white , boiled , fried , or in puddings . potatoes : baked , boiled , fried , roasted , or mashed potatoes . flour and related products : breads , biscuits , cakes , puddings , pies , and popcorn . rice varieties : brown or white , boiled , fried , or in puddings . potatoes : baked , boiled , fried , roasted , or mashed potatoes . increased intake of none - low - starch - containing foods meat : beef , pork , lamb , bacon , salami , corned beef , luncheon mean , potted meat , ham , and veal as well as chicken , turkey duck , or any other poultry meat . fish : white fish such as cod , haddock , plaice , and sole ; shellfish such as crab , lobster , prawns , scampi , cockles , mussels , and oysters ; and other fish such as herring , salmon , mackerel , tuna , and sardines . milk products : fresh , dried , or condensed milk , plain and flavoured yoghurts , and all types of cheese . vegetables : green vegetables such as cabbages , cauliflowers , sprouts , courgettes , peppers , mushrooms , carrots , and spinach or all salad vegetables like lettuce , cucumber , tomatoes , and water cress . meat : beef , pork , lamb , bacon , salami , corned beef , luncheon mean , potted meat , ham , and veal as well as chicken , turkey duck , or any other poultry meat . fish : white fish such as cod , haddock , plaice , and sole ; shellfish such as crab , lobster , prawns , scampi , cockles , mussels , and oysters ; and other fish such as herring , salmon , mackerel , tuna , and sardines . milk products : fresh , dried , or condensed milk , plain and flavoured yoghurts , and all types of cheese . vegetables : green vegetables such as cabbages , cauliflowers , sprouts , courgettes , peppers , mushrooms , carrots , and spinach or all salad vegetables like lettuce , cucumber , tomatoes , and water cress . the total colony counts in the bacterial population of bifidobacteria , lactobacilli , streptococci , and enterbacteria species were found to be increased significantly in the caecum and faeces of a group of rats which have been fed resistant potato starch when compared to those rats taking a diet made of rapidly digestible waxy maize devoid of resistant starch . in an in vitro study , it has been found that the mean number of klebsiella was ten times higher when incubated with simple carbohydrate products such as sucrose , lactose , and glucose than with eleven different amino acids . furthermore , klebsiella microbes do not seem to survive or grow on plant cellulose materials . in a recent experimental study , rats which have been fed potato starch diet had higher colonic bacterial loads than those on cellulose only diet . these results indicate that complex carbohydrates such as starch products are necessary for the growth , replication , and persistence of klebsiella as well as other enterobacterial agents in the gut . the current treatment involving the use of anti - inflammatory , immunosuppressive , and biologic modalities has significant limitations due to lack of treatment response in some patients as well as the occurrence of adverse side effects , such as increased risk of infection and malignancy among some other patients especially in those taking antitumor necrosis factors . some patients with cd , however , may also need surgical interventions . sustained therapeutic responses in patients with cd , however , may require more radical and comprehensive approaches involving strategies which help in the modification of the intestinal bacterial microenvironments . a solution for this medical predicament is the use of an alternative therapy , which should be harmless and aimed at the elimination of the most plausible microbial agent , such as klebsiella , in order to improve or even halt the inflammatory and pathological damage occurring in patients with cd . to achieve this therapeutic goal , the most likely strategy which could be added to the management of this disease is the use of antimicrobial agents and dietary manipulation . manipulation of luminal content of the gut using antibiotics may represent a potentially effective therapeutic option . whilst antimicrobials were shown to be clinically effective mainly in preventing postoperative recurrence , there could also be potential for their inclusion in the primary treatment of cd . in a review analysis , the trial of an antibacterial agent , rifaximin , has been shown to provide promising results in inducing remission of cd in up to 69% in open studies and significantly higher rates than placebo in double blind trials . in another meta - analysis review of randomized placebo - controlled studies , it has been shown that there was a statistically significant effect of antibiotics being superior to placebo in patients with active and quiescent cd as well as active uc . furthermore , the results of another study have shown that administration of 800 mg of the extended intestinal release form of rifaximin twice daily for 12 weeks has induced remission in patients with moderately active cd . it is possible that the use of klebsiella sensitive antibiotics could reduce the bacterial loads in the bowel of cd patients and prevent production of further anti - klebsiella antibodies and tissue damaging autoantibodies . although no studies have yet been carried out on patients with cd in regard to the use of low - starch diet , clinical trials , however , have been carried out in as patients with some encouraging results . in a longitudinal clinical study carried out on a group of patients with as receiving low - starch diet , there was a significant drop in the erythrocyte sedimentation rate and total serum iga by the end of the 9-month period of the study . most of these patients have reported positive responses to the diet involving partial to complete recovery , ranging from disappearance of symptoms to a drop or even cessation in the intake of anti - inflammatory drugs . from experience of recommending the low - starch diet in patients with as for the last 3 decades and more recently in cd , it is concluded that normally it takes around six to eight months for the diet to show its effects . it is plausible that a drop of the starch intake in the daily dietary consumption might reduce the bowel microflora by depleting the substrates necessary for enterobacterial growth . for example , reducing starch intake could stop the growth of klebsiella , with a possibility of having beneficial effects on the disease outcome . it is concluded that klebsiella has a pivotal role in the aetiopathogenesis of cd , as evidenced by enhanced anti - klebsiella immune responses and significant cross - reactivity and cytotoxic reactions . low - starch diet could help in the eradication of klebsiella in the bowel and , hence , decreasing the disease activity and progress with eventual halt or regression of the pathological process in patients with cd and as .

there is a general consensus that crohn 's disease ( cd ) develops as the result of immune - mediated tissue damage triggered by infections with intestinal microbial agents . based on the results of existing microbiological , molecular , and immunological studies , klebsiella microbe seems to have a key role in the initiation and perpetuation of the pathological damage involving the gut and joint tissues in patients with cd . six different gastroenterology centres in the uk have reported elevated levels of antibodies to klebsiella in cd patients . there is a relationship between high intake of starch - containing diet , enhanced growth of gut microbes , and the production of pullulanases by klebsiella . it is proposed that eradication of these microbes by the use of antibiotics and low starch diet , in addition to the currently used treatment , could help in alleviating or halting the disease process in cd .

1. Introduction 2. Aetiopathogenesis 3. Starch Digestion and Its Relation to Gut Microbes 4. Proposal for the Use of Antimicrobial Measures in Treatment of CD 5. Conclusion

crohn 's disease ( cd ) is a chronic , progressive , and potentially disabling disease , characterised by relapsing and remitting episodes of transmural inflammation of the gastrointestinal tract which might be associated with arthritic manifestations . patients with either cd or uc , however , are likely to have associated extraintestinal manifestations . furthermore , patients with cd seem to share more genetic , clinical , laboratory , immunological , and pathological features with as patients [ 57 ] . it has a high impact on the psychological condition , as well as the social status and work abilities in patients with this disease . based on these negative effects on the general health and welfare of patients with cd and the drawbacks of the currently used medical treatments as well as the increased likelihood of surgical complications in these patients , a search for the causative factor(s ) together with a proposal for the use of an alternative therapeutic strategy involving eradication of the causative factors could be of crucial importance in the management of this disease . the same increase in the incidence of cd these rises in the occurrence of cd could largely be explained by altered lifestyles and environmental exposures and/or changes in the dietary habits among the patients with this disease . at least three interacting elements including genetic factors , intestinal microbes , and immune - mediated inflammation are involved in the pathogenesis of cd . the most popular theory , however , is that the disease usually results from an abnormal immune response to certain enterobacterial pathogens present in the gut lumen . although many of the non - hla genes such as nod2/card15 , il23r , and ctla4 have been suggested to play a role in the susceptibility to develop cd until now , however , scientists have not been able to discover a gene which might solely contribute to the aetiology of this disease . aetiopathogenetic evidence for the roles of genetic / microbial ( especially klebsiella ) factors in cd evidence of the genetic involvement positive family history.a considerable increase in the risk of developing the disease among monozygotic twin of patients with cd.higher risk of developing the disease among relatives of patients with cd or other entities of spas.presence of hla - b27 genetic markers in more than 50% of patients with cd having spondylitis.significant association with nod2/card15 and some other genetic mutations.transgenic rats carrying human hla - b27 genes are more prone to develop spontaneous bowel lesions . a considerable increase in the risk of developing the disease among monozygotic twin of patients with cd . higher risk of developing the disease among relatives of patients with cd or other entities of spas . evidence of the environmental / microbial factors more than 80% of dizygotic twins of patients with cd fail to develop the disease.increased incidence of cd among friends and clustering of unrelated people as well as immigrants moving from low to high risk areas.associated bouts of remissions and exacerbations with spatial and temporal variations in the incidence and the clinical course of the disease in cd.increased gut mucosal leukocyte trafficking and enhanced microbial innate immune responses , as well as increased risk of developing the disease after appendectomy in patients with cd . more than 80% of dizygotic twins of patients with cd fail to develop the disease . associated bouts of remissions and exacerbations with spatial and temporal variations in the incidence and the clinical course of the disease in cd . increased gut mucosal leukocyte trafficking and enhanced microbial innate immune responses , as well as increased risk of developing the disease after appendectomy in patients with cd . evidence of the role of klebsiella and crossreactive self antigens increased load of klebsiella culture from fecal matter in the large bowel.occurrence of intercurrent klebsiella intestinal colitis in patients with cd.significantly enhanced antibody responses to klebsiella microbes were found on many occasions in patients with cd when compared to healthy subjects . increased antibody levels against collagens i , iii , and iv , all crossreactive with klebsiella pullulanase enzymes , were found in patients with cd when compared to healthy controls.molecular similarities were found between different antigens present in klebsiella nitrogenase and pullulanase puld enzymes and hla - b27 self - antigens . occurrence of intercurrent klebsiella intestinal colitis in patients with cd . significantly enhanced antibody responses to klebsiella microbes were found on many occasions in patients with cd when compared to healthy subjects . increased antibody levels against collagens i , iii , and iv , all crossreactive with klebsiella pullulanase enzymes , were found in patients with cd when compared to healthy controls . enzymes were found to possess a molecular structure similar to the trihelical shape of collagen i , iii , and iv fibres . the link between hla - b27 and cd on the other hand is relatively more obvious especially in patients with associated spondylitis . for example , more than forty percent of patients with cd , especially those having spondylitis / sacroiliitis , were found to be positive for the hla - b27 markers , but the prevalence of this gene in patients with cd without spondylitis / sacroiliitis merely parallels its frequency in the caucasian general population . moreover , in an experimental study by a group from dallas , transgenic rats with high level of human hla - b27 expression were found to be more prone to develop bowel inflammation with arthritis , and the severity of intestinal features was correlating with concentration of the large bowel microbial flora . extensive evidence supports the role of certain environmental and/or microbial factors in the aetiopathogenesis of ibd and particularly in the causation of cd ( as shown in the first three lists).a negative family history in more than 95% and a low concordance rate among twins of patients with cd support the role of environmental factors in the development of this disease.there is a report for an increased clustering of cd among unrelated individuals and closely living friends , as well as amid second - generation immigrants moving from low to high risk areas . in a previous study on a large moroccan family and one parent with cd emigrated to belgium , 4 out of the 8 children were found to have subsequently developed the disease . patients with cd show intermittent variations in the clinical onset and activity during the disease course as well as during different seasons of the year with associated concurrent intestinal bacterial infections . increased incidence of cd after appendectomy , together with involvement of neutrophils and lymphocytes in the gut inflammation as well as enhanced general immune response to enterobacterial antigens in these patients , supports the role of gut microbes in the aetiopathogenesis of this disease.transgenic rats do not develop colitis under germ - free conditions , but when these rats are transferred into conventional environments , they develop signs of intestinal inflammation and colitis . these results indicate that colonic microflora plays a key role in the development or exacerbation of the intestinal inflammation resembling that occurring in patients with cd . a negative family history in more than 95% and a low concordance rate among twins of patients with cd support the role of environmental factors in the development of this disease . in a previous study on a large moroccan family and one parent with cd emigrated to belgium , failure to find significant genetic variants with high incidence of cd among this family suggests the direct role of a major environmental factor which probably has been encountered in the new residing area and is not related to the sanitation in childhood . patients with cd show intermittent variations in the clinical onset and activity during the disease course as well as during different seasons of the year with associated concurrent intestinal bacterial infections . increased incidence of cd after appendectomy , together with involvement of neutrophils and lymphocytes in the gut inflammation as well as enhanced general immune response to enterobacterial antigens in these patients , supports the role of gut microbes in the aetiopathogenesis of this disease . these results indicate that colonic microflora plays a key role in the development or exacerbation of the intestinal inflammation resembling that occurring in patients with cd . during the last few decades , various microbial agents , including also klebsiella pneumonia , have been implicated to have a role in the aetiopathogenesis of cd . a considerable amount of microbiological , molecular , and immunological evidence exists which supports the role of klebsiella microbes in the aetiology of cd ( figure 1 ) ( as shown in the first three lists ) . it was reported that , in more than 20% of patients with cd , klebsiella microorganisms have been isolated from the large bowel specimens ( hring et al . the disease relapses in patients with cd were found also to be associated with attacks of klebsiella oxytoca colitis . in a study on the biopsy specimens taken from 12 patients with cd , it has been shown that invasion of the bowel mucosa by microbial agents including also enterobacteriaceae spp . was evident in 55.6% of the ileal and in 25% of the colonic specimens from the cd patients , but no bacteria were detected in the tissues of the non - cd controls . in another study using immunohistochemistry , however , the majority of biopsy specimens taken from the bowel mucosa of patients with ibd have shown negative staining for escherichia , listeria , and even klebsiella microbes . this latter finding could indicate that it is the microbial bulk in intestinal lumen rather than at the sites of the pathological lesions , which is important in evoking both local mucosal and general antibacterial immune responses . it was reported that , in more than 20% of patients with cd , klebsiella microorganisms have been isolated from the large bowel specimens ( hring et al . the disease relapses in patients with cd were found also to be associated with attacks of klebsiella oxytoca colitis . in a study on the biopsy specimens taken from 12 patients with cd , it has been shown that invasion of the bowel mucosa by microbial agents including also enterobacteriaceae spp . was evident in 55.6% of the ileal and in 25% of the colonic specimens from the cd patients , but no bacteria were detected in the tissues of the non - cd controls . this latter finding could indicate that it is the microbial bulk in intestinal lumen rather than at the sites of the pathological lesions , which is important in evoking both local mucosal and general antibacterial immune responses . elevated levels of humoral immune responses to klebsiella microbes in patients with cd as well as uc have been shown in several studies carried out at six different gastroenterology centres in the united kingdom.significantly increased levels of antibodies against klebsiella and yersinia enterobacterial agents were observed in patients with cd and uc from birmingham when compared to corresponding healthy controls .iga antibody levels against klebsiella microbes were found to be significantly increased in patients with ibd and as from glasgow when compared to corresponding control subjects .anti - klebsiella antibody levels were found to be significantly higher in patients with cd and as from edinburgh when compared to corresponding controls .in three consecutive studies carried out at different gastroenterology centres in london and winchester , similar results have been reported . in one study , klebsiella antibody levels were found to be significantly higher in patients with ibd and as when compared to healthy or other disease controls , whilst no such elevations were observed in the antibody levels against e. coli or other anaerobic intestinal microbes . in a second study , the levels of class - specific isotypic antibodies against many klebsiella antigens were found to be significantly elevated in patients with cd and as when compared to patients with coeliac disease or to healthy controls . in a third study , the levels of class - specific antibodies against klebsiella microbes and crossreactive collagens i , iii , iv , and v were found to be significantly increased in patients with early and late cd as well as in patients with as when compared to healthy controls . moreover , a positive correlation was demonstrated between antibody levels to collagen types i , iii , and iv and klebsiella in both groups of patients with cd and as . significantly increased levels of antibodies against klebsiella and yersinia enterobacterial agents were observed in patients with cd and uc from birmingham when compared to corresponding healthy controls . anti - klebsiella antibody levels were found to be significantly higher in patients with cd and as from edinburgh when compared to corresponding controls . in three consecutive studies carried out at different gastroenterology centres in london and winchester , klebsiella antibody levels were found to be significantly higher in patients with ibd and as when compared to healthy or other disease controls , whilst no such elevations were observed in the antibody levels against e. coli or other anaerobic intestinal microbes . in a second study , the levels of class - specific isotypic antibodies against many klebsiella antigens were found to be significantly elevated in patients with cd and as when compared to patients with coeliac disease or to healthy controls . in a third study , the levels of class - specific antibodies against klebsiella microbes and crossreactive collagens i , iii , iv , and v were found to be significantly increased in patients with early and late cd as well as in patients with as when compared to healthy controls . moreover , a positive correlation was demonstrated between antibody levels to collagen types i , iii , and iv and klebsiella in both groups of patients with cd and as . it is , therefore , plausible that cd disease could result from repeated subclinical infections by klebsiella microbes in the large bowel with the consequent inflammations and tissue damage in the bowel and joints resulting from the binding of anti - klebsiella and anti - self tissue antibodies to the crossreactive targeted antigens . it is composed of two types of structurally distinct -glucan polymer , amylase , and amylopectin . amylose , which constitutes around 2030% of the starch particle , consists of long chains of several thousand -glucosyl units joined by (14)-linkages , whilst amylopectin , which constitutes the other 7080% part of starch , is a branched molecule comprising short chains of (14)-linked -glucosyl units , joined by (16 ) branch linkages . degradation of the starch molecules is carried out by various catalytic enzymes , which are produced by plants , microbes , and human body . klebsiella microbes can survive in harsh environments exploiting some of their enzymatic products , which are required for the protection , maintenance , and survival of these microbes . apart from nitrogenase reductases these microbes can utilize starch as the sole carbon and energy source via two metabolic routes . the first one involves the extracellular degradation into linear maltodextrins by hydrolysis of the glycosidic bonds via the cell - surface - associated pullulanase and then the subsequent cleavage of the glycosidic linkages by the action of the extracellular glycosyltransferase . for example , it has been reported that a high intake of carbohydrate , particularly oligosaccharides , can stimulate the growth of bifidobacterium spp . recommended diet for crohn 's disease and ankylosing spondylitis patients decreased intake of high - starch - containing foods flour and related products : breads , biscuits , cakes , puddings , pies , and popcorn . increased intake of none - low - starch - containing foods meat : beef , pork , lamb , bacon , salami , corned beef , luncheon mean , potted meat , ham , and veal as well as chicken , turkey duck , or any other poultry meat . these results indicate that complex carbohydrates such as starch products are necessary for the growth , replication , and persistence of klebsiella as well as other enterobacterial agents in the gut . the current treatment involving the use of anti - inflammatory , immunosuppressive , and biologic modalities has significant limitations due to lack of treatment response in some patients as well as the occurrence of adverse side effects , such as increased risk of infection and malignancy among some other patients especially in those taking antitumor necrosis factors . sustained therapeutic responses in patients with cd , however , may require more radical and comprehensive approaches involving strategies which help in the modification of the intestinal bacterial microenvironments . a solution for this medical predicament is the use of an alternative therapy , which should be harmless and aimed at the elimination of the most plausible microbial agent , such as klebsiella , in order to improve or even halt the inflammatory and pathological damage occurring in patients with cd . to achieve this therapeutic goal , the most likely strategy which could be added to the management of this disease is the use of antimicrobial agents and dietary manipulation . manipulation of luminal content of the gut using antibiotics may represent a potentially effective therapeutic option . in another meta - analysis review of randomized placebo - controlled studies , it has been shown that there was a statistically significant effect of antibiotics being superior to placebo in patients with active and quiescent cd as well as active uc . furthermore , the results of another study have shown that administration of 800 mg of the extended intestinal release form of rifaximin twice daily for 12 weeks has induced remission in patients with moderately active cd . it is possible that the use of klebsiella sensitive antibiotics could reduce the bacterial loads in the bowel of cd patients and prevent production of further anti - klebsiella antibodies and tissue damaging autoantibodies . although no studies have yet been carried out on patients with cd in regard to the use of low - starch diet , clinical trials , however , have been carried out in as patients with some encouraging results . in a longitudinal clinical study carried out on a group of patients with as receiving low - starch diet , there was a significant drop in the erythrocyte sedimentation rate and total serum iga by the end of the 9-month period of the study . most of these patients have reported positive responses to the diet involving partial to complete recovery , ranging from disappearance of symptoms to a drop or even cessation in the intake of anti - inflammatory drugs . from experience of recommending the low - starch diet in patients with as for the last 3 decades and more recently in cd , it is concluded that normally it takes around six to eight months for the diet to show its effects . it is plausible that a drop of the starch intake in the daily dietary consumption might reduce the bowel microflora by depleting the substrates necessary for enterobacterial growth . for example , reducing starch intake could stop the growth of klebsiella , with a possibility of having beneficial effects on the disease outcome . it is concluded that klebsiella has a pivotal role in the aetiopathogenesis of cd , as evidenced by enhanced anti - klebsiella immune responses and significant cross - reactivity and cytotoxic reactions . low - starch diet could help in the eradication of klebsiella in the bowel and , hence , decreasing the disease activity and progress with eventual halt or regression of the pathological process in patients with cd and as .

following unilateral damage to the visual cortex , patients experience clinical blindness in both halves of each eye in their contralesional visual hemifield ( homonymous hemianopia ) . despite clinical blindness , some patients retain nonconscious visual abilities for processing unseen stimuli in the blind hemifield ( ajina et al . , 2015 , barbur et al . , 1993 , blythe et al . , 1987 , celeghin et al . , 2015a , corbetta et al . , 1990 , marzi , 1986 , pizzamiglio et al . , 1984 , pppel et al . , 1973 , rafal et al . , 1990 , singer et al . , 1977 , stoerig , 1987 , stoerig et al . , 1985 , , 2013 , torjussen , 1976 , torjussen , 1978 , weiskrantz et al . ( 1974 ) , have been described for different visual functions such as stimulus detection ( sahraie et al . , 1997 , 1995 ) , shape or category - specific processing ( trevethan et al . , 2007 , van den stock et al . , 2015 , van den stock et al . , 2014 ) , color and motion discrimination ( hervais - adelman et al . , 2015 , , 2007 , morland et al . , 1999 ) , recognition of facial and bodily expressions ( bertini et al . moreover , preserved processing of such visual properties has been described under a variety of task demands , such as visually guided or goal directed behaviour ( buetti et al . , 2013 , , 2015c , pppel et al . , 1973 , rafal et al . , 1990 , spering and carrasco , 2015 ) , yes - no or alternative forced - choice tasks ( azzopardi and cowey , 1997 , tamietto et al . , 2009 ) , and perceptual completion requirements ( torjussen , 1978 ) ( see tamietto & morrone , 2016 , for a recent review ) two kinds of strategies have been typically employed to assess blindsight : direct and indirect methods . the former makes use of forced - choice paradigms where the subjects make an explicit decision regarding unseen attributes of the stimulus presented to their blind hemifield ( danckert and rossetti , 2005 , stoerig and cowey , 1989 , weiskrantz , 1990 ) . above chance performance , despite absence of awareness , is taken as indicative of blindsight . in contrast , the latter methods rely on the effect exerted by unseen stimuli presented to the blind hemifield on stimuli simultaneously presented to the intact counterpart . one of the indirect methods most often used for testing blindsight is the redundant target effect ( rte ) ( marzi , tassinari , aglioti , & lutzemberger , 1986 ) . in healthy participants , the tachistoscopic presentation of two or more synchronous stimuli to both visual hemifields ( bvf ) across the vertical meridian results in faster reaction times ( rts ) than a single stimulus presentation to one visual hemifield , either left ( lvf ) or right ( rvf ) . this effect , also known as bilateral summation or redundancy gain , has been reported in many studies in healthy participants as well as in blindsight patients ( celeghin et al . the main advantage of indirect methods is that patients make a choice about visual attributes they do not consciously acknowledge without being forced to do so , but are only required to respond as quickly as possible to the stimulus in their intact hemifield in a simple rt paradigm . therefore , since patients have to respond to stimuli they can normally perceive , the range of visual operations that can potentially be investigated is wide and may include high - order visual operations . ( 2015 ) introduced a variant of the rte to obtain insights on the influence of stimulus numerosity and configuration on visuo - motor responses in blindsight patients . participants were presented with either unilateral or bilateral black dots . for each of these two conditions , the latter were presented in either a variable random spatial configuration or a fixed one wherein the four dots were arranged in a diamond - like shape . notably , the two configurations subtended the same visual angle and had the same luminance . orthogonal to the replication of the common rte in the comparison between unilateral and bilateral conditions , the authors also found an additive effect of stimulus configuration with a speeding up of rt when the gestalt - like , but not the random shape , quadruple pattern was projected to the patients ' blind field . these novel findings have allowed the establishment of a solid approach to study the influence of stimulus configuration on blindsight and its underlying neural structures , an issue that in the past has come under only desultory scrutiny . these results have provided initial support for the notion that nonconscious vision might be sensitive to perceptual organization , thereby enhancing the processing of gestalt - like or structured over meaningless or randomly assembled stimuli . concerning the neuro - functional mechanisms of rte , several studies in patients with unilateral destruction of the primary visual cortex ( v1 ) or with removal of the entire cortical mantle in one hemisphere ( hemispherectomy ) have provided convincing evidence that the superior colliculus ( sc ) is necessary and sufficient for the rte to occur ( leh et al . this raises the interesting , entirely unexplored , possibility that the sc responds differentially to higher - order perceptual properties , such as those involved in stimulus configuration , even in the absence of the geniculo - striate pathway that deprives vision of its conscious component . although suggestive in a number of aspects , the previous results by celeghin , savazzi , et al . , 2015 are not conclusive for two reasons . firstly , the patients had intact portions of extrastriate visual areas as well as spared retino - recipient subcortical structures besides the sc , such as the lateral geniculate nucleus ( lgn ) and the pulvinar ( pulv ) . all these subcortical structures have been shown to receive direct input from the retina and to send ( mainly ) ipsilateral efferents to several extrastriate visual areas bypassing v1 ( ajina et al . , 2008 , lyon et al . , 2010 , schmid et al . , 2010 , , 2004 , tamietto and morrone , 2016 , tamietto et al . , 2012 ) . therefore , the relative contribution of the sc could not be disentangled from that of the other subcortical centers or their extrastriate targets , so that the sc specific role remains unresolved . secondly , while a variety of random configurations were used in the original study , only one diamond - shape dot pattern was presented , thereby leaving the possibility that the effect found for the latter condition was due to familiarity and/or to spatially fixed versus variable stimulus configuration rather than to the presence of a gestalt - like dot pattern per se . the aim of the present study is to tackle these questions by partially modifying the original experimental paradigm and by testing patients with hemispherectomy and blindsight . these patients had undergone removal of an entire cerebral hemisphere or of all the temporo - occipito - parietal cortices . moreover , the lgn and pulv in the affected hemisphere have been both removed leaving only the sc intact among retino - recipient subcortical structures . therefore , testing the rte in these patients has offered the unprecedented opportunity to examine the impact of perceptual configuration in nonconscious visually guided behaviour under the most stringent conditions in order to determine the putative crucial role of the sc . patient dr is a right - handed woman ( 40 years old at the time of testing ) who presented with left hemiparesis since birth and began suffering from epileptic seizures at the age of 5 years . ct and mri scans performed at the age of 17 years revealed severe atrophy of the right cerebral hemisphere and eeg recording showed epileptiform activity over the right frontal - parietal - temporal regions . cognitive testing indicated borderline intelligence scores : full scale intelligence quotient ( fisq ) , 77 ; verbal iq , 92 and performance iq , 65 . at the same age , she underwent a functional hemispherectomy , which consisted of removing the temporal lobe including the mesial structures , the amygdala , the hippocampus , and a frontal - parietal corticectomy . the remaining cortical regions were left in situ but were disconnected from the rest of the brain by sectioning the white matter anteriorly and laterally , as well as posteriorly and laterally along the falx . follow - up assessments , at the age of 19 years , indicated that her level of intellectual function had increased to the low average range : fisq , 83 ; verbal iq , 87 and performance iq , 83 . mri scans postoperatively , as well as further scans performed afterwards for research purposes and published elsewhere , showed the presence of intact left and right sc , whereas the presence of the pulv was limited to the left ( intact ) hemisphere ( fig . 1a ) ( leh et al . , 2008 , leh et al . , 2006a , leh et al . , 2010 , complete contralateral ( left ) hemianopia without macular sparing has been confirmed by computerized perimetry ( allergan , humphrey ) , and her visual acuity was 20/25 . patient se is a right - handed man ( 49 years old at the time of testing ) whose left hemiparesis was noted at birth . seizure onset occurred at the age of 7 years . at the age of 23 years , ct and mri scans showed a porencephalic cyst occupying the right temporal - parietal - occipital regions . eeg recordings detected epileptiform activity in the right occipital cortex alongside independent foci over the right temporo - parietal cortex . neuropsychological testing revealed an fsiq of 78 ; verbal iq of 80 and performance iq of 79 . at the age of 25 , he underwent a surgery to remove the congenital porencephalic cyst , and a temporal - parietal - occipital lobectomy was carried out to alleviate intractable seizures . the lobectomy included the hippocampus and the amygdala but spared the anterior portion of the frontal lobe . mri scans postoperatively , as well as further scans performed afterwards for research purposes and published elsewhere , showed the presence of intact left and right sc , but only presence of the pulv on the left ( intact ) side ( fig . , 2008 , leh et al . , 2006a , leh et al . , 2010 , follow - up cognitive testing , at the age of 26 years , showed an increase in iq to an average range : fsiq , 93 ; verbal iq , 90 and performance iq , 99 . contralateral hemianopia without macular sparing was confirmed by computerized perimetry ( allergan , humphrey ) , and his visual acuity was 20/30 . the stimuli were black dots presented against a uniform gray background of 11.42 cd / m luminance ( rgb values = 126 , 126 , 126 ) . the dots were presented either unilaterally , to the seeing rvf , or to bvf . for each of these two presentation conditions , there were three possible display types : a single dot , a quadruple pattern composed by the same dots organized in gestalt - like configurations , or a quadruple pattern of dots organized in random configurations . quadruple arrays were displayed with the innermost dot at 6.5 of eccentricity with respect to the central fixation along the horizontal meridian ( the same for single dot displays ) and with the outermost dot at 8.5. gestalt - like configurations were of four different shapes : diamond , square , rectangles with longer vertical sides and rectangles with longer horizontal sides . random configurations also consisted of the same dots organized in four different but equally meaningless combinations . in all bvf presentations with quadruple stimuli , the two patterns of stimuli projected to the two visual fields were of the same type , but not physically identical ( e.g. a diamond shape in the lvf and a square shape in the rvf ) , in order to avoid any interpretation of the results in terms of bilateral symmetry ( fig . 2 ) . the stimuli were projected on a 17 lcd monitor ( refresh rate : 16.7 hz ) using a macbook pro notebook with exposure duration of 80 ms ( 5 refresh rates ) . the observer 's eyes were at a distance of 57 cm from the monitor . stimulus presentation and response recording were controlled by means of the presentation 16 software ( neurobehavioral systems , albany , ca ) . participants ' head movements were minimized through the use of a head and chin rest . they were required to maintain fixation on a small black circle ( diameter .3 , luminance : .82 cd / m ) in the center of the screen and to refrain from moving their eyes . eye movements were also monitored online by one experimenter through an infra - red camera connected with an eye - tracking system , and trials were removed in the event of unsteady fixation . in this rare case ( < 5% ) an additional trial was added to the end of the block . moreover , to ensure fixation during stimulus presentation , each trial started with a warning acoustic signal ( duration : 150 ms ; frequency : 1000 hz ) followed by the visual stimulus after a randomized temporal interval ( varying between 300 and 700 ms ) . the patients were tested monocularly with the ( left ) contralesional eye while an eye patch covered the ( right ) ipsilesional eye , and response was performed by pressing the space bar of the notebook with the ( right ) ipsilesional hand . first , in both patients the non - dominant ( right ) eye ipsilateral to the damaged hemisphere tended to deviate independently from the gaze direction of the dominant eye . this had potentially undermined the correct lateralization of the stimuli during fixation , as two different locations could had been represented in the fovea of the two eyes and a stimulus assumed to be projected in the left blind hemifield might have actually fallen into the seeing field of the right eye . for example , stimuli in the temporal hemifield ( the left hemifield of the left eye ) induce preferential gaze orienting or summon attention more readily than stimuli in the nasal hemifield ( the right hemifield of the left eye ) ( rafal et al . these behavioural asymmetries have been proposed to indicate the contribution of the sc in such tasks . anatomically , indeed , the superficial layers of the sc receive visual input predominantly from the nasal hemiretina , which samples the temporal hemifield , whereas the connections from the temporal hemiretina constitute a relatively weaker retino - tectal pathway ( hubel et al . this has been confirmed in an fmri study showing higher activation of the sc following stimulation of the temporal rather than nasal hemifield ( sylvester , josephs , driver , & rees , 2007 ) . therefore , testing the patients monocularly with the left eye ensured that the stimuli projected peripherally to the ( left ) blind temporal hemifield during bilateral conditions were processed uniquely by the nasal hemiretina , and thus relayed to the right sc , ipsilateral to the damaged hemisphere , through the stronger of the two retino - tectal pathways ( fig 3 ) . in contrast , the stimuli projected to the ( right ) intact nasal hemifield reached the left sc , ipsilateral to the intact hemisphere , through the weaker connections involving the temporal hemiretina pathway . this was done to counterbalance the potentially weaker representation of the ( left ) unseen over ( right ) seen stimulus during bilateral stimulation , as well as the overall weaker response in the ( right ) ipsilesional sc compared to the ( left ) sc in the intact side , which might have compromised the rte . the stimuli were presented in two blocks , each containing 84 randomized trials . within each block , the six stimulation conditions and display types were equiprobable , and each was repeated for 14 trials ( unilateral : single , quadruple gestalt - like , quadruple random ; bilateral : single , quadruple gestalt - like , quadruple random ) . in total , each patient received 28 stimulus presentations per condition . based on previous reports on the same patients , only rts in the time - range 2001000 ms were considered ( leh et al . , 2006b , leh et al . , 2010 , patient dr responded to all 168 trials within the accepted range , whereas se did not respond to 3 trials ( 1.8% ) , and had one anticipation ( .6% ) , while the responses to the remaining trials ( 97.6% ) were within the accepted range . mean rts as a function of the six stimulus conditions are shown separately for patient dr and se in fig . visual inspection reveals that rts decreased in bilateral compared to unilateral presentations irrespective of the different display types and for both patients , although this decrease of rts was particularly pronounced for gestalt - like configurations . initially , a 2 3 repeated - measures anova was carried out on rts data with the within - subjects factors of presentation condition ( unilateral vs bilateral ) and configuration ( single , gestalt - like , random ) . patient dr showed a significant main effect of presentation condition [ f(1 , 27 ) = 47.507 , p < the main effect of configuration was also significant [ f(2 , 54 ) = 38.133 , p < .0001 ) , but there was no significant presentation condition configuration interaction [ f(2 , 54 ) = 1.777 , p < .178 ] . a post - hoc bonferroni comparison performed on the configuration factor revealed that rts were significantly faster for gestalt - like patterns than for either single or quadruple random dot shapes [ t(55 ) 6.31 p .0001 ] , which in turn did not differ from each other significantly [ t(55 ) = 1.92 , p = .149 ] . two additional anovas were computed separately for the unilateral and bilateral presentation conditions , each with the factor configuration and the usual three levels ( single , gestalt - like , random ) . the aim of this additional analysis was to determine whether the presentation of gestalt - like configurations in the blind hemifield was pivotal for the effect to occur . this is because in unilateral trials the stimuli were projected in the patients ' intact hemifield and any effect potentially observed thus reflects sensitivity for consciously seen stimuli . conversely , in bilateral displays a stimulus was added in the blind hemifield , and any significant difference arising among conditions in this case can only be accounted for by the nature of the unseen stimulus . on unilateral trials there was a significant difference between display conditions [ f(2 , 54 ) = 12.47 , p < .0001 ] . post - hoc tests showed that rts for gestalt - like configurations in the intact hemifield were significantly faster than for single or quadruple random patterns [ t(27 ) 3.22 p .002 ] , while the latter two configurations did not differ from each other ( p = .864 ) . the anova performed on bilateral trials was also highly significant [ f(2 , 54 ) = 22.93 , p < .0001 ] . post - hoc comparisons revealed a significant speeding up of rts with bilateral gestalt - like patterns with respect to single or bilateral random configurations [ t(27 ) 6.08 p < .0001 ] , while there was no significant difference between single and random patterns ( p = .334 ) . patient se displayed a significant main effect of presentation condition [ f(1 , 25 ) = 12.41 , p = .002 ] indicative of an rte . the configuration factor was also significant [ f(2 , 50 ) = 41.26 , p < .0001 ] , as well as the presentation condition configuration interaction [ f(2 , 50 ) = 40.48 , p < .0001 ] . post - hoc comparisons on the interaction showed that the rts for gestalt - like configurations were significantly faster than for either single or random patterns , but only in bilateral trials [ t(25 ) 7.73 p .0001 ] . this significant interaction made it unnecessary to compute the additional anovas separately for unilateral and bilateral trials . indeed , the interaction already indicates unambiguously that , unlike dr who was sensitive to gestalt - like patterns in both her intact hemifield ( during unilateral presentation ) and blind hemifield ( during bilateral presentation ) , patient se was differentially responsive to gestalt - like configurations only when the stimuli were projected bilaterally , and hence to his blind hemifield as well . additionally , we plotted the cumulative distribution functions ( cdfs ) of rts for all six stimulation conditions and for both patients separately . this detailed graphical description enabled us to check whether the bilateral gain observed on mean values occurred throughout the whole rts distribution . smirnov test of the cdfs , which represents a nonparametric version suitable for carrying out a single - subject analysis of miller 's inequality test ( miller , 1982 ) , a mathematical tool to assess whether the rte is more likely to be related to probability or neural summation . this further analysis is important , because only the latter type of bilateral gain postulates the existence of a neural centre where the visual input from the two hemifields is summed . in fact , observation of rte on mean values is not per se conclusive of neural summation . separate - activation or race models account for a bilateral gain by simply relying on the fact that the probability of a fast detection increases with the number of stimuli ( raab , 1962 , townsend and ashby , 1983 ) . since speed of processing is a random variable , multiple stimuli are on average more likely to yield a faster rt than single stimuli for purely probabilistic reasons . in contrast , co - activation models assume the presence of a functional interaction or neural summation between perceptual channels that result in a reduction of rts larger than that predicted by probability summation alone ( colonius , 1986 , colonius and diederich , 2006 , miller , 1982 ) . note that violation of the inequality test unambiguously supports neural summation , whereas no conclusion can be reached if the inequality is not violated , owing to the conservative nature of the test ( miller , 1982 ) . as displayed in fig . 5 , when gestalt - like configurations were presented , rts for the bilateral condition were faster than for the unilateral condition throughout the entire distribution and in both patients dr and se ( p < .001 by kolmogorov smirnov test ) , thereby providing convincing evidence for an interpretation of the rte in terms of neural summation . conversely , the cdfs for unilateral and bilateral presentations when a single or random dot configurations were displayed overlapped substantially and crossed , thus failing to support an interpretation of the rte in terms of neural summation ( p .1 by kolmogorov smirnov test ) . this latter finding confirms a previous study showing that it is not always possible to attribute the rte for single dots to neural summation ( turatto , mazza , savazzi , & marzi , 2004 ) , but that its nature depends on stimulus and task factors . nevertheless , the present results using cdfs indicate that , under identical conditions , neural summation for gestalt - like configurations was significantly more likely to occur than that for single or random dot configurations in both patients . in the present study , we investigated the sensitivity of two blindsight patients with hemispherectomy to stimulus perceptual organization when the display is presented to the blind hemifield . we found that , in addition to the overall rte often reported in previous studies in hemianopic patients , there was a rte specific for gestalt - like stimulus configurations but not for spatially random patterns . these findings confirm and extend previous observations in patients with blindsight following lesions restricted to portions of the visual cortex ( celeghin , savazzi , et al . , 2015 ) , and also rule out extant alternative interpretations not related to stimulus configuration . a difference in stimulus familiarity or variability between gestalt - like and random configurations can not account for the present results , since there were four different patterns counterbalanced for each of the two display types . moreover , gestalt - like and random patterns were randomly intermingled and presented within the same block of trials , whereas in the previous study by celeghin , savazzi et al . ( 2015 ) , trials with these different configurations were administered in separate blocks always starting with gestalt - like configurations . hence , this new procedure also rules out the possibility that the original findings were partly due to fatigue or habituation determining the lack of effect for random patterns . while both patients exhibited similar overall results , they differed in that dr showed a speeding up of rts also when gestalt - like patterns were presented unilaterally in her intact hemifield , whereas patient se did not . this is possibly related to individual differences in sensitivity to consciously perceived gestalt - like configurations also present in the healthy population ( wagemans et al . , 2012 ) . our present study provides the first causal evidence of the sensitivity of the sc for overall stimulus configuration , as witnessed by the facilitation exerted when a structured perceptual organization is translated into motor output . since the sc in our patients is the only intact visual structure remaining in the ipsilateral side of the otherwise damaged hemisphere , the contribution of other subcortical structures such as the lgn and the pulv can be ruled out . this does not necessarily imply that the ipsilesional sc is solely responsible for the observed effect ( i.e. , does not support sufficiency of the sc for the effect to occur ) . in fact , visual information might well have been transferred from the ( right ) sc , ipsilateral to the damaged hemisphere , to the corresponding contralateral sc in the ( left ) intact side via the inter - collicular commissure or through other inter - hemispheric tracts , and from there to other subcortical structures such as the pulv as well as extrastriate visual areas in the ( left ) intact hemisphere . prior positron emission tomography ( pet ) ( ptito , johannsen , faubert , & gjedde , 1999 ) and fmri ( bittar , ptito , faubert , dumoulin , & ptito , 1999 ) studies on the two patients tested here demonstrated activation in extrastriate visual areas of the ( left ) intact hemisphere following stimulation of the ipsilateral ( left ) hemifield , thereby documenting substantial neuronal plasticity and reorganization . importantly , however , this activation does not seem to originate from cortical reorganization , e.g. owing to the expansion of the visual receptive fields in cortical areas of the intact hemisphere , but rather from the development of aberrant fibre tracts that connect the sc in the ( right ) damaged hemisphere to cortical areas in the opposite intact hemisphere ( leh , johansen - berg , et al . hence , the critical point here is that the visual information concerning stimulus configuration must have been initially processed by the right sc ipsilateral to the damaged hemisphere , thus indicating its necessity in processing stimulus configuration and in visuo - motor integration , as other alternatives are not possible . in keeping with this notion , the crucial involvement of the sc in the rte has been convincingly demonstrated behaviourally ( leh et al . , 2006b , tomaiuolo et al . , 1997 ) , and with combined behavioural and brain imaging studies in hemispherectomized patients ( leh et al . , 2010 ) as well as in an hemianopic patient with lesion confined to v1 ( patient gy ) ( tamietto et al . , 2010 ) . however , it should be stressed that all prior investigations used simple stimuli , whereas the present study used different perceptual configurations matched for stimulus intensity and position . according to a traditional view , it may appear surprising that the sc is able to represent complex stimulus configurations and respond differentially to a gestalt - like perceptual organization . the sc is indeed a phylogenetically ancient visual structure considered to have coarse retinotopy , which receives visual information only from magnocellular ( m ) and koniocellular ( k ) , but not from parvocellular ( p ) ganglion cells , and has a relative differential sensitivity to low spatial frequencies ( merigan and maunsell , 1993 , stone , 1984 ) . however , recent neurophysiological evidence as well as previously somewhat overlooked findings from single - cell recordings in monkeys and rats clearly indicate otherwise . for example , several types of neurons in the superficial ( i.e. , retino - recipient ) layers of macaque monkeys ' sc respond very poorly to simple visual stimuli and their activation requires real objects or certain two - dimensional patterns ( rizzolatti , buchtel , camarda , & scandolara , 1980 ) . likewise , neurons in the monkey sc can separately encode faces or face - like patterns ( nguyen et al . , 2014 ) . furthermore , neurons in the most superficial lamina of the rat 's sc perform sophisticated analysis of visual information and exhibit complex properties previously thought to be characteristic of visual cortical neurons only ( girman & lund , 2007 ) . therefore , the sc seems to participate in early stages of figure - ground segmentation and the combined interaction of m and k channels can enable encoding of complex and high - level properties of the visual input . moreover , early evidence of visuo - spatial localization and discrimination surviving hemidecortication has been provided by the seminal neuropsychological work of perenin and jeannerod ( perenin , 1978 , perenin and jeannerod , 1978 ) . these studies clearly underline the possibility that some degree of perceptual functions can be carried out by subcortical centers in the absence of the visual cortical mantle . lastly , one interesting issue concerns possible inter - hemispheric specialization for global versus local processing , which can contribute to the encoding of gestalt - like configurations . deficits in global processing following right hemisphere damage ( hugdahl & davidson , 2004 ) , or due to unbalancing of the complementary functions of the two hemispheres ( negro et al . , 2015 ) , have been repeatedly reported . in principle , the preserved sensitivity for gestalt - like configuration observed here despite right hemispherectomy could arise from the sc mirroring lateralized functions thus far reported primarily at the cortical level , or from lack of hemispheric specialization at the level of the sc . in the present study , both patients had undergone right hemispherectomy and it was thus not possible to compare the effects of right versus left hemispherectomy ; an issue that awaits further investigation . in conclusion , the present findings offer a clear demonstration that hemianopic patients as a result of hemispherectomy can be selectively sensitive to complex stimulus configuration within the context of the rte task . the sc can act as an interface between structured perceptual organization and motor processing , thereby providing an essential contribution to visually guided behavior despite being functionally and anatomically segregated from the geniculo - striate or extrastriate pathway , and therefore entirely outside conscious visual experience . an important avenue for future research is to try to examine other higher - order visual functions that can be carried out in the absence of striate and extrastriate cortical areas and whether such sensitivity can be proficiently exploited to foster rehabilitation of cortical blindness ( chokron et al . , 2008 , perez and chokron , 2014 ) .

patients with cortical blindness following a lesion to the primary visual cortex ( v1 ) may retain nonconscious visual abilities ( blindsight ) . one intriguing , though largely unexplored question , is whether nonconscious vision in the blind hemifield of hemianopic patients can be sensitive to higher - order perceptual organization , and which v1-independent structure underlies such effect . to answer this question , we tested two rare hemianopic patients who had undergone hemispherectomy , and in whom the only post - chiasmatic visual structure left intact in the same side of the otherwise damaged hemisphere was the superior colliculus ( sc ) . by using a variant of the redundant target effect ( rte ) , we presented single dots , patterns composed by the same dots organized in quadruple gestalt - like configurations , or patterns of four dots arranged in random configurations , either singly to the intact visual hemifield or bilaterally to both hemifields . as reported in a number of prior studies on blindsight patients , we found that bilateral stimulation yielded faster reaction times ( rts ) than single stimulation of the intact field for all conditions ( i.e. , there was an implicit rte ) . in addition to this effect , both patients showed a further speeding up of rts when the gestalt - like , but not the random shape , quadruple patterns were projected to their blind hemifield during bilateral stimulation . because other retino - recipient subcortical and cortical structures in the damaged hemisphere are absent , the sc on the lesioned side seems solely responsible for such an effect . the present results provide initial support to the notion that nonconscious vision might be sensitive to perceptual organization and stimulus configuration through the pivotal contribution of the sc , which can enhance the processing of gestalt - like or structured stimuli over meaningless or randomly assembled ones and translate them into facilitatory motor outputs .

Introduction Methods Results Discussion Conflict of interest

following unilateral damage to the visual cortex , patients experience clinical blindness in both halves of each eye in their contralesional visual hemifield ( homonymous hemianopia ) . despite clinical blindness , some patients retain nonconscious visual abilities for processing unseen stimuli in the blind hemifield ( ajina et al . the former makes use of forced - choice paradigms where the subjects make an explicit decision regarding unseen attributes of the stimulus presented to their blind hemifield ( danckert and rossetti , 2005 , stoerig and cowey , 1989 , weiskrantz , 1990 ) . in contrast , the latter methods rely on the effect exerted by unseen stimuli presented to the blind hemifield on stimuli simultaneously presented to the intact counterpart . one of the indirect methods most often used for testing blindsight is the redundant target effect ( rte ) ( marzi , tassinari , aglioti , & lutzemberger , 1986 ) . in healthy participants , the tachistoscopic presentation of two or more synchronous stimuli to both visual hemifields ( bvf ) across the vertical meridian results in faster reaction times ( rts ) than a single stimulus presentation to one visual hemifield , either left ( lvf ) or right ( rvf ) . this effect , also known as bilateral summation or redundancy gain , has been reported in many studies in healthy participants as well as in blindsight patients ( celeghin et al . the main advantage of indirect methods is that patients make a choice about visual attributes they do not consciously acknowledge without being forced to do so , but are only required to respond as quickly as possible to the stimulus in their intact hemifield in a simple rt paradigm . ( 2015 ) introduced a variant of the rte to obtain insights on the influence of stimulus numerosity and configuration on visuo - motor responses in blindsight patients . for each of these two conditions , the latter were presented in either a variable random spatial configuration or a fixed one wherein the four dots were arranged in a diamond - like shape . orthogonal to the replication of the common rte in the comparison between unilateral and bilateral conditions , the authors also found an additive effect of stimulus configuration with a speeding up of rt when the gestalt - like , but not the random shape , quadruple pattern was projected to the patients ' blind field . these novel findings have allowed the establishment of a solid approach to study the influence of stimulus configuration on blindsight and its underlying neural structures , an issue that in the past has come under only desultory scrutiny . these results have provided initial support for the notion that nonconscious vision might be sensitive to perceptual organization , thereby enhancing the processing of gestalt - like or structured over meaningless or randomly assembled stimuli . concerning the neuro - functional mechanisms of rte , several studies in patients with unilateral destruction of the primary visual cortex ( v1 ) or with removal of the entire cortical mantle in one hemisphere ( hemispherectomy ) have provided convincing evidence that the superior colliculus ( sc ) is necessary and sufficient for the rte to occur ( leh et al . this raises the interesting , entirely unexplored , possibility that the sc responds differentially to higher - order perceptual properties , such as those involved in stimulus configuration , even in the absence of the geniculo - striate pathway that deprives vision of its conscious component . although suggestive in a number of aspects , the previous results by celeghin , savazzi , et al . firstly , the patients had intact portions of extrastriate visual areas as well as spared retino - recipient subcortical structures besides the sc , such as the lateral geniculate nucleus ( lgn ) and the pulvinar ( pulv ) . therefore , the relative contribution of the sc could not be disentangled from that of the other subcortical centers or their extrastriate targets , so that the sc specific role remains unresolved . secondly , while a variety of random configurations were used in the original study , only one diamond - shape dot pattern was presented , thereby leaving the possibility that the effect found for the latter condition was due to familiarity and/or to spatially fixed versus variable stimulus configuration rather than to the presence of a gestalt - like dot pattern per se . the aim of the present study is to tackle these questions by partially modifying the original experimental paradigm and by testing patients with hemispherectomy and blindsight . moreover , the lgn and pulv in the affected hemisphere have been both removed leaving only the sc intact among retino - recipient subcortical structures . at the same age , she underwent a functional hemispherectomy , which consisted of removing the temporal lobe including the mesial structures , the amygdala , the hippocampus , and a frontal - parietal corticectomy . mri scans postoperatively , as well as further scans performed afterwards for research purposes and published elsewhere , showed the presence of intact left and right sc , whereas the presence of the pulv was limited to the left ( intact ) hemisphere ( fig . mri scans postoperatively , as well as further scans performed afterwards for research purposes and published elsewhere , showed the presence of intact left and right sc , but only presence of the pulv on the left ( intact ) side ( fig . for each of these two presentation conditions , there were three possible display types : a single dot , a quadruple pattern composed by the same dots organized in gestalt - like configurations , or a quadruple pattern of dots organized in random configurations . quadruple arrays were displayed with the innermost dot at 6.5 of eccentricity with respect to the central fixation along the horizontal meridian ( the same for single dot displays ) and with the outermost dot at 8.5. gestalt - like configurations were of four different shapes : diamond , square , rectangles with longer vertical sides and rectangles with longer horizontal sides . random configurations also consisted of the same dots organized in four different but equally meaningless combinations . in all bvf presentations with quadruple stimuli , the two patterns of stimuli projected to the two visual fields were of the same type , but not physically identical ( e.g. a diamond shape in the lvf and a square shape in the rvf ) , in order to avoid any interpretation of the results in terms of bilateral symmetry ( fig . they were required to maintain fixation on a small black circle ( diameter .3 , luminance : .82 cd / m ) in the center of the screen and to refrain from moving their eyes . first , in both patients the non - dominant ( right ) eye ipsilateral to the damaged hemisphere tended to deviate independently from the gaze direction of the dominant eye . this had potentially undermined the correct lateralization of the stimuli during fixation , as two different locations could had been represented in the fovea of the two eyes and a stimulus assumed to be projected in the left blind hemifield might have actually fallen into the seeing field of the right eye . for example , stimuli in the temporal hemifield ( the left hemifield of the left eye ) induce preferential gaze orienting or summon attention more readily than stimuli in the nasal hemifield ( the right hemifield of the left eye ) ( rafal et al . these behavioural asymmetries have been proposed to indicate the contribution of the sc in such tasks . anatomically , indeed , the superficial layers of the sc receive visual input predominantly from the nasal hemiretina , which samples the temporal hemifield , whereas the connections from the temporal hemiretina constitute a relatively weaker retino - tectal pathway ( hubel et al . this has been confirmed in an fmri study showing higher activation of the sc following stimulation of the temporal rather than nasal hemifield ( sylvester , josephs , driver , & rees , 2007 ) . therefore , testing the patients monocularly with the left eye ensured that the stimuli projected peripherally to the ( left ) blind temporal hemifield during bilateral conditions were processed uniquely by the nasal hemiretina , and thus relayed to the right sc , ipsilateral to the damaged hemisphere , through the stronger of the two retino - tectal pathways ( fig 3 ) . in contrast , the stimuli projected to the ( right ) intact nasal hemifield reached the left sc , ipsilateral to the intact hemisphere , through the weaker connections involving the temporal hemiretina pathway . this was done to counterbalance the potentially weaker representation of the ( left ) unseen over ( right ) seen stimulus during bilateral stimulation , as well as the overall weaker response in the ( right ) ipsilesional sc compared to the ( left ) sc in the intact side , which might have compromised the rte . within each block , the six stimulation conditions and display types were equiprobable , and each was repeated for 14 trials ( unilateral : single , quadruple gestalt - like , quadruple random ; bilateral : single , quadruple gestalt - like , quadruple random ) . based on previous reports on the same patients , only rts in the time - range 2001000 ms were considered ( leh et al . , 2010 , patient dr responded to all 168 trials within the accepted range , whereas se did not respond to 3 trials ( 1.8% ) , and had one anticipation ( .6% ) , while the responses to the remaining trials ( 97.6% ) were within the accepted range . visual inspection reveals that rts decreased in bilateral compared to unilateral presentations irrespective of the different display types and for both patients , although this decrease of rts was particularly pronounced for gestalt - like configurations . initially , a 2 3 repeated - measures anova was carried out on rts data with the within - subjects factors of presentation condition ( unilateral vs bilateral ) and configuration ( single , gestalt - like , random ) . patient dr showed a significant main effect of presentation condition [ f(1 , 27 ) = 47.507 , p < the main effect of configuration was also significant [ f(2 , 54 ) = 38.133 , p < .0001 ) , but there was no significant presentation condition configuration interaction [ f(2 , 54 ) = 1.777 , p < .178 ] . a post - hoc bonferroni comparison performed on the configuration factor revealed that rts were significantly faster for gestalt - like patterns than for either single or quadruple random dot shapes [ t(55 ) 6.31 p .0001 ] , which in turn did not differ from each other significantly [ t(55 ) = 1.92 , p = .149 ] . two additional anovas were computed separately for the unilateral and bilateral presentation conditions , each with the factor configuration and the usual three levels ( single , gestalt - like , random ) . the aim of this additional analysis was to determine whether the presentation of gestalt - like configurations in the blind hemifield was pivotal for the effect to occur . conversely , in bilateral displays a stimulus was added in the blind hemifield , and any significant difference arising among conditions in this case can only be accounted for by the nature of the unseen stimulus . post - hoc tests showed that rts for gestalt - like configurations in the intact hemifield were significantly faster than for single or quadruple random patterns [ t(27 ) 3.22 p .002 ] , while the latter two configurations did not differ from each other ( p = .864 ) . post - hoc comparisons revealed a significant speeding up of rts with bilateral gestalt - like patterns with respect to single or bilateral random configurations [ t(27 ) 6.08 p < .0001 ] , while there was no significant difference between single and random patterns ( p = .334 ) . post - hoc comparisons on the interaction showed that the rts for gestalt - like configurations were significantly faster than for either single or random patterns , but only in bilateral trials [ t(25 ) 7.73 p .0001 ] . indeed , the interaction already indicates unambiguously that , unlike dr who was sensitive to gestalt - like patterns in both her intact hemifield ( during unilateral presentation ) and blind hemifield ( during bilateral presentation ) , patient se was differentially responsive to gestalt - like configurations only when the stimuli were projected bilaterally , and hence to his blind hemifield as well . additionally , we plotted the cumulative distribution functions ( cdfs ) of rts for all six stimulation conditions and for both patients separately . smirnov test of the cdfs , which represents a nonparametric version suitable for carrying out a single - subject analysis of miller 's inequality test ( miller , 1982 ) , a mathematical tool to assess whether the rte is more likely to be related to probability or neural summation . note that violation of the inequality test unambiguously supports neural summation , whereas no conclusion can be reached if the inequality is not violated , owing to the conservative nature of the test ( miller , 1982 ) . 5 , when gestalt - like configurations were presented , rts for the bilateral condition were faster than for the unilateral condition throughout the entire distribution and in both patients dr and se ( p < .001 by kolmogorov smirnov test ) , thereby providing convincing evidence for an interpretation of the rte in terms of neural summation . this latter finding confirms a previous study showing that it is not always possible to attribute the rte for single dots to neural summation ( turatto , mazza , savazzi , & marzi , 2004 ) , but that its nature depends on stimulus and task factors . nevertheless , the present results using cdfs indicate that , under identical conditions , neural summation for gestalt - like configurations was significantly more likely to occur than that for single or random dot configurations in both patients . in the present study , we investigated the sensitivity of two blindsight patients with hemispherectomy to stimulus perceptual organization when the display is presented to the blind hemifield . we found that , in addition to the overall rte often reported in previous studies in hemianopic patients , there was a rte specific for gestalt - like stimulus configurations but not for spatially random patterns . these findings confirm and extend previous observations in patients with blindsight following lesions restricted to portions of the visual cortex ( celeghin , savazzi , et al . a difference in stimulus familiarity or variability between gestalt - like and random configurations can not account for the present results , since there were four different patterns counterbalanced for each of the two display types . moreover , gestalt - like and random patterns were randomly intermingled and presented within the same block of trials , whereas in the previous study by celeghin , savazzi et al . ( 2015 ) , trials with these different configurations were administered in separate blocks always starting with gestalt - like configurations . while both patients exhibited similar overall results , they differed in that dr showed a speeding up of rts also when gestalt - like patterns were presented unilaterally in her intact hemifield , whereas patient se did not . this is possibly related to individual differences in sensitivity to consciously perceived gestalt - like configurations also present in the healthy population ( wagemans et al . our present study provides the first causal evidence of the sensitivity of the sc for overall stimulus configuration , as witnessed by the facilitation exerted when a structured perceptual organization is translated into motor output . since the sc in our patients is the only intact visual structure remaining in the ipsilateral side of the otherwise damaged hemisphere , the contribution of other subcortical structures such as the lgn and the pulv can be ruled out . this does not necessarily imply that the ipsilesional sc is solely responsible for the observed effect ( i.e. , does not support sufficiency of the sc for the effect to occur ) . in fact , visual information might well have been transferred from the ( right ) sc , ipsilateral to the damaged hemisphere , to the corresponding contralateral sc in the ( left ) intact side via the inter - collicular commissure or through other inter - hemispheric tracts , and from there to other subcortical structures such as the pulv as well as extrastriate visual areas in the ( left ) intact hemisphere . prior positron emission tomography ( pet ) ( ptito , johannsen , faubert , & gjedde , 1999 ) and fmri ( bittar , ptito , faubert , dumoulin , & ptito , 1999 ) studies on the two patients tested here demonstrated activation in extrastriate visual areas of the ( left ) intact hemisphere following stimulation of the ipsilateral ( left ) hemifield , thereby documenting substantial neuronal plasticity and reorganization . owing to the expansion of the visual receptive fields in cortical areas of the intact hemisphere , but rather from the development of aberrant fibre tracts that connect the sc in the ( right ) damaged hemisphere to cortical areas in the opposite intact hemisphere ( leh , johansen - berg , et al . hence , the critical point here is that the visual information concerning stimulus configuration must have been initially processed by the right sc ipsilateral to the damaged hemisphere , thus indicating its necessity in processing stimulus configuration and in visuo - motor integration , as other alternatives are not possible . in keeping with this notion , the crucial involvement of the sc in the rte has been convincingly demonstrated behaviourally ( leh et al . according to a traditional view , it may appear surprising that the sc is able to represent complex stimulus configurations and respond differentially to a gestalt - like perceptual organization . the sc is indeed a phylogenetically ancient visual structure considered to have coarse retinotopy , which receives visual information only from magnocellular ( m ) and koniocellular ( k ) , but not from parvocellular ( p ) ganglion cells , and has a relative differential sensitivity to low spatial frequencies ( merigan and maunsell , 1993 , stone , 1984 ) . for example , several types of neurons in the superficial ( i.e. therefore , the sc seems to participate in early stages of figure - ground segmentation and the combined interaction of m and k channels can enable encoding of complex and high - level properties of the visual input . these studies clearly underline the possibility that some degree of perceptual functions can be carried out by subcortical centers in the absence of the visual cortical mantle . lastly , one interesting issue concerns possible inter - hemispheric specialization for global versus local processing , which can contribute to the encoding of gestalt - like configurations . in principle , the preserved sensitivity for gestalt - like configuration observed here despite right hemispherectomy could arise from the sc mirroring lateralized functions thus far reported primarily at the cortical level , or from lack of hemispheric specialization at the level of the sc . in the present study , both patients had undergone right hemispherectomy and it was thus not possible to compare the effects of right versus left hemispherectomy ; an issue that awaits further investigation . in conclusion , the present findings offer a clear demonstration that hemianopic patients as a result of hemispherectomy can be selectively sensitive to complex stimulus configuration within the context of the rte task . the sc can act as an interface between structured perceptual organization and motor processing , thereby providing an essential contribution to visually guided behavior despite being functionally and anatomically segregated from the geniculo - striate or extrastriate pathway , and therefore entirely outside conscious visual experience . an important avenue for future research is to try to examine other higher - order visual functions that can be carried out in the absence of striate and extrastriate cortical areas and whether such sensitivity can be proficiently exploited to foster rehabilitation of cortical blindness ( chokron et al .

various lumbar interbody fusion procedures are used to treat lumbar degenerative disc disease and spinal instability . among them , posterior lumbar interbody fusion ( plif ) and transforaminal lumbar interbody fusion ( tlif ) are representative surgical approaches . however , the disadvantages of wide open posterior approaches to the lumbar spine , such as persistent low back pain caused by iatrogenic muscle denervation resulting in atrophy and decreased trunk extensor strength , large amount of blood loss , and long recuperation time , are issues awaiting solutions4,10,11,14,15,27,29,32 ) . additionally , it has prompted the development of minimally invasive ( mis ) spinal surgery techniques such as mis - tlif and mis - alif . however , most of these reports have focused on surgical techniques and clinically significant results , such as reduced intraoperative blood loss , improvement in post - operative back pain , and shorter hospitalization stays , in comparison with conventional open surgery . very few studies based on radiographic results of mis lumbar interbody fusion have been performed . radiographic results , including interbody fusion , the restoration of disc height , foraminal height , and maintenance of normal lumbar lordosis are very important to post - operative clinical outcomes for lumbar interbody fusion surgery over the long term . the purpose of the present study was to evaluate the radiographic results of mis - alif and mis - tlif and also compare those of mis lumbar interbody fusion with conventional lumbar interbody fusion . we assessed 31 patients who were treated with mis lumbar interbody fusion in our hospital from july 2006 to september 2008 . the mean follow - up period was 31.6 months with a minimum 24 months ' follow - up . the patients consisted of 14 men and 17 women , and the mean age was 54 years ( range , 35 - 70 years ) . patients were diagnosed preoperatively with foraminal stenosis in 12 cases , degenerative spondylolisthesis in 7 cases , disc degenerative disorder ( ddd ) in 6 cases , isthmic spondylolisthesis in 4 cases , and recurrent hnp in 2 cases . twelve patients presenting with back pain or bilateral leg pain dominant pathology were treated with mis - alif , and 19 patients with unilateral radiating leg pain dominant pathology were treated with mis - tlif . percutaneous pedicle screw fixations with the sextant or viper system were used in all patients who underwent mis interbody fusions . thirty - four interbody fusion levels were performed in total : 6 on l3 - 4 , 15 on l4 - 5 , and 13 on the l5-s1 level . we selected 18 patients as a comparative group , with corresponding ages , gender ratio , and preoperative clinical symptoms , who underwent single level open tlif in 2007 and were followed up for at least two years . all patients underwent follow - up ct and x - rays at 1 , 6 , 12 , and 24 months postoperatively . in the present study , the following radiographic parameters were determined : interbody fusion , lumbar lordosis angle ( lla ) , fused segment angle ( fsa ) , sacral slope angle ( ssa ) , disc height ( dh ) and foraminal height ( fh ) , and the subsidence rate of interbody fusion . successful bony fusion was determined as follows : presence of bony bridging , absence of radiolucent lines around the cage on final follow - up ct or x - rays , and no motion on flexion / extension lateral x - rays . lla was estimated as the cobb 's angle between the l1 upper endplate and the s1 upper endplate . fsa was estimated as the cobb 's angle between the upper endplate of the upper vertebra and the lower endplate of the lower vertebra at the fusion level . ssa was determined as the angle between the s1 superior endplate and a horizontal reference line . disc height ( dh ) was calculated as the average of anterior disc height ( adh ) and posterior disc height ( pdh ) . foraminal height ( fh ) was estimated as the distance between the inferior pedicle wall above the index disc space and the superior pedicle wall of the lower vertebra ( fig . a subsidence rate of interbody disc height was calculated as a percentage as follows : [ immediate postoperative disc height - final disc height ] / [ immediate postoperative disc height ] 100 . radiographic parameters of mis lumbar interbody fusion were evaluated preoperatively and postoperatively and statistically analyzed . the changes of preoperative and final radiographic parameters were compared statistically between the mis - alif and mis - tlif groups and the mis - tlif and open tlif groups . statistical comparisons utilized the wilcoxon signed - rank test and the mann - whitney u test . the patient was placed in a prone position on a jackson operation table and taken to a lumbar lordosis position with hip extension . a longitudinal skin incision of approximately 3 cm in length was made lateral to the midline on the affected side . to approach the lesion facet joint precisely and minimize the skin incision , the distance from the spinous process line to the multifidus and longissimus muscle plane was measured on the preoperative axial mri ( fig . generally , the intermuscular plane is located at approximately 3 cm from the midline , and muscle dissection precedes 3 cm lateral to the midline ; however , the skin incision was made at 3 to 4 cm lateral to the midline . it is easier to lay the tubular retractor slightly to the lateral side and to facilitate decompression of opposite disc . an intermuscular plane dissection , which exfoliated the multifidus and longissimus muscles after fascial incision , after complete exfoliation of the facet joint lesion and part of the post lamina , the dilator was inserted to dilate the muscle , and a 22 mm quadrant tubular retractor ( medtronic sofamor danek , memphis , tn ) was subsequently inserted . after adjustments were made , it was then fixed on the operation table ( fig . the soft tissue on the lateral side of the facet joint was totally exfoliated , and a partial inferior vertebra transverse process was exposed . a fine high - speed drill was used to hollow out the groove at the inferior articular facet of the superior vertebra in " " shape , and a bayonet - shaped osteotome was used to break the bone . the osteotome was also used to break the exposed superior articular facet of the inferior vertebra . after total facetectomy was completed , the exposed ligamentum flavum was incrementally removed , and then the spinal canal in the medial , the traversing nerve root in the inferior medial , and the exiting nerve root in the superior lateral side were confirmed ( fig . if a central canal stenosis or contralateral side disc herniation was present , the tubular retractor was leaned more . the maximum amount of disc was removed , and meticulous endplate preparation was executed with diverse shaped and diverse angled reamer , distractors , curettes , scarpers , and pituitary punches . then , autologous and allograft bone were sufficiently inserted at the ventral disc space and was tamped down compactly using a bone impactor . a single large interbody cage was inserted as unilaterally as possible as oblique to the contralateral side while protecting the exiting and traversing nerve root with a root retractor ( fig . after completion of the interbody fusion procedure , in the same incision area , percutaneous pedicle screw fixations were performed , and rods were inserted under fluoroscopic guidance . identical procedures were performed on the opposite side . during rod compression and final tightening procedures under general anesthesia , the patient was placed in a prone position on a jackson operation table and taken to a lumbar lordosis position with hip extension . a longitudinal skin incision of approximately 3 cm in length was made lateral to the midline on the affected side . to approach the lesion facet joint precisely and minimize the skin incision , the distance from the spinous process line to the multifidus and longissimus muscle plane was measured on the preoperative axial mri ( fig . generally , the intermuscular plane is located at approximately 3 cm from the midline , and muscle dissection precedes 3 cm lateral to the midline ; however , the skin incision was made at 3 to 4 cm lateral to the midline . it is easier to lay the tubular retractor slightly to the lateral side and to facilitate decompression of opposite disc . an intermuscular plane dissection , which exfoliated the multifidus and longissimus muscles after fascial incision , after complete exfoliation of the facet joint lesion and part of the post lamina , the dilator was inserted to dilate the muscle , and a 22 mm quadrant tubular retractor ( medtronic sofamor danek , memphis , tn ) was subsequently inserted . after adjustments were made , it was then fixed on the operation table ( fig . the soft tissue on the lateral side of the facet joint was totally exfoliated , and a partial inferior vertebra transverse process was exposed . a fine high - speed drill was used to hollow out the groove at the inferior articular facet of the superior vertebra in " " shape , and a bayonet - shaped osteotome was used to break the bone . the osteotome was also used to break the exposed superior articular facet of the inferior vertebra . after total facetectomy was completed , the exposed ligamentum flavum was incrementally removed , and then the spinal canal in the medial , the traversing nerve root in the inferior medial , and the exiting nerve root in the superior lateral side were confirmed ( fig . if a central canal stenosis or contralateral side disc herniation was present , the tubular retractor was leaned more . the maximum amount of disc was removed , and meticulous endplate preparation was executed with diverse shaped and diverse angled reamer , distractors , curettes , scarpers , and pituitary punches . then , autologous and allograft bone were sufficiently inserted at the ventral disc space and was tamped down compactly using a bone impactor . a single large interbody cage was inserted as unilaterally as possible as oblique to the contralateral side while protecting the exiting and traversing nerve root with a root retractor ( fig . after completion of the interbody fusion procedure , in the same incision area , percutaneous pedicle screw fixations were performed , and rods were inserted under fluoroscopic guidance . identical procedures were performed on the opposite side . during rod compression and final tightening procedures under general anesthesia , the patient was placed in a prone position on a jackson operation table and taken to a lumbar lordosis position with hip extension . a longitudinal skin incision of approximately 3 cm in length was made lateral to the midline on the affected side . to approach the lesion facet joint precisely and minimize the skin incision , the distance from the spinous process line to the multifidus and longissimus muscle plane was measured on the preoperative axial mri ( fig . generally , the intermuscular plane is located at approximately 3 cm from the midline , and muscle dissection precedes 3 cm lateral to the midline ; however , the skin incision was made at 3 to 4 cm lateral to the midline . it is easier to lay the tubular retractor slightly to the lateral side and to facilitate decompression of opposite disc . an intermuscular plane dissection , which exfoliated the multifidus and longissimus muscles after fascial incision , after complete exfoliation of the facet joint lesion and part of the post lamina , the dilator was inserted to dilate the muscle , and a 22 mm quadrant tubular retractor ( medtronic sofamor danek , memphis , tn ) was subsequently inserted . after adjustments were made , it was then fixed on the operation table ( fig . the soft tissue on the lateral side of the facet joint was totally exfoliated , and a partial inferior vertebra transverse process was exposed . a fine high - speed drill was used to hollow out the groove at the inferior articular facet of the superior vertebra in " " shape , and a bayonet - shaped osteotome was used to break the bone . the osteotome was also used to break the exposed superior articular facet of the inferior vertebra . after total facetectomy was completed , the exposed ligamentum flavum was incrementally removed , and then the spinal canal in the medial , the traversing nerve root in the inferior medial , and the exiting nerve root in the superior lateral side were confirmed ( fig . if a central canal stenosis or contralateral side disc herniation was present , the tubular retractor was leaned more . the maximum amount of disc was removed , and meticulous endplate preparation was executed with diverse shaped and diverse angled reamer , distractors , curettes , scarpers , and pituitary punches . then , autologous and allograft bone were sufficiently inserted at the ventral disc space and was tamped down compactly using a bone impactor . a single large interbody cage was inserted as unilaterally as possible as oblique to the contralateral side while protecting the exiting and traversing nerve root with a root retractor ( fig . after completion of the interbody fusion procedure , in the same incision area , percutaneous pedicle screw fixations were performed , and rods were inserted under fluoroscopic guidance . identical procedures were performed on the opposite side . during rod compression and final tightening procedures thirty - one patients who were treated with mis lumbar interbody fusion in our hospital were evaluated in this study . twelve patients underwent mis - alif and 19 patients underwent mis - tlif procedures . in the mis interbody fusion cases , pre- and postoperative radiographic parameters were the following : mean lumbar lordosis angle ( lla ) , 32.812.1 and 38.911.1 ; fused segment angle ( fsa ) , 12.16.3 and 14.86.7 ; sacral slope angle ( ssa ) , 30.17.3 and 32.58.6 ; disc height ( dh ) , 10.03.0 mm and 13.92.5 mm ; and foraminal height ( fh ) , 16.74.5 mm and 19.93.9 mm . all radiographic parameters had statistically significant improvements at the postoperative measurement except for ssa ( table 1 ) . the changes ( final value - preoperative value ) of radiographic parameters between mis - alif and mis - tlif were also analyzed . in the mis - alif group , the changes of radiographic parameters were as follows : lla , fsa , ssa , dh , and fh were 12.17.4 , 5.53.5 , 3.55.2 , 6.32.6 mm , and 4.33.3 mm , respectively . in the mis - tlif group , changes in values in the same order were as follows : 2.19.7 , 0.74.9,1.55.2 , 2.31.9 mm , and 2.43.2 mm . no significant differences in ssa and fh were observed between the two groups , although lla , fsa , and dh showed statistically significant increases in the mis - alif group ( table 2 ) . in 2007 , 18 patients who had single level open tlif with a minimum 12 months x - ray follow - up were analyzed for the same parameters . between the open tlif and mis - tlif groups , the changes ( final value - preoperative value ) of radiographic parameters were compared . the changes of lla , fsa , ssa , dh , and fh in the open tlif group were 5.412.1 , 3.64.5 , 1.710.2 , 4.34.0 mm , and 3.03.2 mm , respectively . only the disc height was significantly increased in the open tlif group ; the other parameters were not significantly different ( table 3 ) . additionally , a mean subsidence rate of interbody disc height was 12.04% ( table 4 ) , and the fusion rate was 96% ( one pseudarthrosis in the mis - alif group ) . the mis - tlif technique was reported by foley et al.6 ) in 2003 and has come into wide use . this technique involves partially dissecting and dilating the paraspinal muscle using a 20 - 24 mm - sized small tubular retractor , performing unilateral facetectomy , interbody bone fusion , and percutaneous pedicle screw fixation . therefore , this procedure has the advantages of minimizing paraspinal muscle and soft tissue injury , no need for transfusion by reducing intraoperative bleeding , makes early ambulation possible because postoperative back pain is minimal , and reduces the number of in - hospital days as compared with standard open lumbar interbody fusion techniques24,28 ) . despite these many merits cm operative field of view , the exposure and resection of the targeted facet joint ( inferior and superior articular facet ) , epidural bleeding control , discectomy , sufficient neural decompression , endplate preparation , and appropriate interbody cage insertion without nerve root injury requires a great deal of time and is uncomfortable for the operator22 ) . however , one level lesion can be finished up successfully within 3 hours after a learning curve period of approximately 15 cases . in our study , unilateral leg pain dominant pathologies , such as disc protrusion with instability , recurrent disc herniation , unilateral foraminal stenosis , unilateral foraminal disc herniation , and grade i spondylolisthesis were treated with mis - tlif . the mis - alif technique was developed for minimizing abdominal soft tissue and internal organ injury , which can lead to the early recovery of the patient by reducing post - operative pain and complications such as wound problems . the greatest advantage of the alif approach may be the direct access and visualization of the intervertebral disc space . as a result , it is generally accepted that this approach can achieve a more complete discectomy , secure a wide fusion bed , and perform a more effective restoration of disc and foraminal height . additionally , it is easier to correct a sagittal balance as lumbar lordosis by anterior column support and stabilization directly . furthermore , alif does not violate the posterior spinal musculature or bony elements , and epidural scarring , nerve root retraction , and perineural fibrosis can be avoided . in this study , back pain dominant or bilateral pathologies , such as lumbar disc degenerative disorder ( ddd ) , unilateral or bilateral foraminal stenosis with significant disc space narrowing , and grade ii and more spondylolisthesis were treated with mis - alif . in the present study , evaluated radiographic parameters were shown as lumbar lordosis angle ( lla ) , fused segment angle ( fsa ) , sacral slope angle ( ssa ) , foraminal height ( fh ) , and disc height ( dh ) . radiographic parameters related to sagittal balance , such as lla , fsa , and ssa , are significantly correlated with low back pain ( lbp)17 ) and adjacent segment degeneration ( asd)21 ) , postoperatively . lazennec et al.19 ) reported that lbp developing after fusion surgery was shown to be significantly related to a decreased sacral tilt , increased pelvic tilt , and decreased lumbar lordosis . additionally , postoperative sagittal lumbar malalignment can accelerate adjacent segment deterioration by loading the motion segment in a nonphysiologic fashion30 ) . the optimal sagittal balance obtained with surgical correction of a spinal deformity may also affect the environment for bony fusion , preservation of the adjacent levels , and clinical outcome over the long term1,5,8,18 ) . unlike other studies , our study includes radiographic results of mis - alif as well as mis - tlif . in the mis - alif group , the change ( final value - preoperative value ) of lla , fsa , and ssa was 12.1 , 5.5 , and 3.5 ; it is an obvious improvement of regional sagittal balance . in the mis - tlif group statistical analysis between the two groups showed that mis - alif was more effective in correcting sagittal balance than mis - tlif ( table 2 ) . moreover , the changes of lla , fsa , and ssa after mis - tlif were comparable to those of open tlif in statistical analysis between open tlif and mis - tlif ( table 3 ) . this indicates that mis lumbar interbody fusion surgery is a viable strategy for sagittal balance correction of lumbar degenerative disc disease . in the present study , evaluated foraminal height ( fh ) and disc height ( dh ) are correlated with radiculopathy . in foraminal stenosis , a nerve root is compressed laterally to the intervertebral foramen by bony structures such as a hypertrophied facet and spur developed by disc degeneration7,31 ) . disc degeneration causes disc height and foraminal height to be narrowed , which are checked on lateral x - ray as the parameters of foraminal stenosis . surgical removal of bony structures and restoration of foraminal and disc height can decrease the radiating leg pain of the patient . kim et al.16 ) reported that by mis - tlif , dh was significantly increased at the final follow - up compared with the preoperative value , but fh was not evaluated . in our cases , dh was increased from 10.0 mm prior to surgery to 13.9 mm at the final follow - up , and fh was increased from 16.7 mm to 19.9 mm . both changes were statistically significant differences ( table 1 ) . in the mis - alif group , the changes ( final value - preoperative value ) of dh and fh were obvious , as increases of 6.3 mm and 4.3 mm , respectively , were observed . in the mis - tlif group , mis - alif was more effective in the restoration of disc height than mis - tlif ( table 2 ) . these differences may be explained that mis - alif is performed anterior opening and release by resection of all , and it can be more restored dh as well as fh compared that of mis - tlif . additionally , both the open tlif and mis - tlif groups showed statistically significant increases in fh ; however , this was not the case for dh(table 3 ) . as a result , the restoration of dh and fh are sufficiently possible with mis lumbar interbody fusion , which can resolve the radiating leg pain of patients successful interbody bone fusion is essential to getting a good clinical outcome ; if successful bony fusion is not achieved , back pain occurs in the long term . previously reported fusion rates after plif ranged from 56 to 100%2,13,23 ) , and fusion rates after tlif have ranged from 86 to 100%9,12,22,25 - 28 ) . the limited exposure inherent to mis techniques that requires fusion has the potential to affect adequate bone grafting and endplate preparation to allow for arthrodesis to occur . potter et al.25 ) have reported that for obtaining firm interbody fusion , exposure of more than 30% of the interbody endplate is required , and clinically , by using the unilateral transforaminal approach , an average of 69% of the disc volume ( 56% of the endplate ) could be removed . it is actually very difficult within the narrow operative field of the mis approach . despite this concern , several articles on mis - tlif have demonstrated good bone fusion rates ranging from 92 to 100%16,20,33 ) . on the other hand , deutsch et al.3 ) reported a fusion rate for mis - tlif of 65% . in the present study , the fusion rates of mis - tlif and mis - alif group were 100% and 91.7%(one psuedoarthrodesis in mis - alif group ) , respectively . we made a ceaseless effort for successful bone fusion in as many patients as possible ; meticulous and precise endplate preparation and grafting a large amount of mixed bone chips to the ventral interbody disc space before cage insertion were performed within a narrow operative field . finally , the subsidence of interbody disc height is important to lumbar interbody fusion surgery postoperatively . progress of subsidence can cause a recurrent foraminal stenosis by narrowing foraminal height and destroy a corrected sagittal balance , which may lead to recurrent radiating leg and back pain . in our study , usually , significant subsidence is defined as a decrease in interbody disc height of more than 3 mm . thus , our subsidence rate of interbody disc height seems remarkably low . for minimizing the subsidence rate of interbody disc height , grafting a lot of mixed bone chips compactly at the ventral disc space for anterior column support and inserting a single large interbody cage as unilaterally as possible as oblique to the contralateral side for equal distribution of axial loading to the cage these kinds of operator 's efforts can bring out a high fusion rate and low subsidence rate . mis lumbar interbody fusion ( mis - alif and mis - tlif ) is successful and safe with the advantages of minimally invasive spine surgery ( miss ) . radiographic results , including fusion rate , restoration of disc and foraminal height , and the improvement of lumbar lordosis were comparable to those with conventional open surgery . additionally , mis - alif is more effective in the restoration of lumbar lordosis and disc height than mis - tlif . to determine the effectiveness of mis lumbar interbody fusion , larger , long - term , prospective studies are needed in the future

objectiveto evaluate the radiographic results of minimally invasive ( mis ) anterior lumbar interbody fusion ( alif ) and transforaminal lumbar interbody fusion ( tlif).methodstwelve and nineteen patients who underwent mis - alif , mis - tlif , respectively , from 2006 to 2008 were analyzed with a minimum 24-months ' follow - up . additionally , 18 patients treated with single level open tlif surgery in 2007 were evaluated as a comparative group . x - rays and ct images were evaluated preoperatively , postoperatively , and at the final follow - up . fusion and subsidence rates were determined , and radiographic parameters , including lumbar lordosis angle ( lla ) , fused segment angle ( fsa ) , sacral slope angle ( ssa ) , disc height ( dh ) , and foraminal height ( fh ) , were analyzed . these parameters were also compared between the open and mis - tlif groups.resultsin the mis interbody fusion group , statistically significant increases were observed in lla , fsa , and dh and fh between preoperative and final values . the changes in lla , fsa , and dh were significantly increased in the mis - alif group compared with the mis - tlif group , but ssa and fh were not significantly different . no significant differences were seen between open and mis - tlif except for dh . the interbody subsidence and fusion rates of the mis groups were 12.04% and 96% , respectively.conclusionradiographic results of mis interbody fusion surgery are as favorable as those with conventional surgery regarding fusion , restoration of disc height , foraminal height , and lumbar lordosis . mis - alif is more effective than mis - tlif for intervertebral disc height restoration and lumbar lordosis .

INTRODUCTION MATERIALS AND METHODS Surgical technique Minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) RESULTS DISCUSSION CONCLUSION

various lumbar interbody fusion procedures are used to treat lumbar degenerative disc disease and spinal instability . among them , posterior lumbar interbody fusion ( plif ) and transforaminal lumbar interbody fusion ( tlif ) are representative surgical approaches . additionally , it has prompted the development of minimally invasive ( mis ) spinal surgery techniques such as mis - tlif and mis - alif . however , most of these reports have focused on surgical techniques and clinically significant results , such as reduced intraoperative blood loss , improvement in post - operative back pain , and shorter hospitalization stays , in comparison with conventional open surgery . very few studies based on radiographic results of mis lumbar interbody fusion have been performed . radiographic results , including interbody fusion , the restoration of disc height , foraminal height , and maintenance of normal lumbar lordosis are very important to post - operative clinical outcomes for lumbar interbody fusion surgery over the long term . the purpose of the present study was to evaluate the radiographic results of mis - alif and mis - tlif and also compare those of mis lumbar interbody fusion with conventional lumbar interbody fusion . we assessed 31 patients who were treated with mis lumbar interbody fusion in our hospital from july 2006 to september 2008 . the mean follow - up period was 31.6 months with a minimum 24 months ' follow - up . patients were diagnosed preoperatively with foraminal stenosis in 12 cases , degenerative spondylolisthesis in 7 cases , disc degenerative disorder ( ddd ) in 6 cases , isthmic spondylolisthesis in 4 cases , and recurrent hnp in 2 cases . twelve patients presenting with back pain or bilateral leg pain dominant pathology were treated with mis - alif , and 19 patients with unilateral radiating leg pain dominant pathology were treated with mis - tlif . percutaneous pedicle screw fixations with the sextant or viper system were used in all patients who underwent mis interbody fusions . thirty - four interbody fusion levels were performed in total : 6 on l3 - 4 , 15 on l4 - 5 , and 13 on the l5-s1 level . we selected 18 patients as a comparative group , with corresponding ages , gender ratio , and preoperative clinical symptoms , who underwent single level open tlif in 2007 and were followed up for at least two years . all patients underwent follow - up ct and x - rays at 1 , 6 , 12 , and 24 months postoperatively . in the present study , the following radiographic parameters were determined : interbody fusion , lumbar lordosis angle ( lla ) , fused segment angle ( fsa ) , sacral slope angle ( ssa ) , disc height ( dh ) and foraminal height ( fh ) , and the subsidence rate of interbody fusion . successful bony fusion was determined as follows : presence of bony bridging , absence of radiolucent lines around the cage on final follow - up ct or x - rays , and no motion on flexion / extension lateral x - rays . fsa was estimated as the cobb 's angle between the upper endplate of the upper vertebra and the lower endplate of the lower vertebra at the fusion level . disc height ( dh ) was calculated as the average of anterior disc height ( adh ) and posterior disc height ( pdh ) . foraminal height ( fh ) was estimated as the distance between the inferior pedicle wall above the index disc space and the superior pedicle wall of the lower vertebra ( fig . a subsidence rate of interbody disc height was calculated as a percentage as follows : [ immediate postoperative disc height - final disc height ] / [ immediate postoperative disc height ] 100 . radiographic parameters of mis lumbar interbody fusion were evaluated preoperatively and postoperatively and statistically analyzed . the changes of preoperative and final radiographic parameters were compared statistically between the mis - alif and mis - tlif groups and the mis - tlif and open tlif groups . an intermuscular plane dissection , which exfoliated the multifidus and longissimus muscles after fascial incision , after complete exfoliation of the facet joint lesion and part of the post lamina , the dilator was inserted to dilate the muscle , and a 22 mm quadrant tubular retractor ( medtronic sofamor danek , memphis , tn ) was subsequently inserted . the soft tissue on the lateral side of the facet joint was totally exfoliated , and a partial inferior vertebra transverse process was exposed . a fine high - speed drill was used to hollow out the groove at the inferior articular facet of the superior vertebra in " " shape , and a bayonet - shaped osteotome was used to break the bone . after total facetectomy was completed , the exposed ligamentum flavum was incrementally removed , and then the spinal canal in the medial , the traversing nerve root in the inferior medial , and the exiting nerve root in the superior lateral side were confirmed ( fig . the maximum amount of disc was removed , and meticulous endplate preparation was executed with diverse shaped and diverse angled reamer , distractors , curettes , scarpers , and pituitary punches . after completion of the interbody fusion procedure , in the same incision area , percutaneous pedicle screw fixations were performed , and rods were inserted under fluoroscopic guidance . during rod compression and final tightening procedures under general anesthesia , the patient was placed in a prone position on a jackson operation table and taken to a lumbar lordosis position with hip extension . an intermuscular plane dissection , which exfoliated the multifidus and longissimus muscles after fascial incision , after complete exfoliation of the facet joint lesion and part of the post lamina , the dilator was inserted to dilate the muscle , and a 22 mm quadrant tubular retractor ( medtronic sofamor danek , memphis , tn ) was subsequently inserted . the soft tissue on the lateral side of the facet joint was totally exfoliated , and a partial inferior vertebra transverse process was exposed . a fine high - speed drill was used to hollow out the groove at the inferior articular facet of the superior vertebra in " " shape , and a bayonet - shaped osteotome was used to break the bone . after total facetectomy was completed , the exposed ligamentum flavum was incrementally removed , and then the spinal canal in the medial , the traversing nerve root in the inferior medial , and the exiting nerve root in the superior lateral side were confirmed ( fig . the maximum amount of disc was removed , and meticulous endplate preparation was executed with diverse shaped and diverse angled reamer , distractors , curettes , scarpers , and pituitary punches . after completion of the interbody fusion procedure , in the same incision area , percutaneous pedicle screw fixations were performed , and rods were inserted under fluoroscopic guidance . during rod compression and final tightening procedures under general anesthesia , the patient was placed in a prone position on a jackson operation table and taken to a lumbar lordosis position with hip extension . an intermuscular plane dissection , which exfoliated the multifidus and longissimus muscles after fascial incision , after complete exfoliation of the facet joint lesion and part of the post lamina , the dilator was inserted to dilate the muscle , and a 22 mm quadrant tubular retractor ( medtronic sofamor danek , memphis , tn ) was subsequently inserted . the soft tissue on the lateral side of the facet joint was totally exfoliated , and a partial inferior vertebra transverse process was exposed . a fine high - speed drill was used to hollow out the groove at the inferior articular facet of the superior vertebra in " " shape , and a bayonet - shaped osteotome was used to break the bone . after total facetectomy was completed , the exposed ligamentum flavum was incrementally removed , and then the spinal canal in the medial , the traversing nerve root in the inferior medial , and the exiting nerve root in the superior lateral side were confirmed ( fig . the maximum amount of disc was removed , and meticulous endplate preparation was executed with diverse shaped and diverse angled reamer , distractors , curettes , scarpers , and pituitary punches . after completion of the interbody fusion procedure , in the same incision area , percutaneous pedicle screw fixations were performed , and rods were inserted under fluoroscopic guidance . during rod compression and final tightening procedures thirty - one patients who were treated with mis lumbar interbody fusion in our hospital were evaluated in this study . twelve patients underwent mis - alif and 19 patients underwent mis - tlif procedures . in the mis interbody fusion cases , pre- and postoperative radiographic parameters were the following : mean lumbar lordosis angle ( lla ) , 32.812.1 and 38.911.1 ; fused segment angle ( fsa ) , 12.16.3 and 14.86.7 ; sacral slope angle ( ssa ) , 30.17.3 and 32.58.6 ; disc height ( dh ) , 10.03.0 mm and 13.92.5 mm ; and foraminal height ( fh ) , 16.74.5 mm and 19.93.9 mm . all radiographic parameters had statistically significant improvements at the postoperative measurement except for ssa ( table 1 ) . the changes ( final value - preoperative value ) of radiographic parameters between mis - alif and mis - tlif were also analyzed . in the mis - alif group , the changes of radiographic parameters were as follows : lla , fsa , ssa , dh , and fh were 12.17.4 , 5.53.5 , 3.55.2 , 6.32.6 mm , and 4.33.3 mm , respectively . in the mis - tlif group , changes in values in the same order were as follows : 2.19.7 , 0.74.9,1.55.2 , 2.31.9 mm , and 2.43.2 mm . no significant differences in ssa and fh were observed between the two groups , although lla , fsa , and dh showed statistically significant increases in the mis - alif group ( table 2 ) . in 2007 , 18 patients who had single level open tlif with a minimum 12 months x - ray follow - up were analyzed for the same parameters . between the open tlif and mis - tlif groups , the changes ( final value - preoperative value ) of radiographic parameters were compared . the changes of lla , fsa , ssa , dh , and fh in the open tlif group were 5.412.1 , 3.64.5 , 1.710.2 , 4.34.0 mm , and 3.03.2 mm , respectively . only the disc height was significantly increased in the open tlif group ; the other parameters were not significantly different ( table 3 ) . additionally , a mean subsidence rate of interbody disc height was 12.04% ( table 4 ) , and the fusion rate was 96% ( one pseudarthrosis in the mis - alif group ) . the mis - tlif technique was reported by foley et al.6 ) in 2003 and has come into wide use . this technique involves partially dissecting and dilating the paraspinal muscle using a 20 - 24 mm - sized small tubular retractor , performing unilateral facetectomy , interbody bone fusion , and percutaneous pedicle screw fixation . therefore , this procedure has the advantages of minimizing paraspinal muscle and soft tissue injury , no need for transfusion by reducing intraoperative bleeding , makes early ambulation possible because postoperative back pain is minimal , and reduces the number of in - hospital days as compared with standard open lumbar interbody fusion techniques24,28 ) . despite these many merits cm operative field of view , the exposure and resection of the targeted facet joint ( inferior and superior articular facet ) , epidural bleeding control , discectomy , sufficient neural decompression , endplate preparation , and appropriate interbody cage insertion without nerve root injury requires a great deal of time and is uncomfortable for the operator22 ) . in our study , unilateral leg pain dominant pathologies , such as disc protrusion with instability , recurrent disc herniation , unilateral foraminal stenosis , unilateral foraminal disc herniation , and grade i spondylolisthesis were treated with mis - tlif . the mis - alif technique was developed for minimizing abdominal soft tissue and internal organ injury , which can lead to the early recovery of the patient by reducing post - operative pain and complications such as wound problems . the greatest advantage of the alif approach may be the direct access and visualization of the intervertebral disc space . as a result , it is generally accepted that this approach can achieve a more complete discectomy , secure a wide fusion bed , and perform a more effective restoration of disc and foraminal height . additionally , it is easier to correct a sagittal balance as lumbar lordosis by anterior column support and stabilization directly . in this study , back pain dominant or bilateral pathologies , such as lumbar disc degenerative disorder ( ddd ) , unilateral or bilateral foraminal stenosis with significant disc space narrowing , and grade ii and more spondylolisthesis were treated with mis - alif . in the present study , evaluated radiographic parameters were shown as lumbar lordosis angle ( lla ) , fused segment angle ( fsa ) , sacral slope angle ( ssa ) , foraminal height ( fh ) , and disc height ( dh ) . radiographic parameters related to sagittal balance , such as lla , fsa , and ssa , are significantly correlated with low back pain ( lbp)17 ) and adjacent segment degeneration ( asd)21 ) , postoperatively . lazennec et al.19 ) reported that lbp developing after fusion surgery was shown to be significantly related to a decreased sacral tilt , increased pelvic tilt , and decreased lumbar lordosis . the optimal sagittal balance obtained with surgical correction of a spinal deformity may also affect the environment for bony fusion , preservation of the adjacent levels , and clinical outcome over the long term1,5,8,18 ) . unlike other studies , our study includes radiographic results of mis - alif as well as mis - tlif . in the mis - alif group , the change ( final value - preoperative value ) of lla , fsa , and ssa was 12.1 , 5.5 , and 3.5 ; it is an obvious improvement of regional sagittal balance . in the mis - tlif group statistical analysis between the two groups showed that mis - alif was more effective in correcting sagittal balance than mis - tlif ( table 2 ) . moreover , the changes of lla , fsa , and ssa after mis - tlif were comparable to those of open tlif in statistical analysis between open tlif and mis - tlif ( table 3 ) . this indicates that mis lumbar interbody fusion surgery is a viable strategy for sagittal balance correction of lumbar degenerative disc disease . in the present study , evaluated foraminal height ( fh ) and disc height ( dh ) are correlated with radiculopathy . disc degeneration causes disc height and foraminal height to be narrowed , which are checked on lateral x - ray as the parameters of foraminal stenosis . surgical removal of bony structures and restoration of foraminal and disc height can decrease the radiating leg pain of the patient . kim et al.16 ) reported that by mis - tlif , dh was significantly increased at the final follow - up compared with the preoperative value , but fh was not evaluated . in our cases , dh was increased from 10.0 mm prior to surgery to 13.9 mm at the final follow - up , and fh was increased from 16.7 mm to 19.9 mm . both changes were statistically significant differences ( table 1 ) . in the mis - alif group , the changes ( final value - preoperative value ) of dh and fh were obvious , as increases of 6.3 mm and 4.3 mm , respectively , were observed . in the mis - tlif group , mis - alif was more effective in the restoration of disc height than mis - tlif ( table 2 ) . these differences may be explained that mis - alif is performed anterior opening and release by resection of all , and it can be more restored dh as well as fh compared that of mis - tlif . additionally , both the open tlif and mis - tlif groups showed statistically significant increases in fh ; however , this was not the case for dh(table 3 ) . as a result , the restoration of dh and fh are sufficiently possible with mis lumbar interbody fusion , which can resolve the radiating leg pain of patients successful interbody bone fusion is essential to getting a good clinical outcome ; if successful bony fusion is not achieved , back pain occurs in the long term . previously reported fusion rates after plif ranged from 56 to 100%2,13,23 ) , and fusion rates after tlif have ranged from 86 to 100%9,12,22,25 - 28 ) . potter et al.25 ) have reported that for obtaining firm interbody fusion , exposure of more than 30% of the interbody endplate is required , and clinically , by using the unilateral transforaminal approach , an average of 69% of the disc volume ( 56% of the endplate ) could be removed . it is actually very difficult within the narrow operative field of the mis approach . despite this concern , several articles on mis - tlif have demonstrated good bone fusion rates ranging from 92 to 100%16,20,33 ) . on the other hand , deutsch et al.3 ) reported a fusion rate for mis - tlif of 65% . in the present study , the fusion rates of mis - tlif and mis - alif group were 100% and 91.7%(one psuedoarthrodesis in mis - alif group ) , respectively . finally , the subsidence of interbody disc height is important to lumbar interbody fusion surgery postoperatively . in our study , usually , significant subsidence is defined as a decrease in interbody disc height of more than 3 mm . for minimizing the subsidence rate of interbody disc height , grafting a lot of mixed bone chips compactly at the ventral disc space for anterior column support and inserting a single large interbody cage as unilaterally as possible as oblique to the contralateral side for equal distribution of axial loading to the cage these kinds of operator 's efforts can bring out a high fusion rate and low subsidence rate . mis lumbar interbody fusion ( mis - alif and mis - tlif ) is successful and safe with the advantages of minimally invasive spine surgery ( miss ) . radiographic results , including fusion rate , restoration of disc and foraminal height , and the improvement of lumbar lordosis were comparable to those with conventional open surgery . additionally , mis - alif is more effective in the restoration of lumbar lordosis and disc height than mis - tlif . to determine the effectiveness of mis lumbar interbody fusion , larger , long - term , prospective studies are needed in the future

among striking developments in history writing towards the end of the twentieth century were the moves to the spatial and the visual in the reconstruction of historical consciousness . global history , accelerated through american hype on globalisation , and visual culture studies propelled by society s increasing reliance on visual communications , became fashionable projects . global history,1 grounded in contemporary economics and partly pitched in reaction to western parochialism , came seriously to challenge nationalistic and structuralist approaches to history.2 at the same time , the pictorial turn , with its epistemological claim for vision as the prime sense in knowledge production , came to defy not only conventional history of art , but the status and value of the discipline of history itself.3 wrestling with these turns proved enormously productive . in the history of medicine it led to foregrounding disease in its global dimension , revivifying , at the same time as challenging , older narratives on the world wide spread of disease.4 in the history of science it led to heightened attention to visual representations in struggles over the production of scientific knowledge and authority.5 both turnings did more than merely provide historians with exciting new conceptual frameworks for comprehending the past . as illustrated through the contemporaneous growth of a literature on the history of material objects in all their global distribution , they also helped broaden the range of objects deemed worthy of historical attention.6 this paper draws on these moves in relation to one particular material object that is both global and visual : the aids poster.7 their worldwide production from the mid-1980s hugely reinvigorated the whole genre of the health poster and , according to one expert , restored the genre s original function as a communications medium.8 certainly , as never before was so much money , aesthetic effort and psychological marketing put into this particular media on the part of voluntary bodies , national governments and international health agencies.9 our concern , however , is with the wider conceptual frameworks that were mobilised to make these objects meaningful . in the 1980s and 1990s , as we have outlined elsewhere , a cohort of western intellectuals concerned themselves with them along with other representations of aids in order to talk about the politics of identity.10 those concerns , in turn , were linked to broader ones emerging at the time over the rights of citizens to equal access to health care , the privatisation of medicine , and the role of the international pharmaceutical industry in the commercialisation of health care . in this paper we focus on another aspect of these ephemeral mass - produced objects : not their active life on the streets and in the corridors of learning but their afterlife when they were turned into items to be collected , exchanged and stored in museums and archives . it is well known that the social life of material objects in such places is not the same as that of their initial culture of production , circulation and consumption.11 museums and archives , like other depositories for images and artifacts , have particular collecting agendas and particular institutional and intellectual traditions into which new acquisitions are fitted . they also inhabit the present , embracing wider conceptual contexts that serve further to shape the organisation and meaning of their artifacts . here , we explore one such afterlife for aids posters : an exhibition entitled against aids : posters from around the world , which was held at the museum fr kunst und gewerbe in hamburg between february and april 2006 . impact of the exhibition ; our interest in it is , rather , as an illustration of the more general trend towards the the museum fr kunst und gewerbe was founded in 1877 in a spirit of aesthetic modernism and german nationalism , and by the 1890s was host one of germany s largest and most prestigious poster collections . the peoples arts and crafts , much like the south kensington museum in london , established in 1852 and renamed the victoria and albert museum in 1899 at the height of british jingoism . as at the hamburg museum fr kunst und gewerbe , so at the victoria and albert museum over the past few decades , objects have been reorganised for exhibitions accentuating the global. the representation of aids posters at the hamburg exhibition provides us with a means to discuss the politics of such global assemblages.12 on the one hand , it permits us to draw out the inherent contradictions and tensions that can be involved in any such institutional mobilisation of the concept of the unity of the globe.13 on the other , it allows us to underline important continuities hidden under the more apparent or insisted upon discontinuities between national and global discourses , and between modern and postmodern politics of aesthetics continuities rooted , we argue , in shared aesthetic values.14 as important , the example permits us to reflect on how the discourse of the global affects the work of historians using material objects in their constructions of historical consciousness . as these aims and objectives should suggest , we are not concerned here with how viewers might have responded to the images or to the exhibition as a whole ( an almost impossible task given the uniqueness of individual psychology and experience).15 nor are we interested in providing a walk - through critique of the exhibition ; our main interest is in the historical context of the museum and how this bears on the politics of aesthetics implicated in its exhibition of aids posters . against aids : posters from around the world was a modest , low - budget affair . primarily , it was staged in order to exploit the museum s recent acquisition of over a thousand hiv / aids and safe sex posters from a private dealer , a purchase that enabled it to join the club of institutions harbouring such collections.16 the organisers of the exhibition selected only a hundred of the posters to display , choosing those that were most visually arresting , and others that , even after three decades in some cases , still had the power to shock , titillate , and/or amuse ( such as that used on the front of the flyer for the exhibition , figure 1 , or condoman , figure 2 ) . in part to enhance these effects , realist anti - homophobic posters ( figure 3 ) , were mixed with erotic semen kit , for example , joined nautical space with a poster of a condom disguised as a life - saving ring,17 which , in turn , was hung alongside a russian poster advert for rubber tyres . these striking juxtapositions were intended to reveal the variety of aesthetic choices that governments , charities , commercial bodies , and private artists employed in their efforts to inform the public of the threat of hiv / aids and incite onlookers to ethical behaviour ( safer sex ) . dramatically , at the entrance to the exhibition , the visitor was confronted with a full billboard - size reproduction of oliviero toscani s iconic image of 1992 : his re - conception of the prize - winning photograph from life depicting the death of the american aids activist david kirby , turned into an advertisement for the united colours of benetton ( figure 5).18 other less dramatic images played on popular solidarities around aids , as prefigured in the socially integrative against aids in the title of the exhibition . these images could serve to counter any charge that might be levelled at the museum for its use of the more erotic and more humorous and ironic ones , namely , that it was denying the pain and suffering of hiv / aids victims , or trivialising the world s most devastating disease . against aids it was through the display of these posters in particular that the exhibition sought to exemplify regional variety and similarities in aesthetic styles . condoms , the viewer might come to see , were globally an unambiguous symbol for , and the warning against , unsafe sex.19figure 1the leaflet ( 10 21 cm ) for against aids : posters from around the world , museum fr kunst and gewerbe , hamburg , 2006 . by permission of the museum fr kunst and gewerbe , hamburg.figure 2issued by the commonwealth department of community services , aboriginal health workers of australia ( queensland ) , 1991 ( 41 28 cm ) . this poster was reproduced in several countries with the english translated.figure 3produced by terrence higgins trust , 1999 . by permission of the terrence higgins trust.figure 4issued by gay men fighting aids , london , 1994 ( 59 42 cm ) , hywel williams photographer . the image was reproduced on a 15 cm card , which was handed out at the gay pride festival in july 1994 . the text on the card mentions the uk government 's poor funding of safe sex campaigns and parliament 's homophobic reaction to the proposal of an equal age of consent of 16 for both hetero- and homosexuals.figure 5oliviero toscani s 1992 billboard image of the death of david kirby for benetton s shock of reality advertising campaign . by permission of the united colors of benetton . the leaflet ( 10 21 cm ) for against aids : posters from around the world , museum fr kunst and gewerbe , hamburg , 2006 . by permission of the museum fr kunst and gewerbe , hamburg . issued by the commonwealth department of community services , aboriginal health workers of australia ( queensland ) , 1991 ( 41 28 cm ) . this poster was reproduced in several countries with the english translated . produced by terrence higgins trust , 1999 . by permission of the terrence higgins trust . issued by gay men fighting aids , london , 1994 ( 59 42 cm ) , hywel williams photographer . the image was reproduced on a 15 cm card , which was handed out at the gay pride festival in july 1994 . the text on the card mentions the uk government 's poor funding of safe sex campaigns and parliament 's homophobic reaction to the proposal of an equal age of consent of 16 for both hetero- and homosexuals . oliviero toscani s 1992 billboard image of the death of david kirby for benetton s shock of reality advertising campaign . by permission of the united colors of benetton . although a flyer ( figure 1 ) , but no catalogue was produced for the show , the aesthetic and intellectual motives behind it are discernable through the catalogue that accompanied a much larger exhibition of posters held at the museum in 1996 ( which was curated by the same person , jrgen dring , and displayed some of same posters).20 the 1996 exhibition marked the centenary of the museum s first - ever exhibition of posters a late nineteenth century entertainment that was in fact the first of its kind in germany and one of the first in europe.21 in many ways the 1996 exhibition was faithful to that of 1896 , its agenda being more or less the same as that articulated by the main advocate and co - founder of the museum fr kunst und gewerbe , the hamburg lawyer and art critic justus brinckmann ( 18431915).22 in his catalogue for the 1896 exhibition , brinckmann opined that posters and their display in museums and galleries performed the ethical task of elevating the masses to aesthetic appreciation : art should be accessible to everyone . it should bring elevation and joy to all ; not only to those who buy it or have the time to visit art galleries . in order to fulfil this purpose art has to go on the street , and has to cross as if by accident the path of the many thousands going to work who have neither time nor money to devote to it otherwise.23 art should be accessible to everyone . it should bring elevation and joy to all ; not only to those who buy it or have the time to visit art galleries . in order to fulfil this purpose art has to go on the street , and has to cross as if by accident the path of the many thousands going to work who have neither time nor money to devote to it otherwise.23 these were not the only politics in the 1896 exhibition with resonance for the one in 1996 . the wider context at the end of the nineteenth century was that of flourishing national rivalries as well as attitudes to competition as a virtue in itself the posters in the 1896 exhibition , from italy , france , russia , the usa and elsewhere , were organised accordingly and , typical of most exhibitions at the time displaying products from different countries , were competitively judged by an international panel . the exhibition thus served to cohere national identities at the same time as sell the ideology of competition along with the virtues of the products of mass - production for mass ( visual ) consumption . more than this , while on the one hand the 1896 exhibition promoted the promise of democracy embedded in the notion of arts and crafts by and for the people , uniting people through aesthetics education , on the other , it sold an litist aesthetic ethics that simultaneously challenged traditional litist views of what constitutes art and who properly can access and comment on it.24 a century later many of brinckmann s views were dusted off . jrgen dring , the curator of the 1996 exhibition ( and subsequently the 2006 one ) similarly revelled in the non - litist engagement of poster art , at the same time as celebrating the particular aesthetic distinction that brinckmann drew between mundane commercial advertising and sophisticated poster design.25 the 1996 exhibition visually illustrated this difference by juxtaposing examples of each . above all , dring celebrated and reproduced brinckmann s ethical mission to teach people how to appreciate and enjoy the visual world through a better understanding of its underlying aesthetic principles and techniques of production.26 this ethical educational work of the poster is all the more important today , he argued in the catalogue , because the fine arts have lost the main task they had when brinckmann was living , namely , to interpret the world in its visual parts and provide the onlooker with an edifying understanding of it.27 the 2006 exhibition of aids posters was conceived in the same intellectual framework , according to dring.28 it , too , was intended to elicit an emotional response from the viewer and , from that , heighten their sensitivity to art.29 but while brinkmann in his day had looked forward to a brighter future for the poster and its onlookers in museums such as the kunst und gewerbe in hamburg , dring and his colleagues found it hard to be quite so optimistic . in their view as maintained in the catalogue for the 1996 exhibition the general quality of the visual language of posters had dramatically decreased due to the increasing flood of pictures in everyday life . discriminating aesthetic appreciation , they felt , was less and less in evidence in contemporary culture because modern education failed to teach the classics and its iconography . consequently , despite the fact that posters were intimately a part of the revolution in graphic design and design technology of the 1980s and 1990s,30 they had become impoverished superficial , because they could no longer be the apparent proof of this lay in the popular media s focus on the human body or , more precisely , on superficial beauty and feelings around the human body . according to dring and his colleagues this was the zeitgeist of our times that exceeds rational understanding.31 today s fashionable heroes hardly transmit any moral values , they lamented ; indeed , they did the opposite dissipating , through preoccupations with individual self - fulfilment and superficial gratification , the cannon of christian ethics and traditions of virtue behind good art.32 aids posters apparently shared this fate in having behind them no shared aesthetic appreciation . according to the 1996 catalogue , they could communicate no meaningful visual language for the deadly disease they referred to because the health educationalists who issued them were themselves unable to formulate one . hence , the iconography of aids posters appealed predominantly only to the feelings of onlookers . all that could really be said in their favour was that they sponsored a positive moral practice , namely caution against the spread of hiv : the onlooker feels attracted by the picture and decides to use a condom.33 although it seems odd that the curators of the museum fr kunst und gewerbe disdained the very objects they were celebrating , they justified themselves on the grounds that such posters were expressive of their times and therefore wholly within the remit of an institution whose agenda was to archive and exhibit the art of the streets and the people . by the 2006 exhibition , the times were global , and hence a new justification was established through the parade of moral values within that discourse . in maintaining that contemporary culture and advertising were saturating the world with meaningless , morally deprived , corporeally fixated images , the curators of the museum fr kunst und gewerbe were maintaining a distinctly modernistas opposed to postmodernist view of art. in fact , in believing that the mechanics of perception operated along the lines of emotional attraction , followed by rational thought , and then enlightened responsible behaviour , they were following nineteenth - century notions of the physiology of seeing and sense perception.34 they were also assuming that onlookers were simply passive vessels for ethical education in visual good taste and , moreover , that good taste was the highest form of human awareness . interestingly , in many of these respects as well as in connection with the understanding of disease they were sharing an outlook with one of the few intellectuals in the twentieth century ever to provide sustained commentary on posters , the late susan sontag ( 19332004)despite the fact that sontag , as a theorist , was to their left in embracing the marxism of the frankfurt school of critical theory.35 sontag s views were made clear in 1970 in posters : advertisement , art , political artifact , commodity.36 posters , inhabiting the low end of high art , she explained , originated in the effort of expanding capitalist productivity to sell surplus or luxury goods. they could not exist : [ b]efore the specific historic conditions of modern capitalism . sociologically , the advent of the poster reflects the development of an industrialised economy whose goal is ever - increasing mass consumption , and ( somewhat later , when posters turned political ) of the modern secular centralised nation - state , with its peculiarly diffuse conception of ideological consensus and its rhetoric of mass political participation.37 [ b]efore the specific historic conditions of modern capitalism . sociologically , the advent of the poster reflects the development of an industrialised economy whose goal is ever - increasing mass consumption , and ( somewhat later , when posters turned political ) of the modern secular centralised nation - state , with its peculiarly diffuse conception of ideological consensus and its rhetoric of mass political participation.37 posters thus served the purpose of aggressively pushing consumption or , in politics , of selling national i d entities and ideologies . the modern concept of public space as a theatre of persuasion.38 this regard of posters , as aim[ing ] to seduce , to exhort , to sell , to educate , to convince , to appeal,39 was predicated on a view of advertising ( of any sort ) as psychologically dangerous . for sontag , posters blinded people to the real world , a view ( shared by the curators of hamburg s museum fur kunst und gewerbe ) that presupposes some unmediated or , one has only to break through the constructions mediated by language or by images to experience a direct understanding of the world though senses and perceptions . although sontag had no reason to comment on health posters until she came to write aids and its metaphors in 1988 , her thinking on them was indistinguishable from her earlier thoughts . aids posters , she maintained , indict the ideology of consumer capitalism that celebrates recreational risk - free sexuality in the name of individual liberty.40 furthermore , as in her illness as metaphor ( 1978 ) , there was no doubt about the biological essence of such a disease . just as posters mediated false - consciousness , so behind the distorting metaphors of any widely feared disease there lurked a real entity . one had only to strip away the metaphors to get at the underlying value - neutral , non - stigmatising nature of any disease.41 the organisers of the exhibitions of aids posters at hamburg s museum fr kunst und gewerbe similarly believed aids to be realthat is , to be a universal medical problem that was simply yet to be solved . as if to underline this understanding of hiv / aids as medical and nothing more , the 2006 exhibition was accompanied by health education information issued by various private and national hiv / aids agencies . significantly , in both the flyer for the 2006 exhibition and in the section on aids in the 1996 catalogue , a medical discussion of hiv / aids preceded that on the aesthetics of the posters . thus , a distinctly modernist view of art was accompanied by a distinctly modernist , as opposed to postmodernist , view of science and medicine as unquestionably superior forms of consciousness and practice , even to art . how people interacted with aids posters during the images active life on the streets on buses , billboards , underground trains , and so on and how the power and fear of hiv / aids operated in relation to identity were simply not parts of the 2006 exhibition in hamburg . through the literal framing of the posters , their hanging according to the conventions of art galleries , their arrangement ( three or four to a single wall ) , and the choice of them in terms of the quality of their visual language ( bildersprache ) , the organisers denuded them of the local contexts in which they were created and in which they were engaged with politically , intellectually and emotionally . this absenting can probably be illustrated by paying close attention to the history of any one of the posters in the exhibition . here , we focus on only a few of them to illustrate the point : semen kit ( figure 4 ) , stand up against homophobia ( figure 3 ) , and toscani s advertisement for benetton ( figure 5 ) . the first two of these were among the five british examples of posters in the exhibition . both were issued by hiv / aids charities ( as were the other three british examples in the show ) , and were relatively recent . semen kit ( 1994 ) was produced by the gmfa [ gay men fighting aids ] ( established in 1992 ) , whilst stand up against homophobia ( 1999 ) was distributed by the terrence higgins trust ( established in 1982 and , by the 1990s , britain s leading hiv and sexual health charity ) . that they were not issued by the state in 1986 , when thatcher s government announced its intention to spend 20 million over the next twelve months on hiv / aids health information , and commissioned the advertising agency tbwa to undertake it , the resulting iceberg and tombstone images were crafted to sell a health message to a general audience and to meet the government s inflated interest in family values , heterosexual sex , and nationhood.42 condoms were not then in the frame , nor were gay men , and nor was the global. the story of how the patriotic heterosexual imaging43 began to be turned around in the 1990s need not concern us here.44 as in most western countries , it was gay men the first victims of the disease who initially confronted the benign , sexually prudish and denying images of the establishment , and then inverted the anti - liberal homophobic rhetoric generated by the gay plague in celebration of their own collective identity ( the gay community ) . it only needs observing that in the uk this development was significantly different from elsewhere ; bearing upon it were the particularities of social and cultural traditions as well as prevailing political , medical and legal circumstances . pre - existing affirmative representation in the public media of gays were far less pronounced than in some other places , while the ability of the tabloid press and the conservative government of the day to stir homophobia was greater.45 prime minister margaret thatcher s homophobic legislation of 1988 ( section 28 of the local government act forbidding the promotion of homosexuality ) in particular , did much for heightening alterity , struggle and confrontation . yet it was precisely these features that the hamburg exhibition eclipsed by having the images hung alongside foreign advertisements and alongside aids posters from china and elsewhere . the exhibition thereby performed not for the struggle for gay identity within a national context , but for world solidarity . stand up against homophobia , by virtue of being both a product of 1999 , and by presenting hiv / aids as a part of a wider social issue , served in itself to mask the historical significance of these images . semen kit performed similarly through its play to a would - be unproblematic history of overt sexual behaviour among men ( inconised in the image of the sailor ) , and by its appropriation from some american aids posters of the by then more - than - a - decade - old image of the gay body beautiful.46 that gay men in britain , well into the 1990s , were still struggling for a viable visual public identity could not be guessed from the hanging of this poster at hamburg . nor could it be known from either of these images that the changes in gay identity that occurred in britain in the 1990s were , in part , largely attributable to the uptake of a slick visual language borrowed from the culture of madison avenue - driven international advertising the same visual language , from the same source , that was simultaneously taken up by thatcher and other politicians around the world in electioneering . but it was not only governments and gays in the 1980s and 1990s who struggled to capture the meaning of aids ; so too did western medicine . it was its lack of success at turning aids into a meaningful scientific category that , in fact , opened the medical profession to pointed confrontation , and opened up aids to the visual politics of identity . in effect , through aids , and in particular through the early debate over whether hiv caused aids , the medical profession was pulled off its pulpit as the authoritative arbiter of modern secular identity.47 arguably , toscani s poster advertisement for benetton reflects this by blurring the normative boundaries between public art and private anatomy / medicine , as well as by confusing the conventional distinction between commercial marketing and medical humanitarianism.48 for toscani , who regarded the david kirby image as the most significant of those he designed for benetton s shock of reality advertising campaign of 1992,49 it was supposed to show how an international corporation was open to the world s influences and engaged in a continuing quest for new frontiers.50 for him , this meant commitment to global social issues , above all , to the problems of poverty , race and disease . however , this was far from how the image was regarded by others . although in different contexts reactions varied , overall it was greeted with howls of moral indignation and in some places to the sacking of benetton shops . the germans took the image to court , french billstickers refused to post it and , in britain , the guardian ( the first newspaper to run it as a full - page advertisement ) was inundated with letters of complaint.51 nor was that all . while many in the gay community responded to it favourably , others criticised it as a form of cultural and economic imperialism . the critique fed a form of political resistance known as culture jamming , or adbusting that hijacks the ads of big brands to talk back to them in order to re - conquer city space.52 toscani s image immediately became the victim of this semiotic robin hoodism : the american aids coalition to unleash power ( act up ) there s only one pullover this photograph should be used to selland pictured a condom.53 here , supposedly , was the subconscious of the benetton campaign x - rayed to uncover not only its opposite meaning , but also ( in the manner of sontag s analysis ) to reveal a deeper truth ostensibly lurking beneath the layers of advertisement euphemism.54 here , then , was yet another field of combat over the meaning of hiv / aids centred on its imagery . but , again , there was no hint of it at the show in hamburg . while toscani s image still had the power to shock , it is doubtful if anyone outside of living memory who viewed it at the exhibition ( such as the school children bussed in ) could have guessed what it meant for many visual theorists and aids activists worrying over images of aids since the late 1980s , namely , that it was further testimony to the ongoing crisis over the entire framing of knowledge about the human body,55 with aids , not just a medical problem but an epidemic of meanings or significations.56 the exhibition in hamburg gave no clue to how images like this had been regarded as testifying to a self - consciously postmodern culture , ethics , aesthetics , disease representation , and politics of identity . nor was the reproduction of toscani s image at the hamburg exhibition intended to illustrate how the lines between the commercial and the medical humanitarian had become so obscured that there was now little to mark the difference between a health poster and an advertisement for the sale of fashion knitwear . instead , despite the fact that many of the images in the hamburg show had been designed simply to sell condoms , and others , such as that on homophobia , to challenge social injustice , prejudice and exclusion rather than caution against hiv / aids itself,57 the exhibition cohered them all into a would - be historically uniform and medically mediated message against hiv / aids . thus although not with conscious intent the show flew in the face of the preoccupations of the visual theorists and aids activists of the 1980s and 1990s . where they had seen in representations of aids the postmodern play of signifiers , had argued for a plurality of subjectivities involved in visual engagements , and had construed visual perceptions of the human body in general as involving an onlooker s unconscious construction of their own body through the immediate act of viewing , the curators of the hamburg exhibition saw only medicine and art in modernity . this was not the only way in which the exhibition displaced the specific and general historical meanings attached to these objects by investing them with others . the title alone of the exhibition , against aids : posters from around the world did as much . first and foremost it constructed a particular framework for their perception , one that above all suggested that aesthetic form can travel the world regardless of local geographies and local histories of ethnicity , religion , race , rights , sexuality and gender not to mention alternative aesthetic traditions . this global aesthetic spin , in effect , harmonised a modernist western transcendent notion of art with the late twentieth - century notion of a spatially transcendent capitalism an economic system supposedly unfettered by place or national boundary.58 the aesthetic spin and the exhibition as a whole thus further performed for notions of homogeneity and universality attributes long associated with modernity and perceived to be at odds with the pluralities and fragmentations associated with postmodernity during the time of aids in the west.59 the exhibition s title also suggested that the history of aids was about everyone the world over being uniformly against aids . but that , too , was hardly the case at the point of production of many of these posters . in the 1980s , christian fundamentalists and other religionists took a rather different line , and gay men and lesbians did not always see government campaigns against aids as being against aids so much as against themselves.60 as noted above , in thatcher s britain the aids campaign was an occasion for the moral high - grounding of heterosexual values.61 to the extent that people ( and international pharmaceutical companies ) were allegedly against aids in the 1980s and 1990s , their concerns emerged from a multitude of different and often conflicting social and economic interests . moreover , the relative power of those interests was hierarchically organised , and differently so over time , as virginia berridge has made clear for the history of aids in the uk.62 also , implicit to the entitling of the hamburg exhibition was the idea that nations around the world were homogeneous in their fight against hiv / aids . this not only collapsed separate national encounters with hiv / aids , such as its very denial by south africa s president thabo mbeki , but effaced the differences between the kinds of media campaigns used in different countries including the often bitter struggles between local , national and international agencies.63 as important , this political gutting of aids posters through their aestheticisation erased national rivalries and pressures involved in medically treating aids victims ( or not , as the case may be ) . through the mixing of posters from different countries , the exhibition dissolved the conventional boundaries between nation - states , while the multitude of images of condoms that it presented served visually to re - unite them around a commercial product . the images of condoms promoted the idea that campaigns for their use had actually united the countries of the world , a message curiously at odds with the bush administration s contemporaneous funding of medical missionaries advocating sexual abstinence instead of the use of condoms.64 in its own small way , therefore , and for its own particular didactic reasons ( as well as , perhaps , discomfort over germany s nationalistic past ) , the hamburg exhibition effected the same kind of historical effacement and rewrite that the major international media and entertainment companies were also coming to effect by 2006 through their take of up hiv / aids for the purpose of reaping public corporate credibility.65 by then , hiv / aids - funding was a fashionable cause , a benign branding resource for various western philanthropic organisations . at local , national and transnational levels then , against aids : posters from around the world can be seen to have effaced the individual history of the objects on display through a particular universalising and seemingly neutral kind of aestheticisation . at a closer look , however , it both appropriated them into an old script ( a local and fondly held modernist epistemology of viewing and aesthetics ) and a new one globalisation . by collapsing two decades of national histories into a singular and would - be unified world fight against hiv / aids , the history of hiv / aids was visually construed in terms of this new global subjectivity . not only were particular constructions of the recent past left out the local struggles around these objects but also the construction of the present the global media industry s selling of itself through the attack on hiv / aids as a real or made true through aesthetic representation of an ostensibly international struggle against hiv / aids , but by this same conceit was medically re - appropriated and humanised no matter that over the meaning of good thing.66 this is not to suggest that hiv / aids was not recognised as a global problem almost from its start . in 1987 , the american aids activist , feminist and visual theorist , paula treichler , for instance , referred to aids as having a potential for global devastation.67 but for her and others in the 1980s the global was only a background problem , and the term was yet without particular epistemic load . as we have indicated , western nations and their intellectuals and aids activists were gripped more by their own campaigns , interests , and ideologies than by in fact , the idea in the west that hiv / aids was a global problem was a viewpoint that itself had to be fought for through a world wide media campaign brokered by organisations with internationalist interests . the campaign can be dated precisely to 27 may 1987 when the world health organisation issued a press release proclaiming that aids is a global epidemic that demands a global attack.68 the who then produced a poster to sell the message ( figure 6)to compete , that is , with other struggles for the meaning of aids.69 the hamburg exhibition abetted that project through a visual rhetoric of shared international struggle against hiv / aids , just as it unwittingly abetted the subsequent take over of aids programs by the media multinationals.70 thus it eclipsed the history that would enable anyone to believe that aids had ever been anything other than global , or for that matter , anything other than mainly a struggle for economic resources for better medical provision for victims of the condition . images intended for , and often produced by , local sub - groups became artefacts for making up global citizens , with thereby , the exhibition did far more than merely reinforce what had become the semantic hegemony of the global , as iterated through such credulities as global warming and global terrorism.71 through aesthetics alone it rendered tangible the universalising concept of the global . although this was not the exhibition s primary intention , it could not help but perform it , and in so doing contribute to a reconstruction of history and consciousness . far from it ; the globally spun institution - serving celebration of the aesthetics of aids posters merely reflected and reinforced much of what everybody in the west had already come to feel about hiv / aids by 2006that it was a serious world - wide problem about which everyone needed to be continually reminded.figure 6who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon . who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon . we have been concerned with a particular moment in the history of aids posters : not that of their initial public life on the streets , or in pubs and gay clubs , doctors offices , and so on , but their afterlife in places where they might be displayed or stored.72 through the analysis of an exhibition at one such afterlife location our intention has not been that of negative dismissal , nor critique for the sake of it . nor has it been merely to expose how these often strikingly visual objects that aimed at protecting individual health had their meaning changed through appropriation into a conceptual framework different from that of their initial contexts of production and consumption . more interesting to us are the intrinsic and unremitting links between these visual objects and wider politics , or how the visual is inherently a part of the latter . while it might have been supposed that aids posters came to political rest once they were retired , categorised , catalogued and stored according to the principles of collecting institutions , the hamburg exhibition proves otherwise . in fact , as collector s items , they entered a space that was no less political than when they were on the streets in the 1980s and 1990s , and when they were appropriated to western discourses on postmodern identity and on the role of the visual in the cultural negotiation of the self . retirement home they necessarily became a part of the institutional agenda of the museum fr kunst und gewerbe . indeed , from the moment such objects become collectors items and are stored and/or displayed as artifacts they become epistemologically loaded through the very process of objectification . hamburg s museum fr kunst und gewerbe demonstrates that these collecting agendas and the accompanying aesthetic guidelines often have deep historical roots . but what the analysis of its 2006 exhibition of aids posters also shows is that old and seemingly apolitical agendas ( invented to express specific national political interests ) are neither lost nor rendered innocuous in the contemporary world . rather , they come to serve new political frameworks linked to the world of today s visitors a world in which aesthetics is the dominant means to a politics constituted on little more than the idea that if it looks good go with it ( an outlook now as pervasive in the practice of science as in the arts of government).73 crucially , this new politics is sustained through , and for , the absenting of critique ; not today the critical outlook entertained by sontag and other pre - postmodern intellectuals , that visual representations ( and popular posters in particular ) covered - up or cloaked lurking ideologies . postmodernists , unconcerned with that view for the most part , in effect opened the space for the new politics of aesthetics that masks something different : the idea of aesthetics as void of political intention . the hamburg exhibition of aids posters was , in fact , an early example of the coming - to - reign of these particular politics , with the visual alone being the vehicle for understanding and creating a ( global ) community without distinction . whereas for brinckmann in the nineteenth century , aesthetics ( in art ) could be consciously used for the political purpose of populist democracy , with aesthetics and politics in clearly separate spheres , for the inheritors of his institution in hamburg aesthetics ( unbeknownst to them ) became the politics , not simple a means to it . ironically , their arrival at these politics their unknowing performance of them through the exhibition was via adherence to brinckmann s legacy . through that , brinckmann s original political agenda was emptied of its original political purpose . installed in its place were the politics of the appearance of political un - intention . thus did an aesthetic concept born in the nineteenth century to serve nationalistic purposes come to operate for the political work of educating national citizens to global citizenship . what does this mean for historians working with material objects stored in global - tending museums and archives and who are themselves now operating within a global framework ? since material objects have no meaning without a framework , and are framed in being collected , the simple answer is that historians have to take into account the afterlives of such objects as much as the object s original lives . harder is the problem of the historian s own place in the global framework , which , in many respects , is not unlike that of the material object in the global - aspiring museum ( and very much like that of the curators of such exhibitions ) . whether avowed explicitly in global history , or embraced implicitly in the practice of history writing in contemporary culture , the global operates politically and epistemologically . just as global history s predecessor world history is now perceived by some of its originators as having been a product of , and agent for , its cold war moment,74 so for our own times , dominated as they are by multi - national corporations and abiding politicians , the take up of global themes in history writing is widely recognised as providing , at the very least , legitimacy to a globalisation discourse , even if , as often the case , the historian s immediate object is the far from reactionary one of provincialising the west and critiquing its hegemony.75 of course , for some historians the global does politically more , overtly serving as a rhetorical strategy for the re - coherence of the discipline of history itself after its pummelling by poststructuralists , deconstructionists and other fragmenting postmodern forces over the past thirty years or more.76 to this end , what could serve better as a reunifying device than the holising connective metaphor of the globe ? thus the global provides a new grand narrative a universalising tool with which to reimpose the meta - narrativity of history . although seemingly mindless of one of postmodernism s cautions , that totalising worldviews can lead to totalitarianism,77 these historians seek more - or - less intentionally what the curators of the museum fr kunst und gewerbe performed innocently through their exhibition of aids posters . in so doing , they also share company with certain art historians anxious to revive older agendas a means ( as bluntly put by one of them ) to counter the deconstructive criticism of historical culture proposed by self - serving postmodern academics who treat the past as a sour land over which to exercise present concerns and anxieties.78 yet neither global history nor visual culture studies need necessarily lead in this direction . to see the global as a discourse tied politically to institutionally specific agendas ( such as the aesthetics entertained at the museum fr kunst und gewerbe in hamburg in 2006 ) offers the possibility to take an alternative political position , or at least to liberate its historical study from such agendas . nevertheless , in terms of the less - visible shaping of historical knowledge production , the global framework can never be other than world - making as opposed to being simply historically descriptive.79 no matter how hard we try to stick to the recovery of some truth of how the world came to be globally conceived ( past or present ) , our knowledge productions implicitly reproduce and foster the unifying construct . just as with material objects ( or words or images ) there is no meaning to historical events outside the conceptual frameworks we wittingly or unwittingly apply to them . in short , there exists no resting place for history writing ; it is always already fashioned and fashioning . like creating a museum exhibition of posters to aestheticise the global nature of hiv / aids , the business of contributing to a global history of anything entails , by that very act , the politics of constructing a necessarily partial representation of the past. history writing , too , in other words , as a product of its time , can not avoid making up historical consciousness . this article can not escape the charge that it too contributes to this process merely by discussing an event that was conceptualised in it might even be seen to compound the problem by drawing attention to spaces where , a priori , the historian is already politically and epistemologically implicated : the museum and the archive . however , in doing so it has sought to move the discussion beyond the tired call for attending merely to historical contexts , especially of material objects globally attributed . our purpose has been to encourage historians to an awareness of their own immediate entanglements in history s constructedness the constructedness of the present mediated in history writing as much as through aesthetic assemblages of the global. the historian aruf dirlik , critically inquiring into the point of writing world history , has observed that an awareness of the variety of world histories that have been constructed at different times and in different places [ must cause ] any world historian worthy of the name [ to ] be uncommonly aware of the constructedness of the past.80 similarly , we submit , all historians need be reflective on their contributions to the present that is , to a culture given to re - enchantment through the global. quite how we should historicise material objects of the sort discussed in this paper may be open to debate ; what is not is the necessity to historicise our own historical projects . otherwise we move perilously close to becoming blind participants in the historically fashioned spaces where memory is increasingly naturalised and neutralised through universalised and universalising concepts mediated aesthetically . we end up , as it were , nave viewers of the exhibition at hamburg : as blind to the nature of the new post - postmodern politics of aesthetics , as to the modernist would - be universal humanity that the global unwittingly espouses through those politics . how people interacted with aids posters during the images active life on the streets on buses , billboards , underground trains , and so on and how the power and fear of hiv / aids operated in relation to identity were simply not parts of the 2006 exhibition in hamburg . through the literal framing of the posters , their hanging according to the conventions of art galleries , their arrangement ( three or four to a single wall ) , and the choice of them in terms of the quality of their visual language ( bildersprache ) , the organisers denuded them of the local contexts in which they were created and in which they were engaged with politically , intellectually and emotionally . this absenting can probably be illustrated by paying close attention to the history of any one of the posters in the exhibition . here , we focus on only a few of them to illustrate the point : semen kit ( figure 4 ) , stand up against homophobia ( figure 3 ) , and toscani s advertisement for benetton ( figure 5 ) . the first two of these were among the five british examples of posters in the exhibition . both were issued by hiv / aids charities ( as were the other three british examples in the show ) , and were relatively recent . semen kit ( 1994 ) was produced by the gmfa [ gay men fighting aids ] ( established in 1992 ) , whilst stand up against homophobia ( 1999 ) was distributed by the terrence higgins trust ( established in 1982 and , by the 1990s , britain s leading hiv and sexual health charity ) . that they were not issued by the state is important , for they were in fact conveying messages alternative to it . in 1986 , when thatcher s government announced its intention to spend 20 million over the next twelve months on hiv / aids health information , and commissioned the advertising agency tbwa to undertake it , the resulting iceberg and tombstone images were crafted to sell a health message to a general audience and to meet the government s inflated interest in family values , heterosexual sex , and nationhood.42 condoms were not then in the frame , nor were gay men , and nor was the global. the story of how the patriotic heterosexual imaging43 began to be turned around in the 1990s need not concern us here.44 as in most western countries , it was gay men the first victims of the disease who initially confronted the benign , sexually prudish and denying images of the establishment , and then inverted the anti - liberal homophobic rhetoric generated by the gay plague in celebration of their own collective identity ( the gay community ) . it only needs observing that in the uk this development was significantly different from elsewhere ; bearing upon it were the particularities of social and cultural traditions as well as prevailing political , medical and legal circumstances . pre - existing affirmative representation in the public media of gays were far less pronounced than in some other places , while the ability of the tabloid press and the conservative government of the day to stir homophobia was greater.45 prime minister margaret thatcher s homophobic legislation of 1988 ( section 28 of the local government act forbidding the promotion of homosexuality ) in particular , did much for heightening alterity , struggle and confrontation . yet it was precisely these features that the hamburg exhibition eclipsed by having the images hung alongside foreign advertisements and alongside aids posters from china and elsewhere . the exhibition thereby performed not for the struggle for gay identity within a national context , but for world solidarity . stand up against homophobia , by virtue of being both a product of 1999 , and by presenting hiv / aids as a part of a wider social issue , served in itself to mask the historical significance of these images . semen kit performed similarly through its play to a would - be unproblematic history of overt sexual behaviour among men ( inconised in the image of the sailor ) , and by its appropriation from some american aids posters of the by then more - than - a - decade - old image of the gay body beautiful.46 that gay men in britain , well into the 1990s , were still struggling for a viable visual public identity could not be guessed from the hanging of this poster at hamburg . nor could it be known from either of these images that the changes in gay identity that occurred in britain in the 1990s were , in part , largely attributable to the uptake of a slick visual language borrowed from the culture of madison avenue - driven international advertising the same visual language , from the same source , that was simultaneously taken up by thatcher and other politicians around the world in electioneering . but it was not only governments and gays in the 1980s and 1990s who struggled to capture the meaning of aids ; so too did western medicine . it was its lack of success at turning aids into a meaningful scientific category that , in fact , opened the medical profession to pointed confrontation , and opened up aids to the visual politics of identity . in effect , through aids , and in particular through the early debate over whether hiv caused aids , the medical profession was pulled off its pulpit as the authoritative arbiter of modern secular identity.47 arguably , toscani s poster advertisement for benetton reflects this by blurring the normative boundaries between public art and private anatomy / medicine , as well as by confusing the conventional distinction between commercial marketing and medical humanitarianism.48 for toscani , who regarded the david kirby image as the most significant of those he designed for benetton s shock of reality advertising campaign of 1992,49 it was supposed to show how an international corporation was open to the world s influences and engaged in a continuing quest for new frontiers.50 for him , this meant commitment to global social issues , above all , to the problems of poverty , race and disease . although in different contexts reactions varied , overall it was greeted with howls of moral indignation and in some places to the sacking of benetton shops . the germans took the image to court , french billstickers refused to post it and , in britain , the guardian ( the first newspaper to run it as a full - page advertisement ) was inundated with letters of complaint.51 nor was that all . while many in the gay community responded to it favourably , others criticised it as a form of cultural and economic imperialism . the critique fed a form of political resistance known as culture jamming , or adbusting that hijacks the ads of big brands to talk back to them in order to re - conquer city space.52 toscani s image immediately became the victim of this semiotic robin hoodism : the american aids coalition to unleash power ( act up ) there s only one pullover this photograph should be used to selland pictured a condom.53 here , supposedly , was the subconscious of the benetton campaign x - rayed to uncover not only its opposite meaning , but also ( in the manner of sontag s analysis ) to reveal a deeper truth ostensibly lurking beneath the layers of advertisement euphemism.54 here , then , was yet another field of combat over the meaning of hiv / aids centred on its imagery . but , again , there was no hint of it at the show in hamburg . while toscani s image still had the power to shock , it is doubtful if anyone outside of living memory who viewed it at the exhibition ( such as the school children bussed in ) could have guessed what it meant for many visual theorists and aids activists worrying over images of aids since the late 1980s , namely , that it was further testimony to the ongoing crisis over the entire framing of knowledge about the human body,55 with aids , not just a medical problem but an epidemic of meanings or significations.56 the exhibition in hamburg gave no clue to how images like this had been regarded as testifying to a self - consciously postmodern culture , ethics , aesthetics , disease representation , and politics of identity . nor was the reproduction of toscani s image at the hamburg exhibition intended to illustrate how the lines between the commercial and the medical humanitarian had become so obscured that there was now little to mark the difference between a health poster and an advertisement for the sale of fashion knitwear . instead , despite the fact that many of the images in the hamburg show had been designed simply to sell condoms , and others , such as that on homophobia , to challenge social injustice , prejudice and exclusion rather than caution against hiv / aids itself,57 the exhibition cohered them all into a would - be historically uniform and medically mediated message against hiv / aids . thus although not with conscious intent the show flew in the face of the preoccupations of the visual theorists and aids activists of the 1980s and 1990s . where they had seen in representations of aids the postmodern play of signifiers , had argued for a plurality of subjectivities involved in visual engagements , and had construed visual perceptions of the human body in general as involving an onlooker s unconscious construction of their own body through the immediate act of viewing , the curators of the hamburg exhibition saw only medicine and art in modernity . this was not the only way in which the exhibition displaced the specific and general historical meanings attached to these objects by investing them with others . the title alone of the exhibition , against aids : posters from around the world did as much . first and foremost it constructed a particular framework for their perception , one that above all suggested that aesthetic form can travel the world regardless of local geographies and local histories of ethnicity , religion , race , rights , sexuality and gender not to mention alternative aesthetic traditions . this global aesthetic spin , in effect , harmonised a modernist western transcendent notion of art with the late twentieth - century notion of a spatially transcendent capitalism an economic system supposedly unfettered by place or national boundary.58 the aesthetic spin and the exhibition as a whole thus further performed for notions of homogeneity and universality attributes long associated with modernity and perceived to be at odds with the pluralities and fragmentations associated with postmodernity during the time of aids in the west.59 the exhibition s title also suggested that the history of aids was about everyone the world over being uniformly against aids . but that , too , was hardly the case at the point of production of many of these posters . in the 1980s , christian fundamentalists and other religionists took a rather different line , and gay men and lesbians did not always see government campaigns against aids as being against aids so much as against themselves.60 as noted above , in thatcher s britain the aids campaign was an occasion for the moral high - grounding of heterosexual values.61 to the extent that people ( and international pharmaceutical companies ) were allegedly against aids in the 1980s and 1990s , their concerns emerged from a multitude of different and often conflicting social and economic interests . moreover , the relative power of those interests was hierarchically organised , and differently so over time , as virginia berridge has made clear for the history of aids in the uk.62 also , implicit to the entitling of the hamburg exhibition was the idea that nations around the world were homogeneous in their fight against hiv / aids . this not only collapsed separate national encounters with hiv / aids , such as its very denial by south africa s president thabo mbeki , but effaced the differences between the kinds of media campaigns used in different countries including the often bitter struggles between local , national and international agencies.63 as important , this political gutting of aids posters through their aestheticisation erased national rivalries and pressures involved in medically treating aids victims ( or not , as the case may be ) . through the mixing of posters from different countries , the exhibition dissolved the conventional boundaries between nation - states , while the multitude of images of condoms that it presented served visually to re - unite them around a commercial product . the images of condoms promoted the idea that campaigns for their use had actually united the countries of the world , a message curiously at odds with the bush administration s contemporaneous funding of medical missionaries advocating sexual abstinence instead of the use of condoms.64 in its own small way , therefore , and for its own particular didactic reasons ( as well as , perhaps , discomfort over germany s nationalistic past ) , the hamburg exhibition effected the same kind of historical effacement and rewrite that the major international media and entertainment companies were also coming to effect by 2006 through their take of up hiv / aids for the purpose of reaping public corporate credibility.65 by then , hiv / aids - funding was a fashionable cause , a benign branding resource for various western philanthropic organisations . at local , national and transnational levels then , against aids : posters from around the world can be seen to have effaced the individual history of the objects on display through a particular universalising and seemingly neutral kind of aestheticisation . at a closer look , however , it both appropriated them into an old script ( a local and fondly held modernist epistemology of viewing and aesthetics ) and a new one globalisation . by collapsing two decades of national histories into a singular and would - be unified world fight against hiv / aids , the history of hiv / aids was visually construed in terms of this new global subjectivity . not only were particular constructions of the recent past left out the local struggles around these objects but also the construction of the present the global media industry s selling of itself through the attack on hiv / aids as a real or made true through aesthetic representation of an ostensibly international struggle against hiv / aids , but by this same conceit was medically re - appropriated and humanised no matter that over the meaning of good thing.66 this is not to suggest that hiv / aids was not recognised as a global problem almost from its start . in 1987 , the american aids activist , feminist and visual theorist , paula treichler , for instance , referred to aids as having a potential for global devastation.67 but for her and others in the 1980s the global was only a background problem , and the term was yet without particular epistemic load . as we have indicated , western nations and their intellectuals and aids activists were gripped more by their own campaigns , interests , and ideologies than by in fact , the idea in the west that hiv / aids was a global problem was a viewpoint that itself had to be fought for through a world wide media campaign brokered by organisations with internationalist interests . the campaign can be dated precisely to 27 may 1987 when the world health organisation issued a press release proclaiming that aids is a global epidemic that demands a global attack.68 the who then produced a poster to sell the message ( figure 6)to compete , that is , with other struggles for the meaning of aids.69 the hamburg exhibition abetted that project through a visual rhetoric of shared international struggle against hiv / aids , just as it unwittingly abetted the subsequent take over of aids programs by the media multinationals.70 thus it eclipsed the history that would enable anyone to believe that aids had ever been anything other than global , or for that matter , anything other than mainly a struggle for economic resources for better medical provision for victims of the condition . images intended for , and often produced by , local sub - groups became artefacts for making up global citizens , with thereby , the exhibition did far more than merely reinforce what had become the semantic hegemony of the global , as iterated through such credulities as global warming and global terrorism.71 through aesthetics alone it rendered tangible the universalising concept of the global . although this was not the exhibition s primary intention , it could not help but perform it , and in so doing contribute to a reconstruction of history and consciousness . far from it ; the globally spun institution - serving celebration of the aesthetics of aids posters merely reflected and reinforced much of what everybody in the west had already come to feel about hiv / aids by 2006that it was a serious world - wide problem about which everyone needed to be continually reminded.figure 6who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon . who poster of 1987 ( 91 61 cm ) selling aids as a global phenomenon . we have been concerned with a particular moment in the history of aids posters : not that of their initial public life on the streets , or in pubs and gay clubs , doctors offices , and so on , but their afterlife in places where they might be displayed or stored.72 through the analysis of an exhibition at one such afterlife location our intention has not been that of negative dismissal , nor critique for the sake of it . nor has it been merely to expose how these often strikingly visual objects that aimed at protecting individual health had their meaning changed through appropriation into a conceptual framework different from that of their initial contexts of production and consumption . more interesting to us are the intrinsic and unremitting links between these visual objects and wider politics , or how the visual is inherently a part of the latter . while it might have been supposed that aids posters came to political rest once they were retired , categorised , catalogued and stored according to the principles of collecting institutions , the hamburg exhibition proves otherwise . in fact , as collector s items , they entered a space that was no less political than when they were on the streets in the 1980s and 1990s , and when they were appropriated to western discourses on postmodern identity and on the role of the visual in the cultural negotiation of the self . retirement home they necessarily became a part of the institutional agenda of the museum fr kunst und gewerbe . indeed , from the moment such objects become collectors items and are stored and/or displayed as artifacts they become epistemologically loaded through the very process of objectification . hamburg s museum fr kunst und gewerbe demonstrates that these collecting agendas and the accompanying aesthetic guidelines often have deep historical roots . but what the analysis of its 2006 exhibition of aids posters also shows is that old and seemingly apolitical agendas ( invented to express specific national political interests ) are neither lost nor rendered innocuous in the contemporary world . rather , they come to serve new political frameworks linked to the world of today s visitors a world in which aesthetics is the dominant means to a politics constituted on little more than the idea that if it looks good go with it ( an outlook now as pervasive in the practice of science as in the arts of government).73 crucially , this new politics is sustained through , and for , the absenting of critique ; not today the critical outlook entertained by sontag and other pre - postmodern intellectuals , that visual representations ( and popular posters in particular ) covered - up or cloaked lurking ideologies . postmodernists , unconcerned with that view for the most part , in effect opened the space for the new politics of aesthetics that masks something different : the idea of aesthetics as void of political intention . the hamburg exhibition of aids posters was , in fact , an early example of the coming - to - reign of these particular politics , with the visual alone being the vehicle for understanding and creating a ( global ) community without distinction . whereas for brinckmann in the nineteenth century , aesthetics ( in art ) could be consciously used for the political purpose of populist democracy , with aesthetics and politics in clearly separate spheres , for the inheritors of his institution in hamburg aesthetics ( unbeknownst to them ) became the politics , not simple a means to it . ironically , their arrival at these politics their unknowing performance of them through the exhibition was via adherence to brinckmann s legacy . through that , brinckmann s original political agenda was emptied of its original political purpose . installed in its place were the politics of the appearance of political un - intention . thus did an aesthetic concept born in the nineteenth century to serve nationalistic purposes come to operate for the political work of educating national citizens to global citizenship . what does this mean for historians working with material objects stored in global - tending museums and archives and who are themselves now operating within a global framework ? since material objects have no meaning without a framework , and are framed in being collected , the simple answer is that historians have to take into account the afterlives of such objects as much as the object s original lives . harder is the problem of the historian s own place in the global framework , which , in many respects , is not unlike that of the material object in the global - aspiring museum ( and very much like that of the curators of such exhibitions ) . whether avowed explicitly in global history , or embraced implicitly in the practice of history writing in contemporary culture , the global operates politically and epistemologically . just as global history s predecessor world history is now perceived by some of its originators as having been a product of , and agent for , its cold war moment,74 so for our own times , dominated as they are by multi - national corporations and abiding politicians , the take up of global themes in history writing is widely recognised as providing , at the very least , legitimacy to a globalisation discourse , even if , as often the case , the historian s immediate object is the far from reactionary one of provincialising the west and critiquing its hegemony.75 of course , for some historians the global does politically more , overtly serving as a rhetorical strategy for the re - coherence of the discipline of history itself after its pummelling by poststructuralists , deconstructionists and other fragmenting postmodern forces over the past thirty years or more.76 to this end , what could serve better as a reunifying device than the holising connective metaphor of the globe ? thus the global provides a new grand narrative a universalising tool with which to reimpose the meta - narrativity of history . although seemingly mindless of one of postmodernism s cautions , that totalising worldviews can lead to totalitarianism,77 these historians seek more - or - less intentionally what the curators of the museum fr kunst und gewerbe performed innocently through their exhibition of aids posters . in so doing , they also share company with certain art historians anxious to revive older agendas a means ( as bluntly put by one of them ) to counter the deconstructive criticism of historical culture proposed by self - serving postmodern academics who treat the past as a sour land over which to exercise present concerns and anxieties.78 yet neither global history nor visual culture studies need necessarily lead in this direction . to see the global as a discourse tied politically to institutionally specific agendas ( such as the aesthetics entertained at the museum fr kunst und gewerbe in hamburg in 2006 ) offers the possibility to take an alternative political position , or at least to liberate its historical study from such agendas . nevertheless , in terms of the less - visible shaping of historical knowledge production , the global framework can never be other than world - making as opposed to being simply historically descriptive.79 no matter how hard we try to stick to the recovery of some truth of how the world came to be globally conceived ( past or present ) , our knowledge productions implicitly reproduce and foster the unifying construct . just as with material objects ( or words or images ) there is no meaning to historical events outside the conceptual frameworks we wittingly or unwittingly apply to them . in short , there exists no resting place for history writing ; it is always already fashioned and fashioning . like creating a museum exhibition of posters to aestheticise the global nature of hiv / aids , the business of contributing to a global history of anything entails , by that very act , the politics of constructing a necessarily partial representation of the past. history writing , too , in other words , as a product of its time , can not avoid making up historical consciousness . this article can not escape the charge that it too contributes to this process merely by discussing an event that was conceptualised in it might even be seen to compound the problem by drawing attention to spaces where , a priori , the historian is already politically and epistemologically implicated : the museum and the archive . however , in doing so it has sought to move the discussion beyond the tired call for attending merely to historical contexts , especially of material objects globally attributed . our purpose has been to encourage historians to an awareness of their own immediate entanglements in history s constructedness the constructedness of the present mediated in history writing as much as through aesthetic assemblages of the global. the historian aruf dirlik , critically inquiring into the point of writing world history , has observed that an awareness of the variety of world histories that have been constructed at different times and in different places [ must cause ] any world historian worthy of the name [ to ] be uncommonly aware of the constructedness of the past.80 similarly , we submit , all historians need be reflective on their contributions to the present that is , to a culture given to re - enchantment through the global. quite how we should historicise material objects of the sort discussed in this paper may be open to debate ; what is not is the necessity to historicise our own historical projects . otherwise we move perilously close to becoming blind participants in the historically fashioned spaces where memory is increasingly naturalised and neutralised through universalised and universalising concepts mediated aesthetically . we end up , as it were , nave viewers of the exhibition at hamburg : as blind to the nature of the new post - postmodern politics of aesthetics , as to the modernist would - be universal humanity that the global unwittingly espouses through those politics .

drawing on recent visual and spatial turns in history writing , this paper considers aids posters from the perspective of their museum afterlife as collected material objects . museum spaces serve changing political and epistemological projects , and the visual objects they house are not immune from them . a recent globally themed exhibition of aids posters at an arts and crafts museum in hamburg is cited in illustration . the exhibition also serves to draw attention to institutional continuities in collecting agendas . revealed , contrary to postmodernist expectations , is how today s application of aesthetic display for the purpose of making global connections does not radically break with the virtues and morals attached to the visual at the end of the nineteenth century . the historicisation of such objects needs to take into account this complicated mix of change and continuity in aesthetic concepts and political inscriptions . otherwise , historians fall prey to seductive aesthetics without being aware of the politics of them . this article submits that aesthetics is politics .

Introduction The Hamburg Exhibition Aesthetic Framing Re-picturing AIDS Conclusion

among striking developments in history writing towards the end of the twentieth century were the moves to the spatial and the visual in the reconstruction of historical consciousness . as illustrated through the contemporaneous growth of a literature on the history of material objects in all their global distribution , they also helped broaden the range of objects deemed worthy of historical attention.6 this paper draws on these moves in relation to one particular material object that is both global and visual : the aids poster.7 their worldwide production from the mid-1980s hugely reinvigorated the whole genre of the health poster and , according to one expert , restored the genre s original function as a communications medium.8 certainly , as never before was so much money , aesthetic effort and psychological marketing put into this particular media on the part of voluntary bodies , national governments and international health agencies.9 our concern , however , is with the wider conceptual frameworks that were mobilised to make these objects meaningful . in the 1980s and 1990s , as we have outlined elsewhere , a cohort of western intellectuals concerned themselves with them along with other representations of aids in order to talk about the politics of identity.10 those concerns , in turn , were linked to broader ones emerging at the time over the rights of citizens to equal access to health care , the privatisation of medicine , and the role of the international pharmaceutical industry in the commercialisation of health care . here , we explore one such afterlife for aids posters : an exhibition entitled against aids : posters from around the world , which was held at the museum fr kunst und gewerbe in hamburg between february and april 2006 . impact of the exhibition ; our interest in it is , rather , as an illustration of the more general trend towards the the museum fr kunst und gewerbe was founded in 1877 in a spirit of aesthetic modernism and german nationalism , and by the 1890s was host one of germany s largest and most prestigious poster collections . the peoples arts and crafts , much like the south kensington museum in london , established in 1852 and renamed the victoria and albert museum in 1899 at the height of british jingoism . the representation of aids posters at the hamburg exhibition provides us with a means to discuss the politics of such global assemblages.12 on the one hand , it permits us to draw out the inherent contradictions and tensions that can be involved in any such institutional mobilisation of the concept of the unity of the globe.13 on the other , it allows us to underline important continuities hidden under the more apparent or insisted upon discontinuities between national and global discourses , and between modern and postmodern politics of aesthetics continuities rooted , we argue , in shared aesthetic values.14 as important , the example permits us to reflect on how the discourse of the global affects the work of historians using material objects in their constructions of historical consciousness . as these aims and objectives should suggest , we are not concerned here with how viewers might have responded to the images or to the exhibition as a whole ( an almost impossible task given the uniqueness of individual psychology and experience).15 nor are we interested in providing a walk - through critique of the exhibition ; our main interest is in the historical context of the museum and how this bears on the politics of aesthetics implicated in its exhibition of aids posters . primarily , it was staged in order to exploit the museum s recent acquisition of over a thousand hiv / aids and safe sex posters from a private dealer , a purchase that enabled it to join the club of institutions harbouring such collections.16 the organisers of the exhibition selected only a hundred of the posters to display , choosing those that were most visually arresting , and others that , even after three decades in some cases , still had the power to shock , titillate , and/or amuse ( such as that used on the front of the flyer for the exhibition , figure 1 , or condoman , figure 2 ) . dramatically , at the entrance to the exhibition , the visitor was confronted with a full billboard - size reproduction of oliviero toscani s iconic image of 1992 : his re - conception of the prize - winning photograph from life depicting the death of the american aids activist david kirby , turned into an advertisement for the united colours of benetton ( figure 5).18 other less dramatic images played on popular solidarities around aids , as prefigured in the socially integrative against aids in the title of the exhibition . although a flyer ( figure 1 ) , but no catalogue was produced for the show , the aesthetic and intellectual motives behind it are discernable through the catalogue that accompanied a much larger exhibition of posters held at the museum in 1996 ( which was curated by the same person , jrgen dring , and displayed some of same posters).20 the 1996 exhibition marked the centenary of the museum s first - ever exhibition of posters a late nineteenth century entertainment that was in fact the first of its kind in germany and one of the first in europe.21 in many ways the 1996 exhibition was faithful to that of 1896 , its agenda being more or less the same as that articulated by the main advocate and co - founder of the museum fr kunst und gewerbe , the hamburg lawyer and art critic justus brinckmann ( 18431915).22 in his catalogue for the 1896 exhibition , brinckmann opined that posters and their display in museums and galleries performed the ethical task of elevating the masses to aesthetic appreciation : art should be accessible to everyone . the wider context at the end of the nineteenth century was that of flourishing national rivalries as well as attitudes to competition as a virtue in itself the posters in the 1896 exhibition , from italy , france , russia , the usa and elsewhere , were organised accordingly and , typical of most exhibitions at the time displaying products from different countries , were competitively judged by an international panel . the exhibition thus served to cohere national identities at the same time as sell the ideology of competition along with the virtues of the products of mass - production for mass ( visual ) consumption . above all , dring celebrated and reproduced brinckmann s ethical mission to teach people how to appreciate and enjoy the visual world through a better understanding of its underlying aesthetic principles and techniques of production.26 this ethical educational work of the poster is all the more important today , he argued in the catalogue , because the fine arts have lost the main task they had when brinckmann was living , namely , to interpret the world in its visual parts and provide the onlooker with an edifying understanding of it.27 the 2006 exhibition of aids posters was conceived in the same intellectual framework , according to dring.28 it , too , was intended to elicit an emotional response from the viewer and , from that , heighten their sensitivity to art.29 but while brinkmann in his day had looked forward to a brighter future for the poster and its onlookers in museums such as the kunst und gewerbe in hamburg , dring and his colleagues found it hard to be quite so optimistic . in their view as maintained in the catalogue for the 1996 exhibition the general quality of the visual language of posters had dramatically decreased due to the increasing flood of pictures in everyday life . all that could really be said in their favour was that they sponsored a positive moral practice , namely caution against the spread of hiv : the onlooker feels attracted by the picture and decides to use a condom.33 although it seems odd that the curators of the museum fr kunst und gewerbe disdained the very objects they were celebrating , they justified themselves on the grounds that such posters were expressive of their times and therefore wholly within the remit of an institution whose agenda was to archive and exhibit the art of the streets and the people . one had only to strip away the metaphors to get at the underlying value - neutral , non - stigmatising nature of any disease.41 the organisers of the exhibitions of aids posters at hamburg s museum fr kunst und gewerbe similarly believed aids to be realthat is , to be a universal medical problem that was simply yet to be solved . how people interacted with aids posters during the images active life on the streets on buses , billboards , underground trains , and so on and how the power and fear of hiv / aids operated in relation to identity were simply not parts of the 2006 exhibition in hamburg . through the literal framing of the posters , their hanging according to the conventions of art galleries , their arrangement ( three or four to a single wall ) , and the choice of them in terms of the quality of their visual language ( bildersprache ) , the organisers denuded them of the local contexts in which they were created and in which they were engaged with politically , intellectually and emotionally . semen kit performed similarly through its play to a would - be unproblematic history of overt sexual behaviour among men ( inconised in the image of the sailor ) , and by its appropriation from some american aids posters of the by then more - than - a - decade - old image of the gay body beautiful.46 that gay men in britain , well into the 1990s , were still struggling for a viable visual public identity could not be guessed from the hanging of this poster at hamburg . while toscani s image still had the power to shock , it is doubtful if anyone outside of living memory who viewed it at the exhibition ( such as the school children bussed in ) could have guessed what it meant for many visual theorists and aids activists worrying over images of aids since the late 1980s , namely , that it was further testimony to the ongoing crisis over the entire framing of knowledge about the human body,55 with aids , not just a medical problem but an epidemic of meanings or significations.56 the exhibition in hamburg gave no clue to how images like this had been regarded as testifying to a self - consciously postmodern culture , ethics , aesthetics , disease representation , and politics of identity . this global aesthetic spin , in effect , harmonised a modernist western transcendent notion of art with the late twentieth - century notion of a spatially transcendent capitalism an economic system supposedly unfettered by place or national boundary.58 the aesthetic spin and the exhibition as a whole thus further performed for notions of homogeneity and universality attributes long associated with modernity and perceived to be at odds with the pluralities and fragmentations associated with postmodernity during the time of aids in the west.59 the exhibition s title also suggested that the history of aids was about everyone the world over being uniformly against aids . moreover , the relative power of those interests was hierarchically organised , and differently so over time , as virginia berridge has made clear for the history of aids in the uk.62 also , implicit to the entitling of the hamburg exhibition was the idea that nations around the world were homogeneous in their fight against hiv / aids . the images of condoms promoted the idea that campaigns for their use had actually united the countries of the world , a message curiously at odds with the bush administration s contemporaneous funding of medical missionaries advocating sexual abstinence instead of the use of condoms.64 in its own small way , therefore , and for its own particular didactic reasons ( as well as , perhaps , discomfort over germany s nationalistic past ) , the hamburg exhibition effected the same kind of historical effacement and rewrite that the major international media and entertainment companies were also coming to effect by 2006 through their take of up hiv / aids for the purpose of reaping public corporate credibility.65 by then , hiv / aids - funding was a fashionable cause , a benign branding resource for various western philanthropic organisations . we have been concerned with a particular moment in the history of aids posters : not that of their initial public life on the streets , or in pubs and gay clubs , doctors offices , and so on , but their afterlife in places where they might be displayed or stored.72 through the analysis of an exhibition at one such afterlife location our intention has not been that of negative dismissal , nor critique for the sake of it . rather , they come to serve new political frameworks linked to the world of today s visitors a world in which aesthetics is the dominant means to a politics constituted on little more than the idea that if it looks good go with it ( an outlook now as pervasive in the practice of science as in the arts of government).73 crucially , this new politics is sustained through , and for , the absenting of critique ; not today the critical outlook entertained by sontag and other pre - postmodern intellectuals , that visual representations ( and popular posters in particular ) covered - up or cloaked lurking ideologies . the hamburg exhibition of aids posters was , in fact , an early example of the coming - to - reign of these particular politics , with the visual alone being the vehicle for understanding and creating a ( global ) community without distinction . whereas for brinckmann in the nineteenth century , aesthetics ( in art ) could be consciously used for the political purpose of populist democracy , with aesthetics and politics in clearly separate spheres , for the inheritors of his institution in hamburg aesthetics ( unbeknownst to them ) became the politics , not simple a means to it . since material objects have no meaning without a framework , and are framed in being collected , the simple answer is that historians have to take into account the afterlives of such objects as much as the object s original lives . just as global history s predecessor world history is now perceived by some of its originators as having been a product of , and agent for , its cold war moment,74 so for our own times , dominated as they are by multi - national corporations and abiding politicians , the take up of global themes in history writing is widely recognised as providing , at the very least , legitimacy to a globalisation discourse , even if , as often the case , the historian s immediate object is the far from reactionary one of provincialising the west and critiquing its hegemony.75 of course , for some historians the global does politically more , overtly serving as a rhetorical strategy for the re - coherence of the discipline of history itself after its pummelling by poststructuralists , deconstructionists and other fragmenting postmodern forces over the past thirty years or more.76 to this end , what could serve better as a reunifying device than the holising connective metaphor of the globe ? through the literal framing of the posters , their hanging according to the conventions of art galleries , their arrangement ( three or four to a single wall ) , and the choice of them in terms of the quality of their visual language ( bildersprache ) , the organisers denuded them of the local contexts in which they were created and in which they were engaged with politically , intellectually and emotionally . this absenting can probably be illustrated by paying close attention to the history of any one of the posters in the exhibition . semen kit performed similarly through its play to a would - be unproblematic history of overt sexual behaviour among men ( inconised in the image of the sailor ) , and by its appropriation from some american aids posters of the by then more - than - a - decade - old image of the gay body beautiful.46 that gay men in britain , well into the 1990s , were still struggling for a viable visual public identity could not be guessed from the hanging of this poster at hamburg . while toscani s image still had the power to shock , it is doubtful if anyone outside of living memory who viewed it at the exhibition ( such as the school children bussed in ) could have guessed what it meant for many visual theorists and aids activists worrying over images of aids since the late 1980s , namely , that it was further testimony to the ongoing crisis over the entire framing of knowledge about the human body,55 with aids , not just a medical problem but an epidemic of meanings or significations.56 the exhibition in hamburg gave no clue to how images like this had been regarded as testifying to a self - consciously postmodern culture , ethics , aesthetics , disease representation , and politics of identity . moreover , the relative power of those interests was hierarchically organised , and differently so over time , as virginia berridge has made clear for the history of aids in the uk.62 also , implicit to the entitling of the hamburg exhibition was the idea that nations around the world were homogeneous in their fight against hiv / aids . the images of condoms promoted the idea that campaigns for their use had actually united the countries of the world , a message curiously at odds with the bush administration s contemporaneous funding of medical missionaries advocating sexual abstinence instead of the use of condoms.64 in its own small way , therefore , and for its own particular didactic reasons ( as well as , perhaps , discomfort over germany s nationalistic past ) , the hamburg exhibition effected the same kind of historical effacement and rewrite that the major international media and entertainment companies were also coming to effect by 2006 through their take of up hiv / aids for the purpose of reaping public corporate credibility.65 by then , hiv / aids - funding was a fashionable cause , a benign branding resource for various western philanthropic organisations . we have been concerned with a particular moment in the history of aids posters : not that of their initial public life on the streets , or in pubs and gay clubs , doctors offices , and so on , but their afterlife in places where they might be displayed or stored.72 through the analysis of an exhibition at one such afterlife location our intention has not been that of negative dismissal , nor critique for the sake of it . rather , they come to serve new political frameworks linked to the world of today s visitors a world in which aesthetics is the dominant means to a politics constituted on little more than the idea that if it looks good go with it ( an outlook now as pervasive in the practice of science as in the arts of government).73 crucially , this new politics is sustained through , and for , the absenting of critique ; not today the critical outlook entertained by sontag and other pre - postmodern intellectuals , that visual representations ( and popular posters in particular ) covered - up or cloaked lurking ideologies . the hamburg exhibition of aids posters was , in fact , an early example of the coming - to - reign of these particular politics , with the visual alone being the vehicle for understanding and creating a ( global ) community without distinction . whereas for brinckmann in the nineteenth century , aesthetics ( in art ) could be consciously used for the political purpose of populist democracy , with aesthetics and politics in clearly separate spheres , for the inheritors of his institution in hamburg aesthetics ( unbeknownst to them ) became the politics , not simple a means to it . since material objects have no meaning without a framework , and are framed in being collected , the simple answer is that historians have to take into account the afterlives of such objects as much as the object s original lives . just as global history s predecessor world history is now perceived by some of its originators as having been a product of , and agent for , its cold war moment,74 so for our own times , dominated as they are by multi - national corporations and abiding politicians , the take up of global themes in history writing is widely recognised as providing , at the very least , legitimacy to a globalisation discourse , even if , as often the case , the historian s immediate object is the far from reactionary one of provincialising the west and critiquing its hegemony.75 of course , for some historians the global does politically more , overtly serving as a rhetorical strategy for the re - coherence of the discipline of history itself after its pummelling by poststructuralists , deconstructionists and other fragmenting postmodern forces over the past thirty years or more.76 to this end , what could serve better as a reunifying device than the holising connective metaphor of the globe ? our purpose has been to encourage historians to an awareness of their own immediate entanglements in history s constructedness the constructedness of the present mediated in history writing as much as through aesthetic assemblages of the global.

laparoscopic inguinal herniorrhaphy has become widely accepted as an effective alternative to the treatment of inguinal hernias with the anterior approach , because it is minimally invasive , has success rates identical to those of the conventional method , and quickens recovery by decreasing time until return to work or physical activities . in australia , the rate of laparoscopic inguinal herniorrhaphy in 2012 was 48% of the total number of inguinal hernia repairs . in 20102011 , 46 651 separations were performed in hospitals for inguinal hernias , and at least 3711 ( 7.9% ) were for those specified as recurrent , although the statistics do not differentiate between the rates of recurrence of each type of repair . it is generally accepted that the best procedure for an inguinal hernia that recurs after laparoscopic repair is anterior repair and vice versa . however , there is currently no consensus as to the best technique for hernias that recur after both anterior and laparoscopic repairs have failed , partly because not all surgeons who perform laparoscopic inguinal hernia repair also perform laparoscopic ventral hernia repair ( lvhr ) . consequently , there are experts in laparoscopic inguinal hernia repair who have successfully attempted a second laparoscopic repair , but this practice is confined to very few surgeons in specialized hernia centers . on the other hand , surgeons who are confident in performing lvhr and total extraperitoneal ( tep ) or transabdominal preperitoneal ( tapp ) inguinal hernia repair may regard intraperitoneal onlay mesh ( ipom ) repair as merely an extension of lvhr , although detailed knowledge of laparoscopic extraperitoneal inguinal anatomy would be essential . in an attempt to further reduce parietal trauma , sil repair has been touted as the most important innovation in laparoscopic surgical procedures since its inception with the first laparoscopic cholecystectomy in 1988 . indeed , since the first commercial availability of the sil port in 2007 , several different single ports have been made available . prospective randomized controlled trials ( rcts ) , mainly for cholecystectomy and appendectomy and mostly with small samples of patients , but more significantly , comparisons during of the learning curve of single - port and multiport surgery , have shown the single - port approach to be consistently safe and effective , as has single - port laparoscopic inguinal herniorrhaphy . however , data regarding the superiority of single - port over conventional multiport procedures , other than cosmesis , are still lacking , although it is hoped that with increasing experience with sil , more high - powered rcts will provide us with a clearer picture of the place of sil in surgical approaches . our unit has been offering routine laparoscopic herniorrhaphy for inguinal hernias since 1991 and for ventral hernias since 2003 . since december 2009 , we have routinely treated virtually all ventral ( including parastomal ) and inguinal hernias with the single - port approach . after conventional anterior and laparoscopic approaches have failed , the treatment of a recurrent inguinal hernia with laparoscopic ipom repair represents an obvious choice . in addition , parietal trauma can be reduced with single - port compared to multiport surgery . to our knowledge , this is the first case series of sil - ipom repair for the treatment of recurrent inguinal hernias for which conventional anterior and laparoscopic repairs with mesh have both failed . the independent review boards of st luke 's and holroyd private hospitals approved this study . all patients referred with inguinal or femoral hernias from november 1 , 2009 , through june , 30 , 2014 , underwent sil inguinal herniorrhaphy . for this study , the enrollment criterion was the recurrence of an inguinal hernia after failure of both anterior and laparoscopic repairs with mesh . the exclusion criteria were being unfit for general anesthesia or having chronic postherniorrhaphy groin pain . the participants were informed of our practice of performing laparoscopic ipom repair , but that this procedure could now be performed with the sil technique . after induction of general anesthesia , the patients were prepped and draped with iodine from epigastrium to mid thigh and then draped with an iodine - impregnated adhesive cover ( ioband ; 3 m , st paul , minnesota ) , to expose the entire abdomen and both groins ( figure 1 ) . a preoperative intravenous dose of cephalosporin was given , and a urinary catheter was routinely placed . after infiltration with bupivacaine 0.5% with 1:200 000 ephedrine in the umbilical area , a 2- to 2.5-cm ( depending on the laxity of the skin ) crescentic infraumbilical incision was made , the anterior rectus sheath was incised transversely , and the rectus sheath was retracted laterally . the site of entry was on the side contralateral to the previous laparoscopic entry ( if a tep approach was used ) , to avoid scar tissue . the posterior rectus sheath and the peritoneum were then entered for placement of an sil port ( covidien , norwalk , connecticut ) . the patient was placed in a trendelenburg position at 10 to 15 ( figure 1 ) . the procedure was performed with a 52-cm/30 angled laparoscope , to assess the amount of adhesions ( figure 2 ) , and those were meticulously divided by sharp dissection , to avoid electrocautery ( figure 3 ) . modified dissection techniques , namely , chopstick and inline , were used to overcome the relative loss of triangulation . the pubic symphysis was identified , the peritoneum was incised 2 cm superior to it , and the incision was extended laterally , or superior to a direct sac , if present ( figure 4 ) . no attempt was made to incise ( or remove any part of ) the previously placed ( extraperitoneal ) mesh ; rather , the dissection was performed from the inferior aspect of the mesh and continued proximally . care was taken to stay below the inferior epigastric vessels as the dissection continued laterally . the peritoneum was then reflected inferiorly over the pubic symphysis and continued laterally over the spermatic cord and its structures , thus reducing any direct , femoral , and indirect hernia and lipoma of the cord , akin to the dissection during tapp inguinal hernia repair . extreme care was taken to prevent damage to the urinary bladder , external iliac vessels , vas deferens , testicular vessels , and femoral nerve and to preserve other retroperitoneal nerves in the vicinity ( figure 4 ) . no attempt was made to dissect the superior flap of peritoneum overlying the previous laparoscopically placed mesh . often the previously placed extraperitoneal mesh had folded during placement or deflation , causing the recurrence of the hernia , and consequently the inferior peritoneal flap was usually surprisingly easy to lift ( figures 3 and 4 ) . even so , it had to be assumed that , although the previously placed extraperitoneal mesh had been poorly positioned , there would have been some attempt to dissect the peritoneal space below the pubic ramus ; therefore , millimeter - by - millimeter meticulous sharp dissection , with avoidance of electrocautery , was used to minimize damage to the aforementioned retroperitoneal structures and to minimize tearing of the inferior peritoneal flap . after deflation to 8 mm hg , measurements were taken externally for the size of the mesh ( gore - tex dualmesh ; wl gore & associates , flagstaff , arizona ) , which was at least 5 cm longer craniocaudally , extending inferior to the pubic symphysis . a polydioxanone ( pds ) 0 suture ( ethicon , somerville , new jersey ) was placed in the superior medial corner of the mesh to provide transfascial suture fixation , and the mesh was marked 5 cm above its inferior medial corner to correspond to the superior edge of the pubic symphysis ( figure 5 ) . the mesh was rolled inward along its horizontal axis , like a scroll , and placed intraperitoneally via a 12-mm trocar , which temporarily replaced the 5-mm camera trocar . one of the 5-mm trocars was temporarily withdrawn until it was outside the fascial defect , to facilitate insertion of the 12-mm trocar . a stab incision was then made in the midline and inferior to the umbilicus , to retrieve the pds suture in the superior medial corner of the mesh with a suture passer . this method allowed the mesh to be more easily maneuvered into the correct position before nonabsorbable tacks ( protack ; covidien ) were placed onto the pubic bone and along the pubic ramus , taking care to avoid the external iliac vein ( figures 2 and 5 ) . the mesh was then tacked medially and superiorly and , cautiously , laterally , to avoid the nerves in the vicinity . the process was aided by the mesh 's craniocaudal dimension being of sufficient size that its superior edge was well above the previously placed extraperitoneal mesh and within 2 cm of the umbilical sil port , so that the tacks were unlikely to pierce the iliohypogastric nerve , ilioinguinal nerve , genital branch of the genitofemoral nerve , or the lateral cutaneous nerve of the thigh . fibrin sealant ( 2 ml ) ( tisseel duo ; baxter ag , vienna , austria ) was sprayed along the inferior edge of the mesh ( figure 5 ) . the inferior peritoneal flap was then reflected up and tacked lightly onto the mesh , with care taken not to leave any significant gaps that would allow herniation of the bowel loops . fibrin sealant ( 2 ml ) was also sprayed along the mesh peritoneum interface , on the periphery of the mesh , and over the tacks , to minimize the risk of adhesions ( figure 5 ) . the fascial defect in the umbilical wound was closed in layers , subcutaneously and subcuticularly , with interrupted no . 0 pds sutures and absorbable sutures . the urinary catheter was left in place overnight and removed before the patient was discharged home . all patients were seen at 1 week and 4 weeks , with plans to see them annually for 5 years . a single - incision laparoscopic intraperitoneal onlay mesh repair of a right inguinal hernia with multiple recurrences . a , patient with incisions from previous anterior and laparoscopic repair ; b , the setup ( an extra - long laparoscope was used to prevent clashing of the handles of the conventional straight dissecting instruments with the side arm of the scope ) and inset showing close up view of the single - port device . laparoscopic findings in patients with recurrent inguinal hernias after tep or tapp and a mean of 2 anterior repairs . d , a right indirect defect after tapp repair , with the inferolateral aspect of mesh being well above the deep inguinal ring . intraoperative photographs of dissection of a left pantaloon hernia after previous tep and 3 anterior repairs . b , previous extraperitoneal mesh that has been displaced upward , allowing indirect and direct recurrence . c , incision of the peritoneum along the inferior edge of the mesh , which is then extended medially and laterally . d , complete reduction of the direct and indirect defects , with preservation of cord structures . a , extensive adhesions of the sigmoid colon to the rolled up mesh medially of a left recurrent inguinal hernia . c , d , meticulous dissection below the pubic symphysis and along the cords , exposing the bladder and retroperitoneal nerves , which can be at risk of damage during dissection ; hence , there is no fixation of mesh with tacks in these areas . a , single - incision laparoscopic intraperitoneal onlay mesh repair for a right inguinal hernia with multiple recurrences b , mesh fixation into pubic ramus and inferior edge of mesh glued with fibrin sealant . c , d , the inferior peritoneal fold tacked back onto the mesh with fibrin sealant sprayed along the mesh peritoneum interface . between november 1 , 2009 , and june 24 , 2014 , there were 12 patients with recurrent inguinal hernias after previous failed anterior and laparoscopic repairs ; 3 patients with chronic neuropathic pain were excluded from the study . the patients were part of a cohort of 505 , over the same period , who had undergone sil inguinal herniorrhaphy . each of the 9 patients enrolled in the study had had one laparoscopic repair ( 5 tapps and 4 teps ) and a mean of 2 anterior repairs ( range , 14 ) . the mean age was 53 years ( range , 2474 years ) ; all were men . the mean bmi was 26.5 kg / m ( range , 24.528.4 kg / m ) ( table 1 ) . all but 1 patient were found to have direct hernias with 4 having incarcerated hernias containing small bowel / colon / omentum that had to be reduced . furthermore , there were always omental adhesions in the inguinal region that had to be divided ( even in the 5 patients who had undergone the tep approach ) . in all but 1 patient , the mesh was deficient medially , either found within the direct defect or folded , exposing the direct defect . in 4 patients the mesh was also found to be rolled up laterally . one patient also had a contralateral primary hernia , which was treated by ipom repair during the same operation . there were no deaths , morbidities , port - site hernias , or recurrences during a mean follow - up of 24 moths ( range , 248 months ) . mean scar length was 23 mm ( range , 1537 mm ) at the 6-week follow - up . patient demographic and sequence of previous hernia operations all patients were men and age is given in years . bmi , body mass index ( kg / m ) ; ipom , intraperitoneal onlay mesh repair ; tapp , transabdominal preperitoneal , tep , totally extraperitoneal . since the first laparoscopic extraperitoneal inguinal hernia repair by ger et al in 1989 , there has been an exponential increase worldwide in the use of laparoscopic repair for inguinal hernias . data from medicare australia show that , of all hernia repairs , the rates of laparoscopic anterior repair were 9.4% in 1994 , 20.5% in 2000 , and 48% in 2012 . the latter statistic ( for 2012 ) is reflected in the same percentage of surgeons performing laparoscopic repair ( as defined by any surgeon who entered a claim to medicare australia with the code 30609 , which corresponds to laparoscopic inguinal hernia repair ) . this exponential increase in the use of laparoscopic inguinal herniorrhaphy is remarkable , given that most laparoscopic repairs are performed in private hospitals , where surgical trainees are not usually trained , and that the surgery is normally performed only by the consultants . it is possible that many surgeons go overseas to attend hands - on animal workshops in tep / tapp repair , possibly because of the lack of such courses in australia , owing to the country 's stringent animal ethics requirements for surgical training involving animals . it has been estimated that the recurrence rates for inguinal hernias range from 7% to 10% in australia , which means that there are more and more patients with two or more recurrences of inguinal hernia after failed anterior and laparoscopic repairs . the international endohernia society guidelines suggest that the best repair for the treatment of an inguinal hernia recurrence after an anterior procedure is the laparoscopic approach , and vice versa . yet , there are no specific recommendations for the treatment of hernias that recur after a patient has undergone both anterior and laparoscopic procedures . anecdotal reports of a second laparoscopy , usually tapp , have come from highly specialized centers , but the incidence of complications is much higher , even though the success rates are higher than other anterior approaches . van den heuvel and dwars reported a series of 51 patients who underwent tapp repair for recurrent inguinal hernia after previous posterior hernia repair . in two - thirds of the patients , however , there were 8 adverse postoperative events : 4 port - site hernias and 4 chronic postoperative pain that restricted daily activities . the concept of ipom in the management of inguinal hernia is not new . in 1998 , kingsley et al demonstrated the feasibility of inguinal hernia repair by ipom with expanded polytetrafluoroethylene ( eptfe ) mesh ( 1015 cm ) , but the recurrence rate was 43% at the 41-month follow - up . a total of 76 patients underwent tapp and 72 underwent ipom ; 10 7-cm eptfe was used for ipom and 15 12-cm polypropylene mesh was used for tapp . there were no recurrences at 32 months after tapp compared with an 11.1% recurrence rate for ipom . neuralgia was noted in 3 patients who underwent tapp and in 11 who underwent ipom ( p < .05 ) . as a result of these and other studies , the ipom technique has been considered to be inferior to tapp and tep repairs in the treatment of primary inguinal hernias . clearly , multiple factors have contributed to these poor results , including no reduction of the hernia sacs , inadequate mesh size , lack of permanent bony fixation , and lack of tissue glue fixation of the inferior edge of the mesh . central to the conventional laparoscopic approach is the attempt to place the mesh in the extraperitoneal position , which means having to raise the peritoneal flaps sufficiently to cover the new mesh . by and large this is almost impossible , as often the peritoneum adheres so densely to the previous mesh that one ends up with multiple defects in the peritoneal flaps , exposing the normal mesh to bowel and causing adhesions with possible deleterious sequelae . indeed , lo menzo et al reported , in a series of 6 patients with 7 recurrent inguinal hernias after laparoscopic repair , that there were 2 patients in whom the peritoneal flap did not cover the mesh and a tissue - separating mesh with fibrin sealant had to be used to cover the myopectineal orifice . in addition , the tapp repair involves placement of a 10-mm trocar through the linea alba in the umbilical region and of 2 5-mm trocars more inferiorly ( although some surgeons prefer to place these latter trocars laterally on each side of the abdomen ) . all of these trocar sites are at risk of formation of a port - site hernia . an extraperitoneal approach , with the inferiorly placed 5-mm trocars , would be difficult , if not impossible , because the extraperitoneal space is likely to be obliterated from the previous laparoscopic repair . for this reason , most second laparoscopic repairs have been performed as a tapp procedure , in which the 5-mm trocars can be placed laterally on either side of the umbilical camera port . in our study , the umbilical ( the only ) port was placed via the previous infraumbilical scar , with transverse incisions in the anterior and posterior rectus sheaths with retraction of the rectus muscle laterally . these entry and closure techniques prevented port - site hernia formation in our series . with the introduction of the first commercial single port device , sil port ( covidien ) , in 2007 , there has been an exponential increase in the number and variety of sil procedures performed . it has been estimated that the learning curve for sil for an experienced laparoscopic surgeon is between 25 and 50 cases , which means that it should not take more than a year for a general surgeon in australia , who would perform 26 inguinal hernias per annum , on average , to be competent in performing sil . although the conventional laparoscopic ventral hernia repair involves placement of a 10-mm camera port in the upper outer quadrant and 2 5-mm ports more inferiorly in the anterior axillary line , such a configuration of ports would be a considerable distance from the inguinal region , causing poor ergonomics for the dissecting instruments , necessitating the use of longer dissecting instruments , which would further compromise the ergonomics of the dissecting instruments . although sil suffers from a lack of triangulation , this drawback can be overcome by modifying dissection techniques , by using a smaller and longer laparoscope , and with increased experience . our recent prospective randomized controlled study comparing single - port with multiport laparoscopic tep inguinal herniorrhaphy demonstrated safety and efficacy and additional cosmetic and noncosmetic benefits similar to those of the single - port technique beyond the learning curve . to date the principal author has performed in excess of 800 sil - tep and 120 sil - vhr repairs , with the latter including some of the most difficult abdominal wall ( ie , parastomal ) hernias . therefore , it became a natural progression to treat inguinal hernias that recur after the patient has undergone both open and laparoscopic repairs with sil , with the umbilicus used as the only point of access for placement of the single port . the sil port allows for placement of a 12-mm trocar for ease of intraperitoneal placement of antiadhesive mesh . the sil - ipom repair follows closely the dissection of the inferior flap during the tapp repair , as meticulous dissection of the inferior flap , below the inferior border of the previous extraperitoneally placed mesh , is important to reduce any direct , indirect , and femoral hernias , as well as any lipoma of the cord , with preservation of the latter and its structures . since virtually all of these hernias have a direct component , fixation of the mesh into the pubic ramus with nonabsorbable tacks is an important aspect of the repair that ensures permanent fixation and prevents further mesh displacement or eventration into the direct defect . of course , this protection can only be effectively accomplished with permanent tacks that allow adequate fixation of the mesh to the bone . however , unlike a tapp repair of a recurrent inguinal hernia , the sil - ipom does not interfere with the previously placed extraperitoneal mesh , but it aims to cover the defective inferior and medial borders of the previous mesh with an antiadhesive mesh that then extends well above the previous extraperitoneally placed mesh , in an attempt to prevent stapling of the relevant nerves in the groin causing severe chronic postherniorrhaphy pain . furthermore , the fixation of the microporous mesh well above the previously placed extraperitoneal mesh allows for better tissue ingrowth into the rough side of the mesh , as the normal peritoneum above the peritonealized mesh would be healthy live tissue that allows for ingrowth into the mesh . that most hernias that recur two times or more seem to have a direct component suggests an intrinsic weakness of the myopectineal orifice . indeed , henriksen et al found a consistent significant increase in immature type iii collagen compared with the stronger type i collagen in patients with hernias , and the changes were most pronounced in patients with a direct inguinal hernia than in those with an indirect inguinal hernia . furthermore , although the inferior edge of the mesh can not be tacked to avoid damage to vital neurovascular structures , it can be fixed with fibrin sealant . in addition , reflection of the inferior peritoneal fold back onto the mesh and its fixation with tacks prevents any further folding of the mesh , which should minimize the risks of recurrence . lau , in a prospective randomized study of mesh fixation with either fibrin sealant or tacks in laparoscopic inguinal hernia repair , showed that fibrin sealant reduces the incidence of chronic postherniorrhaphy pain . in our study we used fibrin sealant to fix the inferior edge of the mesh ; but in addition we used fibrin sealant for its antiadhesive property , on the basis of findings in our experimental and clinical research . indeed , since 2007 , in all our patients undergoing lvhr we have sprayed fibrin sealant along the periphery of the mesh , where adhesions are likely to take place , as well as on the tacks , which are known to cause adhesions . one important technical aspect in our study relates to the size of the mesh , which had to be long enough in the craniocaudal dimension that the superior edge of the mesh would be tacked well above the relevant nerves in the inguinal region to avoid nerve entrapment , even though , theoretically , only a 5-cm overlap of the defect is normally necessary for a sound repair . the single - port approach not only allowed the instruments to be inserted sufficiently far from the inguinal region , hence facilitating dissection , but it also permitted the mesh to be tacked more superiorly than would have been possible with the multiport approach . in addition , the inferior peritoneal flap had to be meticulously raised , without electrocautery , to prevent accidental damage to the retroperitoneal nerves that otherwise could have caused chronic postoperative pain . in our case series of 9 patients , during 4.5 years all were successfully treated with sil - ipom repair without any complication or recurrence during a mean follow - up of 24 months . these results compare favorably with those obtained with the alternative tapp repair , which would not be suitable for surgeons trained only in tep repair . in contrast , successful sil - ipom repair demands the highest level of competence in laparoscopic surgery . the surgeon must achieve safe adhesiolysis and avoid inadvertent nerve damage or entrapment through detailed knowledge of laparoscopic inguinal anatomy and the use of a sufficiently large piece of antiadhesive mesh , which must be judiciously fixed to achieve successful repair . in our series all of the patients had extensive adhesions of omentum , bowel , or both to the previously placed extraperitoneal mesh , and their meticulous division added significantly to the average operative time of 125 minutes , whereas a primary sil tep repair , in our experience , can be performed in 50 minutes , on average . furthermore , although the previously placed extraperitoneal flap was poorly positioned , it had to be assumed that there would have been some dissection of the peritoneum from the previous laparoscopic approach , either tep or tapp , and therefore , the dissection of the inferior flap was accomplished by meticulous and sharp dissection , millimeter by millimeter , to prevent damage to retroperitoneal structures , including the bladder , external iliac vessels , and the femoral and retroperitoneal nerves . this time - consuming process added to the relatively prolonged operative time compared to the time needed for a primary laparoscopic repair . finally , our hospital finance departments provided an accurate accounting of the costs of the disposables used as well as the hospital charges for lvhr procedures . the cost of an sil port was us $ 480 compared with us $ 340 for the three disposable ports [ consisting of a structural balloon trocar and inflation bulb ( us $ 280 ; tyco healthcare , norwalk , connecticut ) and two ribbed disposable 5-mm trocars ( us $ 30 each ; kii fios first entry ; applied medical , rancho santa margarita , california ) ] that we would normally use for multiport hernia surgery . the total hospital charges for lvhrs were usually between us $ 8 000 and $ 10 000 , depending on the size of the mesh and the number of tacks used , and therefore the small additional cost of the single - port device represents a very small percentage of the overall cost of the procedure , with the potential to reduce postoperative pain , analgesic requirements , and port - site hernia formation and to improve cosmesis , as demonstrated by our recent rct comparing single - port with multiport inguinal herniorrhaphy . multiple recurrences of inguinal hernias following failed conventional anterior and laparoscopic repairs can be safely and effectively treated with laparoscopic ipom repair . when ipom is combined with sil , the umbilicus can be used as the only incision site , which , apart from having the potential to reduce port - site complications , also allows improved ergonomics of the dissecting instruments , albeit with modified dissection techniques , by being of optimal proximity to the inguinal regions , where bilateral repairs can be performed safely and effectively .

background and objectives : despite the exponential increase in the use of laparoscopic inguinal herniorrhaphy , overall recurrence rates have remained unchanged . therefore , a growing number of patients are presenting with recurrent hernias after conventional anterior and laparoscopic repairs have failed . this study reports our experience with single - incision laparoscopic ( sil ) intraperitoneal onlay mesh ( ipom ) repair of these hernias.methods:patients referred with two or more recurrences of inguinal hernia underwent sil - ipom from november 1 , 2009 , to june 24 , 2014 . a 2.5-cm infraumbilical incision was made , and an sil port was placed intraperitoneally . modified dissection techniques were used : chopstick and inline dissection , 5.5-mm/52-cm/30 angled laparoscope , and conventional straight dissecting instruments . the peritoneum was incised above the pubic symphysis , and dissection was continued laterally and proximally , raising the inferior flap below the previous extraperitoneal mesh while reducing any direct , indirect , femoral , or cord lipoma before placement of antiadhesive mesh , which was fixed to the pubic ramus , as well as superiorly , with nonabsorbable tacks before the inferior border was fixed with fibrin sealant . the inferior peritoneal flap was then tacked back onto the mesh.results:nine male patients underwent sil - ipom . their mean age was 53 years and mean body mass index was 26.8 kg / m2 . mean mesh size was 275 cm2 . mean operation time was 125 minutes , with a hospital stay of 1 day . the umbilical scar length was 23 mm at the 6-week follow - up . there were no intra-/postoperative complications , port - site hernias , chronic groin pain , or recurrence of the hernia during a mean follow - up of 24 months.conclusion:inguinal hernias recurring after two or more failed conventional anterior and laparoscopic repairs can be safely and efficiently treated with sil - ipom .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

laparoscopic inguinal herniorrhaphy has become widely accepted as an effective alternative to the treatment of inguinal hernias with the anterior approach , because it is minimally invasive , has success rates identical to those of the conventional method , and quickens recovery by decreasing time until return to work or physical activities . in australia , the rate of laparoscopic inguinal herniorrhaphy in 2012 was 48% of the total number of inguinal hernia repairs . however , there is currently no consensus as to the best technique for hernias that recur after both anterior and laparoscopic repairs have failed , partly because not all surgeons who perform laparoscopic inguinal hernia repair also perform laparoscopic ventral hernia repair ( lvhr ) . on the other hand , surgeons who are confident in performing lvhr and total extraperitoneal ( tep ) or transabdominal preperitoneal ( tapp ) inguinal hernia repair may regard intraperitoneal onlay mesh ( ipom ) repair as merely an extension of lvhr , although detailed knowledge of laparoscopic extraperitoneal inguinal anatomy would be essential . prospective randomized controlled trials ( rcts ) , mainly for cholecystectomy and appendectomy and mostly with small samples of patients , but more significantly , comparisons during of the learning curve of single - port and multiport surgery , have shown the single - port approach to be consistently safe and effective , as has single - port laparoscopic inguinal herniorrhaphy . however , data regarding the superiority of single - port over conventional multiport procedures , other than cosmesis , are still lacking , although it is hoped that with increasing experience with sil , more high - powered rcts will provide us with a clearer picture of the place of sil in surgical approaches . after conventional anterior and laparoscopic approaches have failed , the treatment of a recurrent inguinal hernia with laparoscopic ipom repair represents an obvious choice . to our knowledge , this is the first case series of sil - ipom repair for the treatment of recurrent inguinal hernias for which conventional anterior and laparoscopic repairs with mesh have both failed . all patients referred with inguinal or femoral hernias from november 1 , 2009 , through june , 30 , 2014 , underwent sil inguinal herniorrhaphy . for this study , the enrollment criterion was the recurrence of an inguinal hernia after failure of both anterior and laparoscopic repairs with mesh . after infiltration with bupivacaine 0.5% with 1:200 000 ephedrine in the umbilical area , a 2- to 2.5-cm ( depending on the laxity of the skin ) crescentic infraumbilical incision was made , the anterior rectus sheath was incised transversely , and the rectus sheath was retracted laterally . the posterior rectus sheath and the peritoneum were then entered for placement of an sil port ( covidien , norwalk , connecticut ) . the procedure was performed with a 52-cm/30 angled laparoscope , to assess the amount of adhesions ( figure 2 ) , and those were meticulously divided by sharp dissection , to avoid electrocautery ( figure 3 ) . modified dissection techniques , namely , chopstick and inline , were used to overcome the relative loss of triangulation . the pubic symphysis was identified , the peritoneum was incised 2 cm superior to it , and the incision was extended laterally , or superior to a direct sac , if present ( figure 4 ) . no attempt was made to incise ( or remove any part of ) the previously placed ( extraperitoneal ) mesh ; rather , the dissection was performed from the inferior aspect of the mesh and continued proximally . the peritoneum was then reflected inferiorly over the pubic symphysis and continued laterally over the spermatic cord and its structures , thus reducing any direct , femoral , and indirect hernia and lipoma of the cord , akin to the dissection during tapp inguinal hernia repair . often the previously placed extraperitoneal mesh had folded during placement or deflation , causing the recurrence of the hernia , and consequently the inferior peritoneal flap was usually surprisingly easy to lift ( figures 3 and 4 ) . even so , it had to be assumed that , although the previously placed extraperitoneal mesh had been poorly positioned , there would have been some attempt to dissect the peritoneal space below the pubic ramus ; therefore , millimeter - by - millimeter meticulous sharp dissection , with avoidance of electrocautery , was used to minimize damage to the aforementioned retroperitoneal structures and to minimize tearing of the inferior peritoneal flap . after deflation to 8 mm hg , measurements were taken externally for the size of the mesh ( gore - tex dualmesh ; wl gore & associates , flagstaff , arizona ) , which was at least 5 cm longer craniocaudally , extending inferior to the pubic symphysis . a polydioxanone ( pds ) 0 suture ( ethicon , somerville , new jersey ) was placed in the superior medial corner of the mesh to provide transfascial suture fixation , and the mesh was marked 5 cm above its inferior medial corner to correspond to the superior edge of the pubic symphysis ( figure 5 ) . the mesh was rolled inward along its horizontal axis , like a scroll , and placed intraperitoneally via a 12-mm trocar , which temporarily replaced the 5-mm camera trocar . a stab incision was then made in the midline and inferior to the umbilicus , to retrieve the pds suture in the superior medial corner of the mesh with a suture passer . this method allowed the mesh to be more easily maneuvered into the correct position before nonabsorbable tacks ( protack ; covidien ) were placed onto the pubic bone and along the pubic ramus , taking care to avoid the external iliac vein ( figures 2 and 5 ) . the mesh was then tacked medially and superiorly and , cautiously , laterally , to avoid the nerves in the vicinity . the process was aided by the mesh 's craniocaudal dimension being of sufficient size that its superior edge was well above the previously placed extraperitoneal mesh and within 2 cm of the umbilical sil port , so that the tacks were unlikely to pierce the iliohypogastric nerve , ilioinguinal nerve , genital branch of the genitofemoral nerve , or the lateral cutaneous nerve of the thigh . fibrin sealant ( 2 ml ) ( tisseel duo ; baxter ag , vienna , austria ) was sprayed along the inferior edge of the mesh ( figure 5 ) . the inferior peritoneal flap was then reflected up and tacked lightly onto the mesh , with care taken not to leave any significant gaps that would allow herniation of the bowel loops . fibrin sealant ( 2 ml ) was also sprayed along the mesh peritoneum interface , on the periphery of the mesh , and over the tacks , to minimize the risk of adhesions ( figure 5 ) . a single - incision laparoscopic intraperitoneal onlay mesh repair of a right inguinal hernia with multiple recurrences . a , patient with incisions from previous anterior and laparoscopic repair ; b , the setup ( an extra - long laparoscope was used to prevent clashing of the handles of the conventional straight dissecting instruments with the side arm of the scope ) and inset showing close up view of the single - port device . c , incision of the peritoneum along the inferior edge of the mesh , which is then extended medially and laterally . a , extensive adhesions of the sigmoid colon to the rolled up mesh medially of a left recurrent inguinal hernia . c , d , meticulous dissection below the pubic symphysis and along the cords , exposing the bladder and retroperitoneal nerves , which can be at risk of damage during dissection ; hence , there is no fixation of mesh with tacks in these areas . a , single - incision laparoscopic intraperitoneal onlay mesh repair for a right inguinal hernia with multiple recurrences b , mesh fixation into pubic ramus and inferior edge of mesh glued with fibrin sealant . c , d , the inferior peritoneal fold tacked back onto the mesh with fibrin sealant sprayed along the mesh peritoneum interface . between november 1 , 2009 , and june 24 , 2014 , there were 12 patients with recurrent inguinal hernias after previous failed anterior and laparoscopic repairs ; 3 patients with chronic neuropathic pain were excluded from the study . the mean age was 53 years ( range , 2474 years ) ; all were men . there were no deaths , morbidities , port - site hernias , or recurrences during a mean follow - up of 24 moths ( range , 248 months ) . mean scar length was 23 mm ( range , 1537 mm ) at the 6-week follow - up . bmi , body mass index ( kg / m ) ; ipom , intraperitoneal onlay mesh repair ; tapp , transabdominal preperitoneal , tep , totally extraperitoneal . since the first laparoscopic extraperitoneal inguinal hernia repair by ger et al in 1989 , there has been an exponential increase worldwide in the use of laparoscopic repair for inguinal hernias . the latter statistic ( for 2012 ) is reflected in the same percentage of surgeons performing laparoscopic repair ( as defined by any surgeon who entered a claim to medicare australia with the code 30609 , which corresponds to laparoscopic inguinal hernia repair ) . this exponential increase in the use of laparoscopic inguinal herniorrhaphy is remarkable , given that most laparoscopic repairs are performed in private hospitals , where surgical trainees are not usually trained , and that the surgery is normally performed only by the consultants . it has been estimated that the recurrence rates for inguinal hernias range from 7% to 10% in australia , which means that there are more and more patients with two or more recurrences of inguinal hernia after failed anterior and laparoscopic repairs . in two - thirds of the patients , however , there were 8 adverse postoperative events : 4 port - site hernias and 4 chronic postoperative pain that restricted daily activities . in 1998 , kingsley et al demonstrated the feasibility of inguinal hernia repair by ipom with expanded polytetrafluoroethylene ( eptfe ) mesh ( 1015 cm ) , but the recurrence rate was 43% at the 41-month follow - up . clearly , multiple factors have contributed to these poor results , including no reduction of the hernia sacs , inadequate mesh size , lack of permanent bony fixation , and lack of tissue glue fixation of the inferior edge of the mesh . central to the conventional laparoscopic approach is the attempt to place the mesh in the extraperitoneal position , which means having to raise the peritoneal flaps sufficiently to cover the new mesh . by and large this is almost impossible , as often the peritoneum adheres so densely to the previous mesh that one ends up with multiple defects in the peritoneal flaps , exposing the normal mesh to bowel and causing adhesions with possible deleterious sequelae . indeed , lo menzo et al reported , in a series of 6 patients with 7 recurrent inguinal hernias after laparoscopic repair , that there were 2 patients in whom the peritoneal flap did not cover the mesh and a tissue - separating mesh with fibrin sealant had to be used to cover the myopectineal orifice . in addition , the tapp repair involves placement of a 10-mm trocar through the linea alba in the umbilical region and of 2 5-mm trocars more inferiorly ( although some surgeons prefer to place these latter trocars laterally on each side of the abdomen ) . for this reason , most second laparoscopic repairs have been performed as a tapp procedure , in which the 5-mm trocars can be placed laterally on either side of the umbilical camera port . in our study , the umbilical ( the only ) port was placed via the previous infraumbilical scar , with transverse incisions in the anterior and posterior rectus sheaths with retraction of the rectus muscle laterally . with the introduction of the first commercial single port device , sil port ( covidien ) , in 2007 , there has been an exponential increase in the number and variety of sil procedures performed . although the conventional laparoscopic ventral hernia repair involves placement of a 10-mm camera port in the upper outer quadrant and 2 5-mm ports more inferiorly in the anterior axillary line , such a configuration of ports would be a considerable distance from the inguinal region , causing poor ergonomics for the dissecting instruments , necessitating the use of longer dissecting instruments , which would further compromise the ergonomics of the dissecting instruments . although sil suffers from a lack of triangulation , this drawback can be overcome by modifying dissection techniques , by using a smaller and longer laparoscope , and with increased experience . therefore , it became a natural progression to treat inguinal hernias that recur after the patient has undergone both open and laparoscopic repairs with sil , with the umbilicus used as the only point of access for placement of the single port . the sil port allows for placement of a 12-mm trocar for ease of intraperitoneal placement of antiadhesive mesh . the sil - ipom repair follows closely the dissection of the inferior flap during the tapp repair , as meticulous dissection of the inferior flap , below the inferior border of the previous extraperitoneally placed mesh , is important to reduce any direct , indirect , and femoral hernias , as well as any lipoma of the cord , with preservation of the latter and its structures . since virtually all of these hernias have a direct component , fixation of the mesh into the pubic ramus with nonabsorbable tacks is an important aspect of the repair that ensures permanent fixation and prevents further mesh displacement or eventration into the direct defect . of course , this protection can only be effectively accomplished with permanent tacks that allow adequate fixation of the mesh to the bone . however , unlike a tapp repair of a recurrent inguinal hernia , the sil - ipom does not interfere with the previously placed extraperitoneal mesh , but it aims to cover the defective inferior and medial borders of the previous mesh with an antiadhesive mesh that then extends well above the previous extraperitoneally placed mesh , in an attempt to prevent stapling of the relevant nerves in the groin causing severe chronic postherniorrhaphy pain . furthermore , the fixation of the microporous mesh well above the previously placed extraperitoneal mesh allows for better tissue ingrowth into the rough side of the mesh , as the normal peritoneum above the peritonealized mesh would be healthy live tissue that allows for ingrowth into the mesh . indeed , henriksen et al found a consistent significant increase in immature type iii collagen compared with the stronger type i collagen in patients with hernias , and the changes were most pronounced in patients with a direct inguinal hernia than in those with an indirect inguinal hernia . furthermore , although the inferior edge of the mesh can not be tacked to avoid damage to vital neurovascular structures , it can be fixed with fibrin sealant . in addition , reflection of the inferior peritoneal fold back onto the mesh and its fixation with tacks prevents any further folding of the mesh , which should minimize the risks of recurrence . in our study we used fibrin sealant to fix the inferior edge of the mesh ; but in addition we used fibrin sealant for its antiadhesive property , on the basis of findings in our experimental and clinical research . indeed , since 2007 , in all our patients undergoing lvhr we have sprayed fibrin sealant along the periphery of the mesh , where adhesions are likely to take place , as well as on the tacks , which are known to cause adhesions . one important technical aspect in our study relates to the size of the mesh , which had to be long enough in the craniocaudal dimension that the superior edge of the mesh would be tacked well above the relevant nerves in the inguinal region to avoid nerve entrapment , even though , theoretically , only a 5-cm overlap of the defect is normally necessary for a sound repair . in addition , the inferior peritoneal flap had to be meticulously raised , without electrocautery , to prevent accidental damage to the retroperitoneal nerves that otherwise could have caused chronic postoperative pain . in our case series of 9 patients , during 4.5 years all were successfully treated with sil - ipom repair without any complication or recurrence during a mean follow - up of 24 months . the surgeon must achieve safe adhesiolysis and avoid inadvertent nerve damage or entrapment through detailed knowledge of laparoscopic inguinal anatomy and the use of a sufficiently large piece of antiadhesive mesh , which must be judiciously fixed to achieve successful repair . in our series all of the patients had extensive adhesions of omentum , bowel , or both to the previously placed extraperitoneal mesh , and their meticulous division added significantly to the average operative time of 125 minutes , whereas a primary sil tep repair , in our experience , can be performed in 50 minutes , on average . furthermore , although the previously placed extraperitoneal flap was poorly positioned , it had to be assumed that there would have been some dissection of the peritoneum from the previous laparoscopic approach , either tep or tapp , and therefore , the dissection of the inferior flap was accomplished by meticulous and sharp dissection , millimeter by millimeter , to prevent damage to retroperitoneal structures , including the bladder , external iliac vessels , and the femoral and retroperitoneal nerves . the total hospital charges for lvhrs were usually between us $ 8 000 and $ 10 000 , depending on the size of the mesh and the number of tacks used , and therefore the small additional cost of the single - port device represents a very small percentage of the overall cost of the procedure , with the potential to reduce postoperative pain , analgesic requirements , and port - site hernia formation and to improve cosmesis , as demonstrated by our recent rct comparing single - port with multiport inguinal herniorrhaphy . multiple recurrences of inguinal hernias following failed conventional anterior and laparoscopic repairs can be safely and effectively treated with laparoscopic ipom repair . when ipom is combined with sil , the umbilicus can be used as the only incision site , which , apart from having the potential to reduce port - site complications , also allows improved ergonomics of the dissecting instruments , albeit with modified dissection techniques , by being of optimal proximity to the inguinal regions , where bilateral repairs can be performed safely and effectively .

methods of recruitment and randomization as well as the demographics of the diad study have been published ( 1 ) . inclusion criteria were type 2 diabetes , age 5075 years , and no symptoms or clinical signs suggestive of coronary artery disease ( cad ) . exclusion criteria included angina pectoris ; stress test or coronary angiography within the previous 3 years ; history of myocardial infarction , heart failure , or coronary revascularization ; abnormal rest electrocardiogram ; any current clinical indication for stress testing ; active bronchospasm ; and limited life expectancy due to comorbidity . the participants were randomized to screening with an adenosine vasodilator tc-99m - sestamibi mpi ( n = 561 ) or no screening ( n = 562 ) ( 1 ) . the diad participants were risk - stratified as follows : framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > 20% ) 10-year risk for symptomatic cad ( 6 ) . participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group.ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > 30% ) 10-year risk for cad ( 7 ) . participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group.alfediam/sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . participants meeting one of these criteria were defined as the higher - risk cohort.metabolic syndrome : metabolic syndrome was defined by at least three of five criteria defined by the international diabetes federation taskforce ( 9 ) and was considered to represent higher cardiovascular risk ( 10,11 ) . the criteria included waist circumference 102 cm ( for men ) or 88 cm ( for women ) , triglycerides 150 mg / dl , hdl < 40 mg / dl ( for men ) or < 50 mg / dl ( for women ) , systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg , and fasting glucose 100 mg / dl . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group . ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > 30% ) 10-year risk for cad ( 7 ) . participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group . alfediam / sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . metabolic syndrome : metabolic syndrome was defined by at least three of five criteria defined by the international diabetes federation taskforce ( 9 ) and was considered to represent higher cardiovascular risk ( 10,11 ) . the criteria included waist circumference 102 cm ( for men ) or 88 cm ( for women ) , triglycerides 150 mg / dl , hdl < 40 mg / dl ( for men ) or < 50 mg / dl ( for women ) , systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg , and fasting glucose 100 mg / dl . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . the participants were risk stratified based on clinical variables documented at enrollment into the study . because it is generally agreed that low - risk patients should not undergo specialized cardiac testing ( 12 ) , only participants defined as having an intermediate / high risk were analyzed for outcomes according to randomization . secondary end points included unstable angina , heart failure , stroke , and coronary revascularization ( 3 ) . statistical analysis was performed with minitab 15 statistical software ( minitab , state college , pa ) . cardiac outcomes were compared in low - risk versus intermediate-/high - risk groups and in intermediate-/high - risk participants randomized to screening versus no screening . cox proportional hazards regression was computed using coxph in r ( www.r-project.org ) in order to determine hazard ratios ( hrs ) comparing events in low - risk versus intermediate-/high - risk groups and in screened versus not screened high-/intermediate - risk participants . the diad participants were risk - stratified as follows : framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > 20% ) 10-year risk for symptomatic cad ( 6 ) . participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group.ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > 30% ) 10-year risk for cad ( 7 ) . participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group.alfediam/sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . participants meeting one of these criteria were defined as the higher - risk cohort.metabolic syndrome : metabolic syndrome was defined by at least three of five criteria defined by the international diabetes federation taskforce ( 9 ) and was considered to represent higher cardiovascular risk ( 10,11 ) . the criteria included waist circumference 102 cm ( for men ) or 88 cm ( for women ) , triglycerides 150 mg / dl , hdl < 40 mg / dl ( for men ) or < 50 mg / dl ( for women ) , systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg , and fasting glucose 100 mg / dl . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > 20% ) 10-year risk for symptomatic cad ( 6 ) . participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group . ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group . alfediam / sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . metabolic syndrome : metabolic syndrome was defined by at least three of five criteria defined by the international diabetes federation taskforce ( 9 ) and was considered to represent higher cardiovascular risk ( 10,11 ) . the criteria included waist circumference 102 cm ( for men ) or 88 cm ( for women ) , triglycerides 150 mg / dl , hdl < 40 mg / dl ( for men ) or < 50 mg / dl ( for women ) , systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg , and fasting glucose 100 mg / dl . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . the participants were risk stratified based on clinical variables documented at enrollment into the study . because it is generally agreed that low - risk patients should not undergo specialized cardiac testing ( 12 ) , only participants defined as having an intermediate / high risk were analyzed for outcomes according to randomization . secondary end points included unstable angina , heart failure , stroke , and coronary revascularization ( 3 ) . statistical analysis was performed with minitab 15 statistical software ( minitab , state college , pa ) . cardiac outcomes were compared in low - risk versus intermediate-/high - risk groups and in intermediate-/high - risk participants randomized to screening versus no screening . cox proportional hazards regression was computed using coxph in r ( www.r-project.org ) in order to determine hazard ratios ( hrs ) comparing events in low - risk versus intermediate-/high - risk groups and in screened versus not screened high-/intermediate - risk participants . overall , 283 ( 25% ) participants would be defined as having low risk , and 840 ( 75% ) as having intermediate ( 542 [ 48% ] ) or high ( 298 [ 27% ] ) cardiovascular risk ( table 1 ) . of 522 screened participants , 387 ( 74% ) were defined as having intermediate / high risk . the prevalence of abnormal mpi in the screened intermediate-/high - risk versus screened low - risk groups was similar ( 21 vs. 23% , p = 0.72 ) ( table 2 ) . overall , primary cardiac events trended to be higher in the intermediate-/high - risk group versus the low - risk group ( 28 [ 3.3% ] vs. 4 [ 1.4% ] , p = 0.09 ) . however , primary cardiac event rates in 418 intermediate-/high - risk participants randomized to screening and in the 422 intermediate-/high - risk participants randomized to no screening were similar ( 3.1 and 3.6% ; log rank p = 0.71 ) table 3 ) . cardiac events in risk groups according to various risk stratification schemes data are n ( % ) , unless otherwise indicated . * p values are shown for low risk versus intermediate / high risk for framingham and ukpds ; low risk versus high risk for alfediam / sfc ; no versus yes for metabolic syndrome . results of stress mpi in 522 participants randomized to screening , grouped according to various risk stratification schemes data are n ( % ) . p values reflect comparison of total abnormal mpi in two risk groups ( see text ) . non - mpi abnormalities = ischemic electrocardiogram changes during adenosine infusion , transient ischemic dilation , or baseline left ventricular dysfunction . cardiac events in intermediate-/high - risk participants randomized to no screening versus screening data are n ( % ) , unless otherwise indicated . because of missing data , 19 participants could not be categorized by the ukpds risk engine . of the remaining 1,104 participants , 515 ( 47% ) were categorized as low risk and 589 ( 53% ) as intermediate ( 447 [ 40.5% ] ) or high ( 142 [ 13% ] ) risk ( table 1 ) . of those screened , 276 ( 53% ) were at intermediate / high risk ( table 2 ) . the prevalence of abnormal mpi in intermediate-/high - risk and low - risk participants was not different ( 24 vs. 19% , p = 0.2 ) ( table 2 ) . however , the incidence of primary cardiac events was higher in the intermediate-/high - risk group compared with the low - risk group ( 25 [ 4.2% ] vs. 6 [ 1.2% ] , p = 0.002 ) ( table 1 ) . primary cardiac event rates were similar in 291 intermediate-/high - risk participants randomized to screening and in 298 intermediate-/high - risk participants randomized to no screening ( 4.8 vs. 3.7% , log rank p = 0.51 ) ( table 3 ) . of 1,123 participants , 713 ( 63% ) met alfediam / sfc high - risk criteria ( table 1 ) . of 522 screened participants , 326 ( 62% ) were high risk ( table 2 ) . the prevalence of abnormal mpi in high - risk and low - risk participants was not different ( 23 vs. 19% , p = 0.27 ) ( table 2 ) . the incidence of primary cardiac events was not different in the high- and low - risk groups ( 24 [ 3.4% ] vs. 8 [ 2.0% ] , p = 0.19 ) ( table 1 ) , but secondary event rates were higher in the high - risk than in the low - risk group ( 30 [ 4.2% ] vs. 5 [ 1.2% ] , p = 0.01 ) ( table 1 ) . however , the primary cardiac event rates were similar in 352 high - risk participants randomized to screening and 361 high - risk participants randomized to no screening ( 3.7 vs. 3.1% , log rank p = 0.61 ) ( table 3 ) . of all participants , 804 ( 72% ) had metabolic syndrome ( table 1 ) . of 522 screened participants , 365 ( 70% ) had metabolic syndrome ( table 2 ) . the prevalence of abnormal mpi in participants with versus without metabolic syndrome was not different ( 21 vs. 24% , p = 0.49 ) ( table 2 ) . overall , primary cardiac event rates were similar in both groups ( metabolic syndrome 24 [ 3.0% ] vs. no metabolic syndrome 8 [ 2.5% ] , p = 0.67 ) ( table 1 ) . primary cardiac event rates in 398 participants with metabolic syndrome randomized to screening and in 406 participants with metabolic syndrome randomized to no screening were similar ( 2.5 vs. 3.5% , log rank p = 0.42 ) ( table 3 ) . overall , 283 ( 25% ) participants would be defined as having low risk , and 840 ( 75% ) as having intermediate ( 542 [ 48% ] ) or high ( 298 [ 27% ] ) cardiovascular risk ( table 1 ) . of 522 screened participants , 387 ( 74% ) were defined as having intermediate / high risk . the prevalence of abnormal mpi in the screened intermediate-/high - risk versus screened low - risk groups was similar ( 21 vs. 23% , p = 0.72 ) ( table 2 ) . overall , primary cardiac events trended to be higher in the intermediate-/high - risk group versus the low - risk group ( 28 [ 3.3% ] vs. 4 [ 1.4% ] , p = 0.09 ) . however , primary cardiac event rates in 418 intermediate-/high - risk participants randomized to screening and in the 422 intermediate-/high - risk participants randomized to no screening were similar ( 3.1 and 3.6% ; log rank p = 0.71 ) table 3 ) . cardiac events in risk groups according to various risk stratification schemes data are n ( % ) , unless otherwise indicated . * p values are shown for low risk versus intermediate / high risk for framingham and ukpds ; low risk versus high risk for alfediam / sfc ; no versus yes for metabolic syndrome . results of stress mpi in 522 participants randomized to screening , grouped according to various risk stratification schemes data are n ( % ) . p values reflect comparison of total abnormal mpi in two risk groups ( see text ) . non - mpi abnormalities = ischemic electrocardiogram changes during adenosine infusion , transient ischemic dilation , or baseline left ventricular dysfunction . cardiac events in intermediate-/high - risk participants randomized to no screening versus screening data are n ( % ) , unless otherwise indicated . because of missing data , 19 participants could not be categorized by the ukpds risk engine . of the remaining 1,104 participants , 515 ( 47% ) were categorized as low risk and 589 ( 53% ) as intermediate ( 447 [ 40.5% ] ) or high ( 142 [ 13% ] ) risk ( table 1 ) . of those screened , 276 ( 53% ) were at intermediate / high risk ( table 2 ) . the prevalence of abnormal mpi in intermediate-/high - risk and low - risk participants was not different ( 24 vs. 19% , p = 0.2 ) ( table 2 ) . however , the incidence of primary cardiac events was higher in the intermediate-/high - risk group compared with the low - risk group ( 25 [ 4.2% ] vs. 6 [ 1.2% ] , p = 0.002 ) ( table 1 ) . primary cardiac event rates were similar in 291 intermediate-/high - risk participants randomized to screening and in 298 intermediate-/high - risk participants randomized to no screening ( 4.8 vs. 3.7% , log rank p = 0.51 ) ( table 3 ) . of 1,123 participants , 713 ( 63% ) met alfediam / sfc high - risk criteria ( table 1 ) . of 522 screened participants , 326 ( 62% ) were high risk ( table 2 ) . the prevalence of abnormal mpi in high - risk and low - risk participants was not different ( 23 vs. 19% , p = 0.27 ) ( table 2 ) . the incidence of primary cardiac events was not different in the high- and low - risk groups ( 24 [ 3.4% ] vs. 8 [ 2.0% ] , p = 0.19 ) ( table 1 ) , but secondary event rates were higher in the high - risk than in the low - risk group ( 30 [ 4.2% ] vs. 5 [ 1.2% ] , p = 0.01 ) ( table 1 ) . however , the primary cardiac event rates were similar in 352 high - risk participants randomized to screening and 361 high - risk participants randomized to no screening ( 3.7 vs. 3.1% , log rank p = 0.61 ) ( table 3 ) . of all participants , 804 ( 72% ) had metabolic syndrome ( table 1 ) . of 522 screened participants , 365 ( 70% ) had metabolic syndrome ( table 2 ) . the prevalence of abnormal mpi in participants with versus without metabolic syndrome was not different ( 21 vs. 24% , p = 0.49 ) ( table 2 ) . overall , primary cardiac event rates were similar in both groups ( metabolic syndrome 24 [ 3.0% ] vs. no metabolic syndrome 8 [ 2.5% ] , p = 0.67 ) ( table 1 ) . primary cardiac event rates in 398 participants with metabolic syndrome randomized to screening and in 406 participants with metabolic syndrome randomized to no screening were similar ( 2.5 vs. 3.5% , log rank p = 0.42 ) ( table 3 ) . this post hoc analysis provides an important perspective on the results of the diad study ( 3 ) by demonstrating that the majority of participants were categorized as being either at intermediate or high cardiovascular risk according to four commonly used cardiac risk - stratification schemes . the ukpds risk engine , specifically designed for type 2 diabetic patients , appeared to best predict the occurrence of cardiac events in diad participants . in contrast , risk stratification did not predict the results of screening - stress mpi . the study was not powered to determine the effect of screening on outcomes in the subgroup of diad participants categorized as having higher risk ; such analysis would have required a three- to fourfold larger sample size . however , screening had no apparent benefit on outcomes in the subgroups as defined by these four separate stratification schemes . this analysis expands upon our previous finding that the overall cardiac event rate in asymptomatic patients with type 2 diabetes is lower in the current era than might be predicted based on historical data . specifically , it shows that the purportedly higher - risk subgroups actually had lower event rates than were predicted by either the framingham or ukpds scores . the average annual risk of participants in the combined intermediate-/high - risk framingham group was lower ( 0.6% per year ) than predicted ( 12% per year for intermediate risk and > 2% per year for high risk ) . similarly , in the combined intermediate-/high - risk ukpds groups , the risk was also lower ( 0.8% per year ) than predicted ( intermediate 1.53% per year and high risk > thus , even these higher - risk participants had observed cardiac event rates that would traditionally been considered to be low risk . only a small subgroup of 142 high - risk participants defined by the ukpds risk engine had an event rate of 2% per year ( table 1 ) , which might have warranted more aggressive risk - reduction strategies . although 14 of these high - risk participants had primary cardiac events , it is important to note that the majority of events ( 17 of 31 ) occurred in participants who were not categorized as high risk according to the ukpds engine ( table 1 ) . the observation that cardiac event rates in the diad were lower than predicted by either the framingham score or the ukpds risk engine likely reflects the fact that these scoring schemes are based on clinical data collected in the 1970s to 1990s ( 13,14 ) . in the intervening years , the awareness of cardiovascular risk in type 2 diabetes has grown ( 15 ) , and primary cardiac prevention measures have been widely endorsed and implemented ( 16 ) . in the diad study , the majority of participants were aggressively treated with statins , ace inhibitors , and aspirin ( 3 ) . one might hypothesize that these interventions prevented cardiac events in the higher - risk diad participants . rather than concluding from this analysis that diabetes does not confer significant cardiac risk , it is more appropriate to emphasize the potential benefit of contemporary medical therapy on the outcomes of these patients . our findings have important implications for the utilization of cardiac screening in asymptomatic diabetic patients . the 2009 appropriate use criteria for cardiac radionuclide imaging , issued by a consortium of professional societies ( 12 ) , considered asymptomatic diabetic patients to be a special group in whom screening was appropriate based on their historically high risk for cardiovascular complications , equivalent to that of patients with established cad ( 16,17 ) . the results of the present analysis raise questions about the appropriateness of screening asymptomatic patients with diabetes who are treated with contemporary risk factor modifying therapies . one interesting observation in the current analysis is that none of the stratification schemes predicted abnormalities on screening - stress mpi . neither the presence nor severity of mpi abnormalities was greater in the higher - risk patients . the reasons for this finding are uncertain , but this lack of correlation reduces the potential impact of screening strategies based on existing clinical risk stratification . for example , since the ukpds risk engine predicts outcome but not mpi screening results , there would be patients who might screen negative but still would be at risk for events . in the diad study , although moderate / large mpi abnormalities were predictive of cardiac events , numerically more than half of the events occurred in the larger cohort of patients with negative screening ( 3 ) . we did not observe an effect of screening on cardiac events in any of the intermediate-/high - risk subgroups . thus , these results buttress the original conclusion of the diad study that screening for inducible ischemia can not be currently advocated in asymptomatic patients with type 2 diabetes . however , because of the limited number of subjects , we can not exclude the possibility that a larger study specifically screening a high - risk subgroup might come to a different conclusion in support of screening . most notably , the diad study was designed to include asymptomatic patients with diabetes regardless of additional clinical risk factors ( 1 ) . because of the relatively small number of participants at higher cardiovascular risk , the subgroup analyses have insufficient power to make definitive statistical conclusions as to whether screening leads to strategies that improve cardiac outcomes . furthermore , the diad cohort was representative of the north american population mix that received aggressive primary cardiac prevention . thus , generalization to other countries with different ethnicities and different approaches to diabetes care might not be appropriate . in conclusion , a substantial portion of the diad population would be defined by commonly used risk - stratification schemes as being at intermediate / high cardiovascular risk . nevertheless , even in these higher - risk participants , the annual cardiac event rates were low and outcome was not affected by routine screening for inducible ischemia .

objectiveto estimate baseline cardiovascular risk of 1,123 participants in the detection of ischemia in asymptomatic diabetics ( diad ) study and to assess cardiac event rates and the effect of screening on outcomes in these higher - risk participants.research design and methodsbaseline cardiovascular risk was assessed using four established methods : framingham score , uk prospective diabetes study ( ukpds ) risk engine , criteria of the french - speaking association for the study of diabetes and metabolic diseases , and the presence or absence of metabolic syndrome . cardiac events ( cardiac death or nonfatal myocardial infarction ) were assessed during the 4.8-year follow - up in participants with intermediate / high cardiovascular risk.resultsby various risk - stratification approaches , 5375% of participants were defined as having intermediate or high cardiovascular risk . the prevalence of inducible ischemia on screening in these individuals ranged from 21 to 24% , similar to lower - risk participants ( 1923% ) . cardiac event rates were greater in intermediate-/high - risk versus low - risk groups , but this was only significant for the ukpds risk engine ( 4.2 vs. 1.2% , p = 0.002 ) . the annual cardiac event rate was < 1% in all risk groups , except in the high - risk ukpds group ( 2% per year ) . in intermediate-/high - risk participants randomized to screening versus no screening , 4.8-year cardiac event rates were similar ( 2.54.8% vs. 3.13.7%).conclusionsa substantial portion of the diad population was defined as having intermediate / high baseline cardiovascular risk . nevertheless , their annual cardiac event rate was low and not altered by routine screening for inducible ischemia .

RESEARCH DESIGN AND METHODS Post hoc risk stratification Statistical analysis RESULTS Framingham risk score UKPDS risk engine ALFEDIAM/SFC high-risk criteria Metabolic syndrome CONCLUSIONS

the participants were randomized to screening with an adenosine vasodilator tc-99m - sestamibi mpi ( n = 561 ) or no screening ( n = 562 ) ( 1 ) . the diad participants were risk - stratified as follows : framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > 20% ) 10-year risk for symptomatic cad ( 6 ) . participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group.ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > 30% ) 10-year risk for cad ( 7 ) . participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group.alfediam/sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . participants meeting one of these criteria were defined as the higher - risk cohort.metabolic syndrome : metabolic syndrome was defined by at least three of five criteria defined by the international diabetes federation taskforce ( 9 ) and was considered to represent higher cardiovascular risk ( 10,11 ) . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group . ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > 30% ) 10-year risk for cad ( 7 ) . participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group . alfediam / sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . because it is generally agreed that low - risk patients should not undergo specialized cardiac testing ( 12 ) , only participants defined as having an intermediate / high risk were analyzed for outcomes according to randomization . cardiac outcomes were compared in low - risk versus intermediate-/high - risk groups and in intermediate-/high - risk participants randomized to screening versus no screening . cox proportional hazards regression was computed using coxph in r ( www.r-project.org ) in order to determine hazard ratios ( hrs ) comparing events in low - risk versus intermediate-/high - risk groups and in screened versus not screened high-/intermediate - risk participants . the diad participants were risk - stratified as follows : framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > 20% ) 10-year risk for symptomatic cad ( 6 ) . participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group.ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > 30% ) 10-year risk for cad ( 7 ) . participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group.alfediam/sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . participants meeting one of these criteria were defined as the higher - risk cohort.metabolic syndrome : metabolic syndrome was defined by at least three of five criteria defined by the international diabetes federation taskforce ( 9 ) and was considered to represent higher cardiovascular risk ( 10,11 ) . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . framingham risk score : on the basis of age , sex , lipid levels , blood pressure , smoking , and presence of diabetes , the participants were categorized as having either a low ( < 10% ) , intermediate ( 1020% ) , or high ( > 20% ) 10-year risk for symptomatic cad ( 6 ) . participants with intermediate or high framingham risk scores were defined as having a higher risk and were compared with the low - risk group . ukpds risk engine : on the basis of age , sex , duration of diabetes , smoking , systolic blood pressure , total cholesterol , hdl , ethnicity , and a1c , the participants were classified into three ukpds risk categories : low ( < 14% ) , intermediate ( 1530% ) , or high ( > participants with an intermediate or high ukpds risk score were defined as having a higher risk and were compared with the low - risk group . alfediam / sfc high - risk criteria : the alfediam recommended screening for inducible myocardial ischemia in patients with type 2 diabetes ( 8) with one of the following : age > 60 years ; duration of diabetes > 10 years and at least two other cardiovascular risk factors ; peripheral arterial disease ; and proteinuria and microalbuminuria with at least two other cardiovascular risk factors . participants with metabolic syndrome were defined as having a higher risk ; their outcome was compared with the cohort without metabolic syndrome . because it is generally agreed that low - risk patients should not undergo specialized cardiac testing ( 12 ) , only participants defined as having an intermediate / high risk were analyzed for outcomes according to randomization . cardiac outcomes were compared in low - risk versus intermediate-/high - risk groups and in intermediate-/high - risk participants randomized to screening versus no screening . cox proportional hazards regression was computed using coxph in r ( www.r-project.org ) in order to determine hazard ratios ( hrs ) comparing events in low - risk versus intermediate-/high - risk groups and in screened versus not screened high-/intermediate - risk participants . overall , 283 ( 25% ) participants would be defined as having low risk , and 840 ( 75% ) as having intermediate ( 542 [ 48% ] ) or high ( 298 [ 27% ] ) cardiovascular risk ( table 1 ) . of 522 screened participants , 387 ( 74% ) were defined as having intermediate / high risk . the prevalence of abnormal mpi in the screened intermediate-/high - risk versus screened low - risk groups was similar ( 21 vs. 23% , p = 0.72 ) ( table 2 ) . overall , primary cardiac events trended to be higher in the intermediate-/high - risk group versus the low - risk group ( 28 [ 3.3% ] vs. 4 [ 1.4% ] , p = 0.09 ) . however , primary cardiac event rates in 418 intermediate-/high - risk participants randomized to screening and in the 422 intermediate-/high - risk participants randomized to no screening were similar ( 3.1 and 3.6% ; log rank p = 0.71 ) table 3 ) . * p values are shown for low risk versus intermediate / high risk for framingham and ukpds ; low risk versus high risk for alfediam / sfc ; no versus yes for metabolic syndrome . results of stress mpi in 522 participants randomized to screening , grouped according to various risk stratification schemes data are n ( % ) . cardiac events in intermediate-/high - risk participants randomized to no screening versus screening data are n ( % ) , unless otherwise indicated . of the remaining 1,104 participants , 515 ( 47% ) were categorized as low risk and 589 ( 53% ) as intermediate ( 447 [ 40.5% ] ) or high ( 142 [ 13% ] ) risk ( table 1 ) . the prevalence of abnormal mpi in intermediate-/high - risk and low - risk participants was not different ( 24 vs. 19% , p = 0.2 ) ( table 2 ) . however , the incidence of primary cardiac events was higher in the intermediate-/high - risk group compared with the low - risk group ( 25 [ 4.2% ] vs. 6 [ 1.2% ] , p = 0.002 ) ( table 1 ) . primary cardiac event rates were similar in 291 intermediate-/high - risk participants randomized to screening and in 298 intermediate-/high - risk participants randomized to no screening ( 4.8 vs. 3.7% , log rank p = 0.51 ) ( table 3 ) . of 1,123 participants , 713 ( 63% ) met alfediam / sfc high - risk criteria ( table 1 ) . the prevalence of abnormal mpi in high - risk and low - risk participants was not different ( 23 vs. 19% , p = 0.27 ) ( table 2 ) . the incidence of primary cardiac events was not different in the high- and low - risk groups ( 24 [ 3.4% ] vs. 8 [ 2.0% ] , p = 0.19 ) ( table 1 ) , but secondary event rates were higher in the high - risk than in the low - risk group ( 30 [ 4.2% ] vs. 5 [ 1.2% ] , p = 0.01 ) ( table 1 ) . however , the primary cardiac event rates were similar in 352 high - risk participants randomized to screening and 361 high - risk participants randomized to no screening ( 3.7 vs. 3.1% , log rank p = 0.61 ) ( table 3 ) . the prevalence of abnormal mpi in participants with versus without metabolic syndrome was not different ( 21 vs. 24% , p = 0.49 ) ( table 2 ) . overall , primary cardiac event rates were similar in both groups ( metabolic syndrome 24 [ 3.0% ] vs. no metabolic syndrome 8 [ 2.5% ] , p = 0.67 ) ( table 1 ) . primary cardiac event rates in 398 participants with metabolic syndrome randomized to screening and in 406 participants with metabolic syndrome randomized to no screening were similar ( 2.5 vs. 3.5% , log rank p = 0.42 ) ( table 3 ) . overall , 283 ( 25% ) participants would be defined as having low risk , and 840 ( 75% ) as having intermediate ( 542 [ 48% ] ) or high ( 298 [ 27% ] ) cardiovascular risk ( table 1 ) . of 522 screened participants , 387 ( 74% ) were defined as having intermediate / high risk . the prevalence of abnormal mpi in the screened intermediate-/high - risk versus screened low - risk groups was similar ( 21 vs. 23% , p = 0.72 ) ( table 2 ) . overall , primary cardiac events trended to be higher in the intermediate-/high - risk group versus the low - risk group ( 28 [ 3.3% ] vs. 4 [ 1.4% ] , p = 0.09 ) . however , primary cardiac event rates in 418 intermediate-/high - risk participants randomized to screening and in the 422 intermediate-/high - risk participants randomized to no screening were similar ( 3.1 and 3.6% ; log rank p = 0.71 ) table 3 ) . * p values are shown for low risk versus intermediate / high risk for framingham and ukpds ; low risk versus high risk for alfediam / sfc ; no versus yes for metabolic syndrome . results of stress mpi in 522 participants randomized to screening , grouped according to various risk stratification schemes data are n ( % ) . cardiac events in intermediate-/high - risk participants randomized to no screening versus screening data are n ( % ) , unless otherwise indicated . of the remaining 1,104 participants , 515 ( 47% ) were categorized as low risk and 589 ( 53% ) as intermediate ( 447 [ 40.5% ] ) or high ( 142 [ 13% ] ) risk ( table 1 ) . the prevalence of abnormal mpi in intermediate-/high - risk and low - risk participants was not different ( 24 vs. 19% , p = 0.2 ) ( table 2 ) . however , the incidence of primary cardiac events was higher in the intermediate-/high - risk group compared with the low - risk group ( 25 [ 4.2% ] vs. 6 [ 1.2% ] , p = 0.002 ) ( table 1 ) . primary cardiac event rates were similar in 291 intermediate-/high - risk participants randomized to screening and in 298 intermediate-/high - risk participants randomized to no screening ( 4.8 vs. 3.7% , log rank p = 0.51 ) ( table 3 ) . of 1,123 participants , 713 ( 63% ) met alfediam / sfc high - risk criteria ( table 1 ) . the prevalence of abnormal mpi in high - risk and low - risk participants was not different ( 23 vs. 19% , p = 0.27 ) ( table 2 ) . the incidence of primary cardiac events was not different in the high- and low - risk groups ( 24 [ 3.4% ] vs. 8 [ 2.0% ] , p = 0.19 ) ( table 1 ) , but secondary event rates were higher in the high - risk than in the low - risk group ( 30 [ 4.2% ] vs. 5 [ 1.2% ] , p = 0.01 ) ( table 1 ) . however , the primary cardiac event rates were similar in 352 high - risk participants randomized to screening and 361 high - risk participants randomized to no screening ( 3.7 vs. 3.1% , log rank p = 0.61 ) ( table 3 ) . the prevalence of abnormal mpi in participants with versus without metabolic syndrome was not different ( 21 vs. 24% , p = 0.49 ) ( table 2 ) . overall , primary cardiac event rates were similar in both groups ( metabolic syndrome 24 [ 3.0% ] vs. no metabolic syndrome 8 [ 2.5% ] , p = 0.67 ) ( table 1 ) . primary cardiac event rates in 398 participants with metabolic syndrome randomized to screening and in 406 participants with metabolic syndrome randomized to no screening were similar ( 2.5 vs. 3.5% , log rank p = 0.42 ) ( table 3 ) . this post hoc analysis provides an important perspective on the results of the diad study ( 3 ) by demonstrating that the majority of participants were categorized as being either at intermediate or high cardiovascular risk according to four commonly used cardiac risk - stratification schemes . the ukpds risk engine , specifically designed for type 2 diabetic patients , appeared to best predict the occurrence of cardiac events in diad participants . the study was not powered to determine the effect of screening on outcomes in the subgroup of diad participants categorized as having higher risk ; such analysis would have required a three- to fourfold larger sample size . this analysis expands upon our previous finding that the overall cardiac event rate in asymptomatic patients with type 2 diabetes is lower in the current era than might be predicted based on historical data . the average annual risk of participants in the combined intermediate-/high - risk framingham group was lower ( 0.6% per year ) than predicted ( 12% per year for intermediate risk and > 2% per year for high risk ) . similarly , in the combined intermediate-/high - risk ukpds groups , the risk was also lower ( 0.8% per year ) than predicted ( intermediate 1.53% per year and high risk > thus , even these higher - risk participants had observed cardiac event rates that would traditionally been considered to be low risk . only a small subgroup of 142 high - risk participants defined by the ukpds risk engine had an event rate of 2% per year ( table 1 ) , which might have warranted more aggressive risk - reduction strategies . although 14 of these high - risk participants had primary cardiac events , it is important to note that the majority of events ( 17 of 31 ) occurred in participants who were not categorized as high risk according to the ukpds engine ( table 1 ) . the observation that cardiac event rates in the diad were lower than predicted by either the framingham score or the ukpds risk engine likely reflects the fact that these scoring schemes are based on clinical data collected in the 1970s to 1990s ( 13,14 ) . in the diad study , the majority of participants were aggressively treated with statins , ace inhibitors , and aspirin ( 3 ) . one might hypothesize that these interventions prevented cardiac events in the higher - risk diad participants . neither the presence nor severity of mpi abnormalities was greater in the higher - risk patients . in the diad study , although moderate / large mpi abnormalities were predictive of cardiac events , numerically more than half of the events occurred in the larger cohort of patients with negative screening ( 3 ) . we did not observe an effect of screening on cardiac events in any of the intermediate-/high - risk subgroups . thus , these results buttress the original conclusion of the diad study that screening for inducible ischemia can not be currently advocated in asymptomatic patients with type 2 diabetes . however , because of the limited number of subjects , we can not exclude the possibility that a larger study specifically screening a high - risk subgroup might come to a different conclusion in support of screening . in conclusion , a substantial portion of the diad population would be defined by commonly used risk - stratification schemes as being at intermediate / high cardiovascular risk . nevertheless , even in these higher - risk participants , the annual cardiac event rates were low and outcome was not affected by routine screening for inducible ischemia .

the t cell coreceptors cd4 and cd8 are known to bind class ii and class i molecules directly and to be critical for development and activation of most t cells 123 . however , the function of these molecules in tcr binding to mhc / peptide ligands is unclear . a key role would be anticipated , as coengagement of the tcr and coreceptor greatly enhance t cell responses 14 , and direct participation of the cd8 coreceptor in tcr however , these studies did not determine if the coreceptor functions to facilitate tcr engagement with ligand , enhance signal transduction , or both . indeed , the model in which cd4 and cd8 assist in forming the tcr mhc / peptide interaction has been repeatedly challenged . xu and littman suggested that efficient cd4 coreceptor function required binding of associated lck with previously assembled tcr recent studies using multivalent peptide / class ii mhc ligands have allowed more direct measurement of mhc / peptide binding to the tcr . these reports all reached the same , surprising conclusion : cd4 plays no role in forming a stable tcr interaction with multimeric ligands 8910 . previous work showed that cd4 is important in interactions with agonist but not antagonist mhc / peptide ligands , concluding that a stable tcr interaction induced by agonist ligands was a prerequisite for recruitment of the coreceptor rather than the other way around 1112 . less had been reported on the role of cd8 in tcr mhc / peptide binding . yet recent work using surface plasmon resonance assays failed to detect any enhancement by cd8 of tcr binding to specific class i mhc / peptide ligands 13 . furthermore , cd8 binding to class i mhc molecules is known to be enhanced by activation of tyrosine kinases through the tcr 71415 . these data have lead to the conclusion that cd8 , like cd4 , might participate in t cell responses only after the tcr has already stably bound and been activated by its ligand 713 . finally , several groups have reported analysis of specific t cell populations using mhc / peptide multimers in the presence of anti - cd8 antibodies 16171819 . this shows that multimer binding can occur in the presence of anti - cd8 antibody , although , importantly , the effect of antibody on cd8 function was not determined . thus , a composite model from these studies is that both cd4 and cd8 are recruited into the tcr peptide / mhc complex only after it has stably assembled and had an opportunity to participate in signal transduction 6789101113202122 . recent studies on the role of coreceptors in t cell activation , however , suggest that there may be key differences between the functional role of cd4 and cd8 . using soluble class i mhc / peptide complexes , it was shown that calcium mobilization could be induced by monomeric class i / peptide complexes , providing that cd8 was available 23 . in the absence of cd8 , boniface et al . showed that cd4 was critical for very early activation events ( the acidification response ) induced by mhc / peptide multimers but that even high concentrations of mhc / peptide multimers were unable to induce a sustained calcium flux response 10 . furthermore , even in the presence of cd4 , mhc / peptide monomers failed to induce any class ii these data further support the model in which cd4 has a critical role for class ii restricted t cell activation and mediates this effect after tcr encounter with multimeric mhc / peptide ligand . but the data from delon et al . suggests that cd8 may play a different role , potentiating the response to rare and/or low - affinity ligands 23 . it is difficult to compare these systems , however , as the approach used by delon et al . did not determine if cd8 facilitated binding of the mhc / peptide complex to the tcr or whether the effect of cd8 could be attributed purely to enhanced signal transduction . to address these issues , we studied the role of cd8 in tcr binding and activation using soluble multimeric mhc / peptide ligands . in contrast to the models discussed above , we demonstrate here that cd8 is critical in class i / peptide multimer binding to tcr in two well defined , class i restricted tcr - transgenic systems . indeed , in one of these systems ( ot - i ) , the coreceptor was not only involved in but absolutely required for significant tcr binding to multimeric mhc / peptide ligands . furthermore , we found that class i multimers , but not monomers , were capable of inducing rapid calcium mobilization and that this response is dependent on cd8 in both tcr - transgenic systems studied . finally , we showed that different antibodies against cd8 had dramatically different results on multimer binding and t cell activation . specifically , although most anti - cd8 antibodies blocked multimer binding to the t cell , one antibody enhanced specific multimer binding . this enhancement was especially dramatic in the case of multimers containing low - affinity tcr ligands , including tcr antagonists . thus , our data is in contrast with that of other groups , who propose that tcr binding to antagonist ligands is coreceptor independent . 612-wk - old ot - i , ot - i recombination activating gene ( rag)-1 , and 2c mice were generated and maintained under specific pathogen free conditions . major lymph nodes were harvested , and a single - cell suspension was generated as previously described 25 . the majority of ln cells in ot - i rag-1 mice are cd8ot - i tcr , and these cells were used without further purification . for most staining experiments and all activation experiments involving normal ot - i and 2c mice , cd8 cellect column ( cytovax ) , which removes b cells and cd4 cells , thus enriching the population for cd8 and/or cd48 t cells . the peptides ovap ( siinfekl ) , siyp ( siyryygl ) , g4 ( siigfekl ) , and e1 ( eiinfekl ) have been described previously 262728 and were synthesized by research genetics . plasmids encoding k molecules with the bira recognition sequence at the cooh terminus and human 2 m molecules ( gifts of j. altman , emory university , atlanta , ga and e. pamer , yale university , new haven , ct , respectively ) were transformed and overexpressed in escherichia coli . the synthesized proteins were purified from inclusion bodies , denatured , and mixed with the appropriate k binding peptide , and the mixture was allowed to renature in suitable oxidoreductive conditions over 48 h. after biotinylation using bira ( avidity ) , the monomeric mhc / peptide complexes were purified via fast protein liquid chromatography ( fplc ) on a superdex 200 column ( pharmacia biotech , inc . ) . the efficiency of biotinylation was assessed by sequential immunoprecipitation with avidin - conjugated beads ( pierce chemical co. ) , followed by anti - k antibody ( y3)-conjugated cnbr sepharose beads ( sigma chemical co. ) , and was routinely estimated to be > 80% efficient using this method . after concentration of the appropriate - sized fractions , the biotinylated k / peptide complexes were mixed with streptavidin ( sa)pe ( molecular probes , inc . ) or sa allophycocyanin ( pharmingen ) at a 4:1 molar ratio . multimers which eluted in the predicted range of tetramers were purified via a second round of fplc size exclusion . however , in some experiments the unpurified multimer preparation was used for staining with no significant difference in staining profiles . multimers were tested over a range of doses for flow cytometry and were typically used at 10 g / ml . t cells ( 5 10 ) were stained in 50 l of facs buffer ( pbs , 1% fcs , and 3 mm azide ) or rp-10 with 3 mm azide at the temperatures and times indicated in the figures . typically , mhc multimers ( 10 g / ml ) and anti - cd8 antibodies ( 50 g / ml unless stated otherwise ) were added simultaneously . in fig . 4 , ot - i t cells were stained with ova / k multimers for 2 h at 4c , washed twice , and reincubated with facs buffer alone or with saturating concentrations of anti - cd8 fitc - conjugated antibodies . the following anti - cd8 antibodies were used : 3.168 ( rat igm ; reference 29 ) , 53.6.7 ( rat igg2a ; references 30 and 31 ) ( pharmingen ) , and ct - cd8a ( rat igg2a ; caltag labs . ) . the anti - cd8 antibody used was 53.5.8 ( rat igg1 ; references 30 and 31 ; pharmingen ) . cells were analyzed using a becton dickinson facscalibur. for ca flux measurements , the t cells were loaded with indo-1am at 37c as described previously 32 , washed , and then incubated on ice with or without fitc - conjugated anti - cd8 antibodies for 3060 min . after short centrifugation , the cells were resuspended into media at 37c and analyzed immediately on a facsvantage instrument . after establishing a baseline for 1 min , monomeric or multimeric mhc / peptide complexes ( to 10 g / ml ) or as the molecular mass of ova / k sa pe multimers is 400 kd , 10 g / ml represents a molar concentration of 25 nm for the multimer and 100 nm for the ovap molecules within the multimer . thus , the concentration of free ovap added in these experiments was double that of ovap included in the multimer . the cells were quickly mixed and then analyzed by flow cytometry for an additional 715 min , as indicated . data were analyzed using both cellquest ( becton dickinson ) and flowjo ( treestar ) software . 612-wk - old ot - i , ot - i recombination activating gene ( rag)-1 , and 2c mice were generated and maintained under specific pathogen free conditions . major lymph nodes were harvested , and a single - cell suspension was generated as previously described 25 . the majority of ln cells in ot - i rag-1 mice are cd8ot - i tcr , and these cells were used without further purification . for most staining experiments and all activation experiments involving normal ot - i and 2c mice , cd8 cellect column ( cytovax ) , which removes b cells and cd4 cells , thus enriching the population for cd8 and/or cd48 t cells . the peptides ovap ( siinfekl ) , siyp ( siyryygl ) , g4 ( siigfekl ) , and e1 ( eiinfekl ) have been described previously 262728 and were synthesized by research genetics . plasmids encoding k molecules with the bira recognition sequence at the cooh terminus and human 2 m molecules ( gifts of j. altman , emory university , atlanta , ga and e. pamer , yale university , new haven , ct , respectively ) were transformed and overexpressed in escherichia coli . the synthesized proteins were purified from inclusion bodies , denatured , and mixed with the appropriate k binding peptide , and the mixture was allowed to renature in suitable oxidoreductive conditions over 48 h. after biotinylation using bira ( avidity ) , the monomeric mhc / peptide complexes were purified via fast protein liquid chromatography ( fplc ) on a superdex 200 column ( pharmacia biotech , inc . ) . the efficiency of biotinylation was assessed by sequential immunoprecipitation with avidin - conjugated beads ( pierce chemical co. ) , followed by anti - k antibody ( y3)-conjugated cnbr sepharose beads ( sigma chemical co. ) , and was routinely estimated to be > 80% efficient using this method . after concentration of the appropriate - sized fractions , the biotinylated k / peptide complexes were mixed with streptavidin ( sa)pe ( molecular probes , inc . ) or sa allophycocyanin ( pharmingen ) at a 4:1 molar ratio . multimers which eluted in the predicted range of tetramers were purified via a second round of fplc size exclusion . however , in some experiments the unpurified multimer preparation was used for staining with no significant difference in staining profiles . multimers were tested over a range of doses for flow cytometry and were typically used at 10 g / ml . t cells ( 5 10 ) were stained in 50 l of facs buffer ( pbs , 1% fcs , and 3 mm azide ) or rp-10 with 3 mm azide at the temperatures and times indicated in the figures . typically , mhc multimers ( 10 g / ml ) and anti - cd8 antibodies ( 50 g / ml unless stated otherwise ) were added simultaneously . in fig . 4 , ot - i t cells were stained with ova / k multimers for 2 h at 4c , washed twice , and reincubated with facs buffer alone or with saturating concentrations of anti - cd8 fitc - conjugated antibodies . the following anti - cd8 antibodies were used : 3.168 ( rat igm ; reference 29 ) , 53.6.7 ( rat igg2a ; references 30 and 31 ) ( pharmingen ) , and ct - cd8a ( rat igg2a ; caltag labs . ) . the anti - cd8 antibody used was 53.5.8 ( rat igg1 ; references 30 and 31 ; pharmingen ) . cells were analyzed using a becton dickinson facscalibur. for ca flux measurements , the t cells were loaded with indo-1am at 37c as described previously 32 , washed , and then incubated on ice with or without fitc - conjugated anti - cd8 antibodies for 3060 min . after short centrifugation , the cells were resuspended into media at 37c and analyzed immediately on a facsvantage instrument . after establishing a baseline for 1 min , monomeric or multimeric mhc / peptide complexes ( to 10 g / ml ) or as the molecular mass of ova / k sa pe multimers is 400 kd , 10 g / ml represents a molar concentration of 25 nm for the multimer and 100 nm for the ovap molecules within the multimer . thus , the concentration of free ovap added in these experiments was double that of ovap included in the multimer . the cells were quickly mixed and then analyzed by flow cytometry for an additional 715 min , as indicated . data were analyzed using both cellquest ( becton dickinson ) and flowjo ( treestar ) software . we sought to determine if cd8 participates in binding of multivalent mhc / peptide ligands to class i restricted tcrs . ot - i and 2c are tcr - transgenic mouse strains that bear receptors specific for the mouse class i molecule k complexed with ovap and siy , respectively 262728 . using standard procedures , k / peptide multimeric complexes were synthesized bearing each of these peptides 1618 . as direct cd8 binding to k molecules has been reported 33 , it was important to determine if interaction of these multimers with cd8 t cells was dependent on the specificity of the tcr . fresh t cells were isolated from the lymph nodes of ot - i and 2c transgenic mice and stained with mhc / peptide multimers for 1 h at 37c in tissue culture media containing azide to block t cell activation ( conditions derived from crawford et al . ) . these binding assays revealed fine tcr specificity in multimer binding , such that the 2c t cells bound the k / siy multimer but not the ova / k multimer , whereas the reciprocal pattern of binding was observed for ot - i cells ( fig . double staining under the same conditions with the anti - cd8 antibody 53.6.7 revealed that specific multimer staining was preserved in the presence of this antibody . it is important for subsequent experiments to note that there is a population of tcr expressing cd8 t cells in 2c animals ( fig . these cells are known to express the clonotypic tcr at levels similar to the cd8 2c cells ( 343536 ; our data not shown ) . both cd8 and cd8 2c cells stain with the k / siy multimer , although staining intensities differ for these two populations ( fig . we next tested a panel of antibodies that recognize distinct epitopes on the cd8 and - chains to determine their effects on the tcr mhc / peptide interaction . in stark contrast to the results using the 53.6.7 antibody , we found that saturating concentrations of two anti - cd8 antibodies , 3.168 ( fig . 2 a ) and ct - cd8a ( data not shown ) , and the anti - cd8 antibody 53.5.8 ( data not shown ) , showed almost total blockade of k / ova multimer binding to ot - i cells ( fig . 2 a and data not shown ) . furthermore , we noted that 53.6.7 actually enhanced binding over that of the multimer without anti - cd8 ( fig . the enhancing effect of 53.6.7 and blocking by the other cd8 antibodies was titratable and covered a similar dose range ( fig . importantly , no staining of ot - i cells with the noncognate k / siy multimer was observed , regardless of the anti - cd8 antibody used ( fig . this indicates that the anti - cd8 antibodies did not change the fine specificity of multimer binding . also , these results were not influenced by the dose of multimer used , as similar enhancement or blockade was observed using higher or lower doses of the ova / k multimer ( data not shown ) . furthermore , similar profiles of enhancement or blockade were observed using unconjugated or allophycocyanin - conjugated anti - cd8 antibodies , arguing against some artifact of facs compensation ( data not shown ) . we considered that staining the cells at 37c could allow effects such as cd8 and/or tcr capping and internalization to occur . furthermore , it is known that even high concentrations of azide do not prevent ligand - triggered tcr internalization at 37c ( 37 , 38 , and our unpublished observations ) . to control for these phenomena , these conditions prevent antibody - induced internalization and tcr downregulation ( 37 , 38 , and our unpublished observations ) . as shown in fig . 3 , multimer binding was slightly improved with staining at 37 versus 4c , as expected from crawford et al . 9 . however , the effects of the anti - cd8 antibodies were identical under both conditions : the antibody 53.6.7 slightly enhanced multimer binding ( fig . 3 ) , ct - cd8a , and 53.5.8 ( not shown ) all blocked multimer binding completely at both temperatures . similar results were obtained when the cells were stained for longer times at these temperatures ( data not shown ) . these data indicate that the cd8 effects observed are not dependent on t cell activation or tcr / cd8 internalization . however , given the potential complications of tcr and/or cd8 internalization and signaling at higher temperatures , subsequent staining was performed at 4c in the presence of 3 mm azide . using this system as we know that the half - life of the ot - i tcr ova / k complex is relatively short , 39 the binding of individual heads of the ova / k multimer to the tcr is expected to be dynamic rather than static , such that each head of the multimer dissociates and reassociates with tcrs over time . if cd8 was involved in this process , then anti - cd8 antibodies might affect the stability of prebound mhc multimers . to test this , we compared the effects of anti - cd8 antibodies on multimer binding to ot - i cells under two conditions : ( a ) when antibodies and ova / k multimers were added simultaneously ( fig . 4 a ) or ( b ) when ova / k multimers were allowed to bind first and the cells were subsequently incubated with anti - cd8 antibodies ( fig . 4 b ) . in the absence of anti - cd8 antibodies at either step , multimer staining appears quite stable , decreasing only slightly in the second incubation ( compare fig . as expected , multimer staining was greatly decreased when the blocking anti - cd8 antibodies ( 3.168 or ct - cd8a ) were added during multimer binding ( fig . 4 a ) , but there was also significant loss of multimer when these antibodies were added only in the second incubation ( fig . 4 b ) . this displacement effect could be observed kinetically , in that multimer binding was not reduced to the same extent after only 45 min ( rather than 2 h ) of incubation with the blocking anti - cd8 antibodies ( data not shown ) . it is also important to note that the blockade of multimer binding was more efficient when multimer and anti - cd8 antibodies were added simultaneously rather than sequentially ( compare fig . 4a and fig . in contrast to these results , the enhancing anti - cd8 antibody ( 53.6.7 ) did not cause loss of prebound multimer but , on the contrary , appeared to stabilize multimer staining at the level observed at the beginning of the second incubation ( compare fig . 4a and fig . , these data support the model of a dynamic nature of multimer binding to the tcr and suggest that cd8 participates in both the initial association of the tcr with mhc / peptide multimer and the stability of this interaction . as mentioned previously , 2c tcr transgenic mice are interesting in that they develop a natural population of t cells that are positive for the 2c tcr yet are cd8 34 . such t cells are functional , as they can respond to tcr ligand , albeit only at high doses 3536 . these results suggest that 2c cells may be relatively coreceptor independent , and hence we were interested in what role cd8 might play in multimer binding to the 2c receptor . all 2c cells stained specifically with the k / siy multimer in the absence of anti - cd8 antibody , but the profile was bi - modal , with a multimer and a multimer population ( fig . 5 a ) . the percentages of multimer and multimer populations correlate with the percentage of cd8 and cd8 cells , respectively ( data not shown ) , suggesting that cd8 plays a role in multimer binding to the 2c . we confirmed a role for cd8 using anti - cd8 antibodies . staining with 3.168 , ct - cd8a , or 53.5.8 caused disappearance of the k / siy multimer population , this group of cells becoming multimer . in contrast , 53.6.7 did not cause this change in multimer staining ( fig . 5 a ) and , in some experiments , slightly increased the staining on the k / siy multimer population . as in the case of ot - i cells , the staining using nonspecific multimer was not significantly affected by use of any anti - cd8 antibody ( fig . more directly , we analyzed k / siy multimer binding on both the 2c cd8 and cd8 populations , identified using either 53.6.7 or 3.168 ( fig . j ) . multimer binding to the cd8 population was higher than to the cd8 population when 53.6.7 was used to reveal cd8 , whereas cd8 t cells stained with 3.168-bound multimer no better than cd8 2c cells ( fig . , there was a slight enhancement of siy / k multimer staining of the cd8 population when 53.6.7 was used ( compare fig . 5c and fig . d ) . these results are thus analogous to the influence of anti - cd8 antibodies on ot - i tcr binding , with the main difference between the systems being the degree of multimer binding in the absence / blockade of cd8 : negligible in the case of the ot - i receptor , but merely reduced in the case of multimer binding to 2c tcr . indicate that tcr binding to tcr antagonist ligands is coreceptor ( cd4 ) independent 1112 . to characterize the role of cd8 in binding tcr antagonists in our system , we used k / peptide multimers containing altered peptide ligands with known affinity for the ot - i tcr . the variant peptide g4 is a weak agonist / antagonist for ot - i , whereas e1 is an antagonist 263240 . the ot - i affinity for these ligands is known and matches their biological function , with the rank order of affinity being ova > g4>e1 ( reference 39 and alam , s.m . , and n.r.j . gascoigne , personal communication ) . staining ot - i cells with these k / peptide multimeric complexes revealed that the order of affinity matches the intensity of staining , such that multimer complexes containing ova stained more intensely than those containing g4 and the e1 multimer stained only slightly ( but reproducibly ) above the negative control level ( fig . 6 ) . that the rank order of staining mirrors tcr affinity is consistent with data from crawford et al . we next tested the effect of anti - cd8 antibodies on binding of these multimers . 6 ) , ct - cd8a , and 53.5.8 ( not shown ) totally negated binding of all multimeric ligands . in contrast , the enhancing effect of 53.6.7 was observed for all of the specific ot - i ligands and was extremely marked for the low - affinity ligand e1/k , bringing staining with this multimer well above the level of the control ( siy / k ; fig . 6 ) . these data demonstrate that binding of multimers containing low - affinity mhc / peptide ligands is still strongly influenced by cd8 participation . furthermore , these data indicate that use of the enhancing 53.6.7 antibody can significantly augment staining of a multimer containing an antagonist ligand ( e1/k ) . 23 indicated that monomeric mhc / peptide ligands could stimulate a ca flux provided that cd8 was available , whereas data from boniface et al . 10 indicated that even multimeric mhc class ii / peptide ligands fail to induce a sustained ca response . hence , it was possible that our experiments using multimer staining would not be predictive of the capacity of these ligands to activate cd8 t cells . we thus wished to explore the capacity of our class i mhc / peptide monomers and multimers to stimulate naive t cells and study the role of cd8 in such stimulation . as an early activation event that can easily be studied in real time , we focused on induction of ca mobilization measured by flow cytometry . use of fluorochrome - labeled multimers allowed us to study multimer binding in real time and correlate this with ca mobilization . 7 , ot - i t cells bound specific multimers rapidly ( within seconds ) , and this slightly preceded the initiation of a ca flux response . the vast majority of ot - i t cells ( 8291% over three experiments ) participated in robust ca mobilization under these conditions , and the level of intracellular ca did not return to baseline over the time course studied . these data therefore indicate that the ca flux response induced by cognate mhc / peptide multimers is synchronous and sustained . in contrast to these results , nonspecific multimers neither bound nor induced ca mobilization ( fig . 7 ) , and neither did the ot - i tcr antagonist e1/k multimer ( data not shown ) . because the ot - i t cells themselves express k , it was possible that ova peptide was released from the multimeric complexes and presented via t cell t cell interactions . as a control for this , we added double the concentration of free ova peptide used in the multimer sample . 8 a ) , indicating that mhc / peptide multimers were responsible for the activation event . in contrast to previous reports in another class i mhc restricted system 23 , we saw no activation of ca flux by monomeric ova / k ( fig . 8 a ) . we went further to test whether we could induce ot - i t cell activation by multimerization of the monomeric ligands on - the - fly by addition of sa this approach showed a slight but noticeable rise in intracellular ca consistent with a presumably inefficient assembly of ova / k multimers and subsequent ot - i activation ( fig . the inefficiency of this response is to be expected , as multimerization takes several hours in our standard protocol , making the weak ca flux observed even more significant . again , the ova / k multimer induced a strong , sustained ca flux in ot - i cells that was not induced by the control ( siy / k ) multimer ( fig . 8b and fig . this flux was similar in magnitude and duration to that induced by cross - linked anti - cd3 antibody 500.a2 ( fig . 8 d ) . to study the role of cd8 in this process , we pretreated the cells with anti - cd8 antibody , either 3.168 or 53.6.7 . fluorochrome - labeled anti - cd8 antibodies and multimers were used so that staining of the responding t cell populations could be studied during the experiment . activation of the ot - i cells was unaffected by prestaining with the 53.6.7 antibody , whereas activation was completely blocked using 3.168 ( fig . 8b and fig . it was possible that pretreatment with 3.168 was inducing more than simple blockade of multimer binding . this was a special concern , as anti - cd8treated cells frequently showed slight elevations in basal levels of intracellular ca not observed with unstained control populations ( fig . however , pretreatment of ot - i cells with the blocking 3.168 antibody had no effect on the response to anti - cd3 ( fig . 8 d ) , which argues against a general inhibitory effect of the anti - cd8 antibody . anti - cd3 antibody cross - linking was able to induce ca flux in both populations of cd8 and cd8 2c cells ( data not shown ) and , as for ot - i cells , activation of 2c cells was observed only using the specific multimer ( in this case siy / k ; fig . intriguingly , from preliminary experiments it was evident that multimer induced activation failed to stimulate all of the 2c cells . to determine if there was a difference in the capacity of cd8 and cd8 populations to respond to siy / k multimers , we used the 53.6.7 antibody to separate these subsets . the cd8 population failed to respond to siy / k multimer stimulation , whereas the cd8 population responded well ( fig . 8 e ) . in the presence of 3.168 , however , neither the cd8 nor cd8 populations responded ( fig . over the entire time course , there was a slight rise in intracellular ca in the cd8 population ( fig . 8e and fig . f ) , and we are investigating the possibility that the response of cd8 cells is kinetically delayed . in any case , the effect of cd8 on t cell activation by multimeric ligands mirrored the staining profile , with the important exception that , although cd8 2c cells can bind well to the siy / k multimer in the staining protocol , they do not respond efficiently to it by ca flux . we also studied a later activation parameter , the upregulation of cd69 , which was induced after 3 h by agonist ligands ( ova / k in the case of ot - i and siy / k for 2c cells ) but not by noncognate or nonagonistic multimeric ligands ( data not shown ) . these data are in keeping with the results of the ca flux experiments and suggest that stimulation with cognate mhc / peptide multimers is capable of inducing new gene transcription . the production of synthetic mhc / peptide multimers has caused a revolution in t cell biology , allowing detection of antigen - specific t cell populations by using increased avidity to compensate for the very low affinity of tcrs for mhc / peptide ligands 1641 . although there have been numerous papers published showing the capacity of multimers to bind antigen - specific tcrs , there has been comparatively little analysis of the role of the coreceptors in binding and t cell activation by these multimeric ligands . a few reports using multimeric class ii mhc / peptide ligands to analyze the minimal requirements for tcr binding and t cell activation reached the unanimous conclusion that the cd4 coreceptor is critical for activation of proximal signal transduction events but not required at all for tcr binding to either dimeric or multimeric mhc / peptide ligands 8910 . our data using class i mhc / peptide multimers differ from these results in several key ways . first , we observe that tcr binding to cognate class i multimers is highly cd8 dependent , as shown by blockade with anti - cd8 antibodies and analysis of cd8 class i restricted t cells . the degree of cd8 dependence differed between the two tcr systems studied , but in both cases the multimer interaction was profoundly influenced by cd8 participation . 10 , we found that ca flux was efficiently induced by low doses of class i / peptide multimers . moreover , the size of the responding population and uniformly raised levels of intracellular ca ( fig . 7 ) suggest that this is a sustained ca flux response , rather than the transient partial agonist like response observed by boniface et al . however , using the flow cytometric approach it is difficult to determine the extent of ca oscillations in individual cells , so accurate resolution of this question will require single - cell analysis . our functional assay data also differs from that of delon et al . and abastado et al . , who showed that monomeric class i mhc / peptide complexes induced sustained ca flux , provided that cd8 was accessible , whereas dimeric mhc / peptide complexes could activate even in the absence of cd8 2324 . in contrast , we found that mhc / peptide monomers fail to activate ca flux and that the activation induced by multimeric mhc / peptide complexes is still highly cd8 dependent . we also showed that generation of multimers from monomer ligands during the time course of the ca flux experiment could induce ot - i t cell activation , albeit inefficiently . in comparing our results with those of delon 23 , it is important to note that they studied primed ctls , whereas we describe responses of naive t cells . the profound effect of cd8 on ova / k multimer binding to ot - i was initially unexpected , as the current literature suggests that multimeric mhc / peptide ligands bind efficiently to the tcr alone and because the affinity of the ot - i receptor is in the same range as class ii mhc restricted tcrs , which evidently do not require coreceptor participation for binding 89104142 . we were concerned that anti - cd8 antibody binding may indirectly influence accessibility to the tcr , for example through some consequence of t cell activation or by induction of tcr internalization . this is unlikely given the consistency in results when parameters such as ig isotype , temperature , and duration of staining and the presence or absence of azide were varied . furthermore , expression of tcrs as assessed by cd3 staining was unaffected by exposure to anti - cd8 antibodies ( data not shown ) , arguing against modulation of the tcr in these experiments . perhaps most convincing are the parallel results obtained using the 2c system , in which multimer staining was not lost after anti - cd8 blockade but merely reduced to approximately the same level as naturally occurring cd8 2c cells . 5 , who showed cd8 dependence for tcr binding to monomeric mhc / peptide ligands , suggests that our results are not an artifact of using multimeric tcr ligands . instead , we are left with the idea that the inherent affinity of the ot - i tcr is insufficient to allow even multimeric ligand binding if cd8 participation is blocked . further work will be required to determine how this obligate role for cd8 for ot - i tcr engagement by multimers is mediated . an interesting result in this context was the capacity of blocking anti - cd8 antibodies to induce loss of prebound mhc / peptide multimers ( fig . this suggests that the mhc / peptide tcr interaction is dynamic in nature and that stable multimer binding reflects a series of tcr ligand release and rebinding , which is influenced by cd8 . in keeping with this , the enhancing antibody 53.6.72 appeared to stabilize prebound mhc / peptide multimer ( fig . 4 ) , raising the possibility that binding by this antibody lengthens the half - life of the mhc / peptide tcr ( cd8 ) interaction . thus , we show that cumulative multimer binding is enhanced by cd8 but can be reversed by sustained blockade of certain cd8 combining sites . on the other hand , recent measurements of cell surface associated 2c tcr binding to the siy / k complex indicate that the affinity of this interaction is extremely high 43 . thus , the interaction of this tcr with the siy / k ligand may inherently be less coreceptor dependent . we also demonstrated that cd8 played a critical role in ot - i tcr binding to multimers containing low - affinity , altered peptide ligands . in keeping with the predictions of crawford et al . 9 , we saw a ranking of multimer binding consistent with multimer affinity for the ot - i tcr . however , in our case this was again strongly influenced by manipulation of the coreceptor . most striking is the effect of anti - cd8 on binding to the e1/k , which we previously showed was a low - affinity tcr antagonist 263940 . binding to this multimeric ligand is detectable but weak on normal ot - i cells but is strongly enhanced by the 53.6 antibody , whereas it is completely blocked by the other anti - cd8 antibodies tested . interestingly , previous work had indicated that high levels of cd8 expression could convert this ligand into a weak agonist 40 . our data is in contrast , however , with the conclusion made by madrenas et al . and hampl et al . , who proposed that the cd4 coreceptor plays no role in binding to tcr antagonists 1112 . aside from potential differences between the roles of cd4 and cd8 in antagonist recognition , it is interesting to note that there are also large differences in the ratio of tcr affinity for agonists versus antagonists in these systems . thus , tcr antagonists bind with only three- to fivefold lower affinity than agonists in the ot - i system 39 , but the difference in the 2b4 system used by lyons et al . was 1050-fold 44 . thus , differences in the involvement of the coreceptor in encounter with antagonists may relate to the core tcr affinity for these ligands . this raises the concern , however , that coreceptor participation in antagonist recognition may vary depending on the affinity of the particular tcr and hence may not be generalizable . we conclude that tcr interactions and activation by multimeric mhc / peptide ligands on the surfaces of living cd8 cells typically involve cd8 . these conclusions are similar to those presented by luescher et al . 5 using a k - restricted t hybridoma system in which tcr binding to mhc / peptide monomers could be detected . thus , data from three different tcr systems involving two mhc class i alleles were strikingly similar and imply that this role of cd8 for tcr mhc / peptide interactions can be generalized , at least in the mouse system . an intriguing outcome of these studies was the diverse effect of different antibodies to cd8 . the cd8 antibody 53.6.7 enhanced tcr association by cognate ( but not noncognate ) mhc / peptide ligands , whereas the cd8 antibodies ct - cd8a and 3.168 and the cd8 antibody 53.5.8 all dramatically blocked tcr binding and , correspondingly , t cell activation mhc / peptide binding and others block it . presumably , 53.6.7 favors encounter between cd8 and the tcr or cd8 and class i , whereas the other antibodies block these interactions . incidentally , our results explain why the role of cd8 in multimer binding had not been appreciated until this report : previous studies in the mouse exclusively used the 53.6.7 antibody to stain for cd8 171819 , which would be expected to augment rather than block multimer binding . it is also of interest that the cd8 antibody tested shows efficient blockade of multimer binding . cd8 expression is known to enhance t cell responses 4546 and development 474849 , but it is unclear whether this chain plays a direct role in mhc class i binding or t cell signaling 2350 . interestingly , 53.5.8 does not appear to occlude the cd8 chain , as determined by antibody binding competition 51 , and has only a mild effect on cd8 binding to immobilized class i molecules 5 , implying a minimal role in cd8-mediated adhesion . such a role for cd8 is supported by experiments indicating that cd8 is more efficient than cd8 at association with the tcr 52 . also in support of our observations is a report describing partial blockade by 53.5.8 of multimer binding to polyclonal antigen although siy / k binding was clearly not entirely dependent on cd8 , overt activation of these cells by multimer ligands did require cd8 . this matches well with reports that anti - cd8treated 2c cells and/or cd8 2c cells fail to respond to physiological densities of mhc / peptide antigen expressed on apcs 35 , although this cd8 requirement could be overcome by very high antigen density 36 . thus , our multimeric system appears to mirror the response of 2c to physiological levels of antigen expressed by apcs . 33 , who used surface plasmon resonance to show a role for cd8 in enhancing tcr mhc / peptide interactions , including the 2c receptor . 13 , who suggested that cd8 multimers contributed to the evident enhancement of tcr binding observed by garcia et al . as we deliberately used multimeric mhc / peptide ligands in this work , it may not be surprising that we observed a similar enhancing role for cd8 in our system . lastly , a technical consequence of our studies is that the use of cd8 antibodies in flow cytometric analysis can drastically influence tcr binding to mhc / peptide ligands . moreover , our data using low - affinity tcr ligands together with the enhancing anti - cd8 antibody 53.6.7 indicates that significant multimer staining may be seen using ligands that fail to induce a functional response . human cd8 antibodies will exert , but the effects documented here raise a cautionary note .

recent data using mhc / peptide tetramers and dimers suggests that the t cell coreceptors , cd4 and cd8 , although important for t cell activation , do not play a direct role in facilitating t cell receptor ( tcr ) binding to multivalent mhc / peptide ligands . instead , a current model proposes that coreceptors are recruited only after a stable tcr mhc / peptide complex has already formed and signaled . in contrast , we show using multimeric class i mhc / peptide ligands that cd8 plays a critical ( in some cases obligatory ) role in antigen - specific tcr binding . t cell activation , measured by calcium mobilization , was induced by multimeric but not monomeric ligands and also showed cd8 dependency . our analysis using anti - cd8 antibodies revealed that binding to different epitopes of cd8 can either block or augment tcr mhc / peptide interaction . these effects on tcr binding to high - affinity agonist ligands were even more pronounced when binding to multimeric low - affinity ligands , including tcr antagonists , was studied . our data have important implications for the role of cd8 in tcr binding to mhc / peptide ligands and in t cell activation . in addition , our results argue against the view that multimeric mhc / peptide ligands bind directly and solely to the tcr ; rather , our data highlight a pivotal contribution of cd8 for this association .

Introduction Materials and Methods Mice and Cells. Peptides and MHC Multimers. Antibodies and Flow Cytometry. Results Discussion

the t cell coreceptors cd4 and cd8 are known to bind class ii and class i molecules directly and to be critical for development and activation of most t cells 123 . however , the function of these molecules in tcr binding to mhc / peptide ligands is unclear . a key role would be anticipated , as coengagement of the tcr and coreceptor greatly enhance t cell responses 14 , and direct participation of the cd8 coreceptor in tcr however , these studies did not determine if the coreceptor functions to facilitate tcr engagement with ligand , enhance signal transduction , or both . indeed , the model in which cd4 and cd8 assist in forming the tcr mhc / peptide interaction has been repeatedly challenged . xu and littman suggested that efficient cd4 coreceptor function required binding of associated lck with previously assembled tcr recent studies using multivalent peptide / class ii mhc ligands have allowed more direct measurement of mhc / peptide binding to the tcr . previous work showed that cd4 is important in interactions with agonist but not antagonist mhc / peptide ligands , concluding that a stable tcr interaction induced by agonist ligands was a prerequisite for recruitment of the coreceptor rather than the other way around 1112 . less had been reported on the role of cd8 in tcr mhc / peptide binding . yet recent work using surface plasmon resonance assays failed to detect any enhancement by cd8 of tcr binding to specific class i mhc / peptide ligands 13 . furthermore , cd8 binding to class i mhc molecules is known to be enhanced by activation of tyrosine kinases through the tcr 71415 . these data have lead to the conclusion that cd8 , like cd4 , might participate in t cell responses only after the tcr has already stably bound and been activated by its ligand 713 . finally , several groups have reported analysis of specific t cell populations using mhc / peptide multimers in the presence of anti - cd8 antibodies 16171819 . thus , a composite model from these studies is that both cd4 and cd8 are recruited into the tcr peptide / mhc complex only after it has stably assembled and had an opportunity to participate in signal transduction 6789101113202122 . recent studies on the role of coreceptors in t cell activation , however , suggest that there may be key differences between the functional role of cd4 and cd8 . using soluble class i mhc / peptide complexes , it was shown that calcium mobilization could be induced by monomeric class i / peptide complexes , providing that cd8 was available 23 . furthermore , even in the presence of cd4 , mhc / peptide monomers failed to induce any class ii these data further support the model in which cd4 has a critical role for class ii restricted t cell activation and mediates this effect after tcr encounter with multimeric mhc / peptide ligand . suggests that cd8 may play a different role , potentiating the response to rare and/or low - affinity ligands 23 . did not determine if cd8 facilitated binding of the mhc / peptide complex to the tcr or whether the effect of cd8 could be attributed purely to enhanced signal transduction . to address these issues , we studied the role of cd8 in tcr binding and activation using soluble multimeric mhc / peptide ligands . in contrast to the models discussed above , we demonstrate here that cd8 is critical in class i / peptide multimer binding to tcr in two well defined , class i restricted tcr - transgenic systems . indeed , in one of these systems ( ot - i ) , the coreceptor was not only involved in but absolutely required for significant tcr binding to multimeric mhc / peptide ligands . furthermore , we found that class i multimers , but not monomers , were capable of inducing rapid calcium mobilization and that this response is dependent on cd8 in both tcr - transgenic systems studied . specifically , although most anti - cd8 antibodies blocked multimer binding to the t cell , one antibody enhanced specific multimer binding . this enhancement was especially dramatic in the case of multimers containing low - affinity tcr ligands , including tcr antagonists . thus , our data is in contrast with that of other groups , who propose that tcr binding to antagonist ligands is coreceptor independent . the following anti - cd8 antibodies were used : 3.168 ( rat igm ; reference 29 ) , 53.6.7 ( rat igg2a ; references 30 and 31 ) ( pharmingen ) , and ct - cd8a ( rat igg2a ; caltag labs . ) for ca flux measurements , the t cells were loaded with indo-1am at 37c as described previously 32 , washed , and then incubated on ice with or without fitc - conjugated anti - cd8 antibodies for 3060 min . after establishing a baseline for 1 min , monomeric or multimeric mhc / peptide complexes ( to 10 g / ml ) or as the molecular mass of ova / k sa pe multimers is 400 kd , 10 g / ml represents a molar concentration of 25 nm for the multimer and 100 nm for the ovap molecules within the multimer . typically , mhc multimers ( 10 g / ml ) and anti - cd8 antibodies ( 50 g / ml unless stated otherwise ) were added simultaneously . for ca flux measurements , the t cells were loaded with indo-1am at 37c as described previously 32 , washed , and then incubated on ice with or without fitc - conjugated anti - cd8 antibodies for 3060 min . after establishing a baseline for 1 min , monomeric or multimeric mhc / peptide complexes ( to 10 g / ml ) or as the molecular mass of ova / k sa pe multimers is 400 kd , 10 g / ml represents a molar concentration of 25 nm for the multimer and 100 nm for the ovap molecules within the multimer . we sought to determine if cd8 participates in binding of multivalent mhc / peptide ligands to class i restricted tcrs . fresh t cells were isolated from the lymph nodes of ot - i and 2c transgenic mice and stained with mhc / peptide multimers for 1 h at 37c in tissue culture media containing azide to block t cell activation ( conditions derived from crawford et al . ) we next tested a panel of antibodies that recognize distinct epitopes on the cd8 and - chains to determine their effects on the tcr mhc / peptide interaction . in stark contrast to the results using the 53.6.7 antibody , we found that saturating concentrations of two anti - cd8 antibodies , 3.168 ( fig . 2 a ) and ct - cd8a ( data not shown ) , and the anti - cd8 antibody 53.5.8 ( data not shown ) , showed almost total blockade of k / ova multimer binding to ot - i cells ( fig . furthermore , we noted that 53.6.7 actually enhanced binding over that of the multimer without anti - cd8 ( fig . this indicates that the anti - cd8 antibodies did not change the fine specificity of multimer binding . furthermore , similar profiles of enhancement or blockade were observed using unconjugated or allophycocyanin - conjugated anti - cd8 antibodies , arguing against some artifact of facs compensation ( data not shown ) . however , the effects of the anti - cd8 antibodies were identical under both conditions : the antibody 53.6.7 slightly enhanced multimer binding ( fig . these data indicate that the cd8 effects observed are not dependent on t cell activation or tcr / cd8 internalization . to test this , we compared the effects of anti - cd8 antibodies on multimer binding to ot - i cells under two conditions : ( a ) when antibodies and ova / k multimers were added simultaneously ( fig . this displacement effect could be observed kinetically , in that multimer binding was not reduced to the same extent after only 45 min ( rather than 2 h ) of incubation with the blocking anti - cd8 antibodies ( data not shown ) . it is also important to note that the blockade of multimer binding was more efficient when multimer and anti - cd8 antibodies were added simultaneously rather than sequentially ( compare fig . , these data support the model of a dynamic nature of multimer binding to the tcr and suggest that cd8 participates in both the initial association of the tcr with mhc / peptide multimer and the stability of this interaction . the percentages of multimer and multimer populations correlate with the percentage of cd8 and cd8 cells , respectively ( data not shown ) , suggesting that cd8 plays a role in multimer binding to the 2c . we confirmed a role for cd8 using anti - cd8 antibodies . these results are thus analogous to the influence of anti - cd8 antibodies on ot - i tcr binding , with the main difference between the systems being the degree of multimer binding in the absence / blockade of cd8 : negligible in the case of the ot - i receptor , but merely reduced in the case of multimer binding to 2c tcr . to characterize the role of cd8 in binding tcr antagonists in our system , we used k / peptide multimers containing altered peptide ligands with known affinity for the ot - i tcr . we next tested the effect of anti - cd8 antibodies on binding of these multimers . in contrast , the enhancing effect of 53.6.7 was observed for all of the specific ot - i ligands and was extremely marked for the low - affinity ligand e1/k , bringing staining with this multimer well above the level of the control ( siy / k ; fig . these data demonstrate that binding of multimers containing low - affinity mhc / peptide ligands is still strongly influenced by cd8 participation . 23 indicated that monomeric mhc / peptide ligands could stimulate a ca flux provided that cd8 was available , whereas data from boniface et al . we thus wished to explore the capacity of our class i mhc / peptide monomers and multimers to stimulate naive t cells and study the role of cd8 in such stimulation . these data therefore indicate that the ca flux response induced by cognate mhc / peptide multimers is synchronous and sustained . in contrast to previous reports in another class i mhc restricted system 23 , we saw no activation of ca flux by monomeric ova / k ( fig . we went further to test whether we could induce ot - i t cell activation by multimerization of the monomeric ligands on - the - fly by addition of sa this approach showed a slight but noticeable rise in intracellular ca consistent with a presumably inefficient assembly of ova / k multimers and subsequent ot - i activation ( fig . to study the role of cd8 in this process , we pretreated the cells with anti - cd8 antibody , either 3.168 or 53.6.7 . fluorochrome - labeled anti - cd8 antibodies and multimers were used so that staining of the responding t cell populations could be studied during the experiment . anti - cd3 antibody cross - linking was able to induce ca flux in both populations of cd8 and cd8 2c cells ( data not shown ) and , as for ot - i cells , activation of 2c cells was observed only using the specific multimer ( in this case siy / k ; fig . to determine if there was a difference in the capacity of cd8 and cd8 populations to respond to siy / k multimers , we used the 53.6.7 antibody to separate these subsets . in any case , the effect of cd8 on t cell activation by multimeric ligands mirrored the staining profile , with the important exception that , although cd8 2c cells can bind well to the siy / k multimer in the staining protocol , they do not respond efficiently to it by ca flux . the production of synthetic mhc / peptide multimers has caused a revolution in t cell biology , allowing detection of antigen - specific t cell populations by using increased avidity to compensate for the very low affinity of tcrs for mhc / peptide ligands 1641 . although there have been numerous papers published showing the capacity of multimers to bind antigen - specific tcrs , there has been comparatively little analysis of the role of the coreceptors in binding and t cell activation by these multimeric ligands . a few reports using multimeric class ii mhc / peptide ligands to analyze the minimal requirements for tcr binding and t cell activation reached the unanimous conclusion that the cd4 coreceptor is critical for activation of proximal signal transduction events but not required at all for tcr binding to either dimeric or multimeric mhc / peptide ligands 8910 . our data using class i mhc / peptide multimers differ from these results in several key ways . first , we observe that tcr binding to cognate class i multimers is highly cd8 dependent , as shown by blockade with anti - cd8 antibodies and analysis of cd8 class i restricted t cells . 10 , we found that ca flux was efficiently induced by low doses of class i / peptide multimers . , who showed that monomeric class i mhc / peptide complexes induced sustained ca flux , provided that cd8 was accessible , whereas dimeric mhc / peptide complexes could activate even in the absence of cd8 2324 . in contrast , we found that mhc / peptide monomers fail to activate ca flux and that the activation induced by multimeric mhc / peptide complexes is still highly cd8 dependent . we also showed that generation of multimers from monomer ligands during the time course of the ca flux experiment could induce ot - i t cell activation , albeit inefficiently . the profound effect of cd8 on ova / k multimer binding to ot - i was initially unexpected , as the current literature suggests that multimeric mhc / peptide ligands bind efficiently to the tcr alone and because the affinity of the ot - i receptor is in the same range as class ii mhc restricted tcrs , which evidently do not require coreceptor participation for binding 89104142 . we were concerned that anti - cd8 antibody binding may indirectly influence accessibility to the tcr , for example through some consequence of t cell activation or by induction of tcr internalization . furthermore , expression of tcrs as assessed by cd3 staining was unaffected by exposure to anti - cd8 antibodies ( data not shown ) , arguing against modulation of the tcr in these experiments . 5 , who showed cd8 dependence for tcr binding to monomeric mhc / peptide ligands , suggests that our results are not an artifact of using multimeric tcr ligands . instead , we are left with the idea that the inherent affinity of the ot - i tcr is insufficient to allow even multimeric ligand binding if cd8 participation is blocked . an interesting result in this context was the capacity of blocking anti - cd8 antibodies to induce loss of prebound mhc / peptide multimers ( fig . this suggests that the mhc / peptide tcr interaction is dynamic in nature and that stable multimer binding reflects a series of tcr ligand release and rebinding , which is influenced by cd8 . 4 ) , raising the possibility that binding by this antibody lengthens the half - life of the mhc / peptide tcr ( cd8 ) interaction . on the other hand , recent measurements of cell surface associated 2c tcr binding to the siy / k complex indicate that the affinity of this interaction is extremely high 43 . we also demonstrated that cd8 played a critical role in ot - i tcr binding to multimers containing low - affinity , altered peptide ligands . most striking is the effect of anti - cd8 on binding to the e1/k , which we previously showed was a low - affinity tcr antagonist 263940 . binding to this multimeric ligand is detectable but weak on normal ot - i cells but is strongly enhanced by the 53.6 antibody , whereas it is completely blocked by the other anti - cd8 antibodies tested . , who proposed that the cd4 coreceptor plays no role in binding to tcr antagonists 1112 . we conclude that tcr interactions and activation by multimeric mhc / peptide ligands on the surfaces of living cd8 cells typically involve cd8 . 5 using a k - restricted t hybridoma system in which tcr binding to mhc / peptide monomers could be detected . thus , data from three different tcr systems involving two mhc class i alleles were strikingly similar and imply that this role of cd8 for tcr mhc / peptide interactions can be generalized , at least in the mouse system . the cd8 antibody 53.6.7 enhanced tcr association by cognate ( but not noncognate ) mhc / peptide ligands , whereas the cd8 antibodies ct - cd8a and 3.168 and the cd8 antibody 53.5.8 all dramatically blocked tcr binding and , correspondingly , t cell activation mhc / peptide binding and others block it . incidentally , our results explain why the role of cd8 in multimer binding had not been appreciated until this report : previous studies in the mouse exclusively used the 53.6.7 antibody to stain for cd8 171819 , which would be expected to augment rather than block multimer binding . cd8 expression is known to enhance t cell responses 4546 and development 474849 , but it is unclear whether this chain plays a direct role in mhc class i binding or t cell signaling 2350 . interestingly , 53.5.8 does not appear to occlude the cd8 chain , as determined by antibody binding competition 51 , and has only a mild effect on cd8 binding to immobilized class i molecules 5 , implying a minimal role in cd8-mediated adhesion . this matches well with reports that anti - cd8treated 2c cells and/or cd8 2c cells fail to respond to physiological densities of mhc / peptide antigen expressed on apcs 35 , although this cd8 requirement could be overcome by very high antigen density 36 . 33 , who used surface plasmon resonance to show a role for cd8 in enhancing tcr mhc / peptide interactions , including the 2c receptor . 13 , who suggested that cd8 multimers contributed to the evident enhancement of tcr binding observed by garcia et al . as we deliberately used multimeric mhc / peptide ligands in this work , it may not be surprising that we observed a similar enhancing role for cd8 in our system . lastly , a technical consequence of our studies is that the use of cd8 antibodies in flow cytometric analysis can drastically influence tcr binding to mhc / peptide ligands . moreover , our data using low - affinity tcr ligands together with the enhancing anti - cd8 antibody 53.6.7 indicates that significant multimer staining may be seen using ligands that fail to induce a functional response .

the human body is potentially so highly nutritious that it can well nourish some bacteria . however , it maintains an iron - restricted condition , as most iron is bound to iron - withholding glycoproteins such as transferrin and lactoferrin , or is sequestered within cells . this low iron - availability provides a non - specific defense mechanism that limits bacterial growth within the human body . conversely , in order for bacteria to grow within the human body , they must be able to acquire iron effectively . for this purpose , most bacteria have developed their specific high - affinity iron - uptake systems . among these systems , the siderophore - mediated iron - uptake system is basic and essential in most bacteria ( 1 , 2 ) . the recent increase in the incidence of infections caused by multidrug - resistant bacteria including staphylococci , underlines the importance of identifying novel preventive or therapeutic agents . bacterial iron - uptake systems are attractive targets for the development of preventive or therapeutic vaccines , especially for multidrug - resistant bacteria . similarly , iron - chelation therapies capable of preventing bacterial iron - uptake have also received attention as attractive novel preventive or therapeutic modalities ( 3 - 7 ) . in vivo iron - availability increases under some pathological conditions . in vivo bacterial growth can be stimulated by increased iron - availability , and conversely , bacterial growth can be suppressed by reducing iron - availability . coagulase - negative staphylococci ( cons ) , especially s. epidermidis , are known to be major causative agents of septicemia in patients who have received anticancer chemotherapy or stem cell transplantation , because serum iron levels are elevated in these patients ( 8 - 10 ) . recently , it was reported that s. epidermidis infections in such patients can be prevented by administering human apotransferrin , which is a major iron - withholding protein ( 11 ) . moreover , it was reported that the growth of coagulase - positive staphylococcus aureus in human serum is suppressed by administrating lactoferrin , another iron - withholding protein ( 12 ) . these findings suggest a possibility that chemical iron - chelating agents can also be used to prevent staphylococcal infections , as well as to improve iron overload in such patients . deferoxamine ( c25h48n6o8ch4o3s ) is the best known hydroxamate siderophore derived from streptomyces pilosus , and is currently being used as the standard parenteral iron - chelator for the treatment of iron - overload ( 13 ) . in addition , deferoxamine is known to have an antimicrobial effect , mainly because it competes with bacteria for available iron ( 14 ) . in vitro studies have demonstrated that deferoxamine has bacteriostatic activity against some pathogenic bacteria including s. epidermidis , especially in the presence of ascorbic acid ( 14 - 18 ) . however , one of the drawbacks of deferoxamine is that some pathogenic bacteria , including yersinia enterocolitica , vibrio vulnificus and s. aureus , are able to utilize the drug for iron - uptake , in the same manner as they use their own siderophores via specific receptors ( 19 - 23 ) . this cautions that deferoxamine therapy in iron - overloaded patients can facilitate fatal infections caused by y. enterocolitica , v. vulnificus and s. aureus . recently , a new synthetic oral iron - chelator deferiprone ( 1,2-dimethyl-3-hydroxypyrid-4-one ) became clinically available ( 24 , 25 ) . deferiprone captures iron in vitro from transferrin , lactoferrin , ferritin , and hemosiderin , and in vivo after its parenteral or intragastric administration , and the effect of deferiprone is similar to or greater than that of parenteral deferoxamine . moreover , deferiprone has been found to inhibit the growths of y. enterocolitica and v. vulnificus capable of utilizing deferoxamine because structurally it is completely unlike deferoxamine ( 20 , 26 ) . however , whether deferiprone can inhibit or facilitate the growth of staphylococci , especially s. aureus , has not been determined yet . accordingly , in this study , we determined the effect of deferiprone on the in vitro growth of staphylococci . s. aureus kctc1927 ( actc6538 ) , s. epidermidis kctc1917 ( actc12228 ) and s. saprophyticus kctc3345 ( actc15305 ) strains were purchased from the korean collection for type cultures ( http://kctc.kribb.re.kr/ ) . cons ( n=25 ) and s. aureus strains ( n=25 ) isolated from various clinical samples were used in this study . production of coagulase was tested using staphaurex plus kits ( murex biotech limited , dartford , u.k . ) . cops included methicillin - susceptible strains ( n=11 ) and methicillin - resistant strains ( n=14 ) . brain heart infusion ( bhi , bd , sparks , md , u.s.a . ) agar was used to subculture all staphylococci . staphylococcal siderophore detection ( ssd ) medium , which was recommended as a minimal medium containing low phosphate and citrate for studying staphylococcal iron - uptake systems ( 27 ) , was used without deferration , because its iron concentration was less than 1 g / dl ( 28 ) . when necessary , 1 m of ferric chloride ( fc ) as a non - transferrin - bound - iron source or 0.5 mg / ml of human holotransferrin ( ht ; 1,200 - 1,600 g of iron per 1 g of protein ) as a transferrin - bound - iron source was added to ssd agars or broths . deferoxamine was purchased from ciba - geigy ( basel , switzerland ) and deferiprone from apotex inc . staphylococci were preconditioned in bhi broth containing 200 m dipyridyl , an iron chelator , at 37 overnight in order to adapt them to iron - restricted conditions and to reduce intracellular iron stores . in order to observe the effect of deferoxamine on staphylococcal growth , about 110 cfu of the preconditioned staphylococci were spread onto ssd agars supplemented with 1 m fc ( or 0.5 mg / ml ht ) . paper discs containing 30 l of deferoxamine solution ( 100 m ) or phosphate - buffered saline were then placed on agar surfaces . because s. aureus strains generally grew more rapidly than cons on ssd agars , we observed s. aureus growths after 24 hr but cons growths after 48 hr . response to deferoxamine was determined by the presence of a growth - stimulatory zone or a growth - inhibitory zone around discs containing deferoxamine , as shown in fig . 1 . staphylococci , preconditioned as above , were inoculated into ssd broths containing 1 m fc ( or 0.5 mg / ml ht ) or into ssd broths containing 1 m fc ( or 0.5 mg / ml ht ) plus 0.5 - 1.5 mm deferiprone at about 110 cfu / ml , and then cultured with vigorous shaking ( 220 rpm ) for 6 hr or 24 hr at 37. during culture , aliquots were withdrawn at appropriate times , and bacterial growths were measured by optical densities at a wavelength of 600 nm ( od600 ) . response to deferiprone was determined by comparing the od600 ratios of strains after culture for 6 hr in broths with / without deferiprone . response was arbitrarily expressed using od ratios , as ; less than 0.8 ( growth inhibition ) , 0.8 - 1.2 ( no response ) and more than 1.2 ( growth stimulation ) . the responses of s. aureus kctc1927 , s. epidermidis kctc1917 , and s. saprophyticus kctc3345 strains are presented as growth kinetics over 24 hr . during culture in ssd broths containing ht , culture supernatants were obtained by centrifuging culture aliquots obtained at appropriate times at 10,000 rpm for 5 min . equal volumes ( 20 l ) of culture supernatants were reacted with sample buffer containing 8 m urea , but not sds or mercaptoethanol , at 37 for 30 min , and then electrophoresed on 5% stacking gel and 6% running gel containing 6 m urea ( 28 ) . proteins were visualized by staining with coomassie brilliant blue r-250 . on 6 m urea - gel , transferrin molecules are separated into four forms according to their iron - saturation levels , i.e. , apoferric ( ap ) , c - terminal monoferric ( mc ) , n - terminal monoferric ( mn ) , and diferric ( df ) forms . s. aureus kctc1927 ( actc6538 ) , s. epidermidis kctc1917 ( actc12228 ) and s. saprophyticus kctc3345 ( actc15305 ) strains were purchased from the korean collection for type cultures ( http://kctc.kribb.re.kr/ ) . cons ( n=25 ) and s. aureus strains ( n=25 ) isolated from various clinical samples were used in this study . production of coagulase was tested using staphaurex plus kits ( murex biotech limited , dartford , u.k . ) . cops included methicillin - susceptible strains ( n=11 ) and methicillin - resistant strains ( n=14 ) . brain heart infusion ( bhi , bd , sparks , md , u.s.a . ) agar was used to subculture all staphylococci . staphylococcal siderophore detection ( ssd ) medium , which was recommended as a minimal medium containing low phosphate and citrate for studying staphylococcal iron - uptake systems ( 27 ) , was used without deferration , because its iron concentration was less than 1 g / dl ( 28 ) . when necessary , 1 m of ferric chloride ( fc ) as a non - transferrin - bound - iron source or 0.5 mg / ml of human holotransferrin ( ht ; 1,200 - 1,600 g of iron per 1 g of protein ) as a transferrin - bound - iron source was added to ssd agars or broths . deferoxamine was purchased from ciba - geigy ( basel , switzerland ) and deferiprone from apotex inc . staphylococci were preconditioned in bhi broth containing 200 m dipyridyl , an iron chelator , at 37 overnight in order to adapt them to iron - restricted conditions and to reduce intracellular iron stores . in order to observe the effect of deferoxamine on staphylococcal growth , about 110 cfu of the preconditioned staphylococci were spread onto ssd agars supplemented with 1 m fc ( or 0.5 mg / ml ht ) . paper discs containing 30 l of deferoxamine solution ( 100 m ) or phosphate - buffered saline were then placed on agar surfaces . because s. aureus strains generally grew more rapidly than cons on ssd agars , we observed s. aureus growths after 24 hr but cons growths after 48 hr . response to deferoxamine was determined by the presence of a growth - stimulatory zone or a growth - inhibitory zone around discs containing deferoxamine , as shown in fig . 1 . staphylococci , preconditioned as above , were inoculated into ssd broths containing 1 m fc ( or 0.5 mg / ml ht ) or into ssd broths containing 1 m fc ( or 0.5 mg / ml ht ) plus 0.5 - 1.5 mm deferiprone at about 110 cfu / ml , and then cultured with vigorous shaking ( 220 rpm ) for 6 hr or 24 hr at 37. during culture , aliquots were withdrawn at appropriate times , and bacterial growths were measured by optical densities at a wavelength of 600 nm ( od600 ) . response to deferiprone was determined by comparing the od600 ratios of strains after culture for 6 hr in broths with / without deferiprone . response was arbitrarily expressed using od ratios , as ; less than 0.8 ( growth inhibition ) , 0.8 - 1.2 ( no response ) and more than 1.2 ( growth stimulation ) . the responses of s. aureus kctc1927 , s. epidermidis kctc1917 , and s. saprophyticus kctc3345 strains are presented as growth kinetics over 24 hr . during culture in ssd broths containing ht , culture supernatants were obtained by centrifuging culture aliquots obtained at appropriate times at 10,000 rpm for 5 min . equal volumes ( 20 l ) of culture supernatants were reacted with sample buffer containing 8 m urea , but not sds or mercaptoethanol , at 37 for 30 min , and then electrophoresed on 5% stacking gel and 6% running gel containing 6 m urea ( 28 ) . proteins were visualized by staining with coomassie brilliant blue r-250 . on 6 m urea - gel , transferrin molecules are separated into four forms according to their iron - saturation levels , i.e. , apoferric ( ap ) , c - terminal monoferric ( mc ) , n - terminal monoferric ( mn ) , and diferric ( df ) forms . we first tested the responses of staphylococci to deferoxamine ( fig . 1 , table 1 ) . the growth of s. aureus kctc1927 was enhanced but those of s. epidermidis kctc1917 and s. saprophyticus kctc3345 were inhibited by deferoxamine . the growths of 15 s. aureus strains were enhanced around discs containing deferoxamine , but the other 10 strains did not respond . in contrast , only two cons ( s. epidermidis ) strains showed ' growth enhancement ' by deferoxamine , whereas the growths of the remaining 23 strains were inhibited . the response of one of the two cons strains to deferoxamine was shown in the fig . similar results were observed when ssd broths containing ht plus deferoxamine were used ( data not shown ) . moreover , the responses of s. aureus strains to deferoxamine were not associated with their susceptibilities to methicillin ( data not shown ) . as shown in the fig . 1 , some s. aureus and cons strains appeared to respond to phosphate - buffered saline alone , indicating that they can use phosphate for iron - uptake . to determine whether deferiprone can inhibit the growths of staphylococci on non - transferrin - bound - iron , we cultured s. aureus kctc1927 , s. epidermidis kctc1917 and s. saprophyticus kctc3345 strains in ssd broths only , in ssd broths containing 1 m fc , or in ssd broths containing 1 m fc plus 0.5 - 1.5 mm deferiprone . the growths of the three strains were stimulated by the addition of fc ( fig . s. aureus kctc1927 grew more actively than s. epidermidis kctc1917 or s. saprophyticus kctc3345 in iron - limited ssd broths or in ssd broths containing fc . deferiprone showed a greater growth - inhibitory effect against s. epidermidis kctc1917 and s. saprophyticus kctc3345 than against s. aureus kctc1927 . at less than 0.5 mm , deferiprone did not affect the growths of all the three staphylococci ; that is to say , it did not facilitate the growths of all the three staphylococci in contrast with deferoxamine ( data not shown ) . to determine whether deferiprone inhibits the growths of s. aureus ( n=25 ) and cons ( n=25 ) strains on non - transferrin - bound - iron , we cultured all 50 staphylococci in ssd broths containing 1 m fc or in ssd broths containing 1 m fc plus 1.5 mm deferiprone at 37 for 6 hr . deferiprone inhibited the growths of all s. aureus and cons strains on non - transferrin - bound - iron regardless of the ability to utilize deferoxamine ( table 2 ) and methicillin susceptibility ( data not shown ) . to determine whether deferiprone can also inhibit the growths of s. aureus kctc1927 , s. epidermidis kctc1917 and s. saprophyticus kctc3345 strains on transferrin - bound - iron , we cultured these strains in ssd broths only , in ssd broths containing 0.5 mg / ml ht or in ssd broths containing 0.5 mg / ml ht plus 0.5 - 1.5 mm deperiprone for 24 hr . the growths of all three strains were stimulated by the addition of ht ( fig . s. aureus kctc1927 grew more actively than s. epidermidis kctc1917 or s. saprophyticus kctc3345 in ssd broths containing ht ( fig . 3 ) , and it also utilized iron from transferrin more efficiently than the other two staphylococci ( fig . , transferrin bands were changed from df to mn or mc to ap forms as the result of the removal of iron from transferrin molecules by staphylococci or deferiprone . the growth stimulations of s. epidermidis kctc1917 and s. saprophyticus kctc3345 by ht were inhibited dose - dependently by deferiprone . in contrast , the growth stimulation of s. aureus kctc1927 by ht was not inhibited by deferiprone . as shown at 0 hr in fig . 4 , deferiprone evidently reduced the iron - saturation level of transferrin by removing iron from diferric transferrin . nevertheless , s. aureus kctc1927 still utilized iron from transferrin more efficiently than the other two staphylococci . to determine whether deferiprone inhibits the growths of s. aureus ( n=25 ) and cons ( n=25 ) strains on transferrin - bound - iron , we cultured all 50 staphylococci in ssd broths containing 0.5 mg / ml ht or in ssd broths containing 0.5 mg / ml ht plus 1.5 mm deferiprone at 37 for 6 hr . in contrast , the growths of all s. aureus strains were not inhibited by deferiprone . hartzen et al . reported that deferoxamine can inhibit the growths of staphylococci , especially in the presence of ascorbic acid ( 16 - 18 ) . however , according to our results , the responses of staphylococci to deferoxamine appear to vary among staphylococcal species and strains . deferoxamine was found to stimulate the growths of a few cons strains as well as of most s. aureus strains ( table 1 ) . our results are supported by recent reports , in which s. aureus was found to utilize deferoxamine for iron - acquisition and growth via a specific atp - binding cassette ( abc ) transporter system ( 23 , 29 ) . one iron - regulated abc transporter has also been reported in s. epidermidis ( 30 ) . however , it is not determined whether this transporter system is involved in the uptake of deferoxamine - iron complex by s. epidermidis . overall , it appears that deferoxamine therapy in patients with iron - overload can facilitate staphylococcal infections , especially s. aureus infections . moreover , deferoxamine is known to stimulate the growths of other pathogenic bacteria including y. enterocolitica and v. vulnificus ( 19 - 22 , 26 ) . these problems severely limit the use of deferoxamine for the treatment of iron - overload , although the drug has been used for some time as a standard iron - chelator for the treatment of iron - overload ( 13 ) . s. aureus appear to possess more efficient iron - uptake systems than cons . in our previous studies ( 31 - 34 ) , s. aureus was found to acquire iron efficiently from partially iron - saturated transferrin as well as highly iron - saturated transferrin , whereas s. epidermidis and s. saprophyticus were able to acquire iron only from highly iron - saturated transferrin , but less efficiently than s. aureus . lindsay et al . found that s. aureus is able to actively grow and produce siderophores in severely iron - limited ssd medium , whereas cons grew poorly and did not produce siderophores ( 27 ) . trivier and courcol reported that s. aureus could grow in severely iron - limited media containing 0.04 m fe whereas other bacteria could not ( 35 ) . martinaho et al . also found that the growth initiation of s. epidermidis is dependent on the presence of readily - available iron , i.e. , non - transferrin - bound - iron ( 10 ) . according to the results of the present study , s. aureus grew more actively in ssd broths containing fc or ht and utilized iron from transferrin more efficiently than s. epidermidis or s. saprophyticus ( fig . 2 - 4 ) . in addition , deferiprone was found to inhibit the growths of all cons strains , but not of s. aureus strains , on transferrin - bound - iron . overall , all these findings indicate that s. aureus possess more efficient iron - uptake systems that enable them to grow more actively in severely iron - limited conditions and to utilize transferrin - bound - iron more efficiently than cons . cons , but not s. aureus , are known to be major causative agents of septicemia in iron - overloaded patients who have received anticancer chemotherapy or stem cell transplantation . cons growth proved to be stimulated by elevated non - transferrin - bound - iron or transferrin - bound iron in such iron - overloaded patients ( 8 , 9 ) . according to our results , deferiprone can inhibit significantly the growths of staphylococci , especially cons , by reducing iron - availability at high doses ; moreover , at least it does not promote the growths of staphylococci , especially s. aureus , at low doses and on transferrin - bound - iron . as shown in fig . 4 , the ability of deferiprone to remove iron from transferrin in vitro and in vivo has been confirmed independently by others under similar conditions ( 36 - 38 ) . accordingly , deferiprone can be useful for the prevention of cons infections along with the treatment of iron - overload by reducing both the level of non - transferrin - bound - iron and the iron - saturation level of transferrin . it has been reported that apotransferrin can prevent s. epidermidis growth in patients with iron - overload ( 11 ) and that lactoferrin or apotransferrin can prevent s. aureus growth in human serum or human peritoneal dialysate ( 12 , 32 ) . however , our results show that s. aureus can still efficiently acquire iron from low iron - saturated transferrin ( fig . 3 , 4 ) . in addition , apotransferrin and lactoferrin are inevitably destroyed in vivo and must be administered intravenously because of the proteineous natures . in contrast , deperiprone is a simple chemical oral agent and thus is free of these limitations of apotransferrin and lactoferrin ( 24 , 25 ) . it was reported that deferiprone can also inhibit the growth of y. enterocolitica by decreasing iron - availability ( 20 ) . in addition , we recently found that deferiprone can also inhibit the growth of v. vulnificus , which causes fatal septicemia with an associated mortality rate of over 50% ( 26 ) . moreover , no one has yet found a bacterium capable of utilizing deferiprone for iron uptake . according to our results , deferiprone did not promote the growths of staphylococci at less than 0.5 mm in contrast with deferoxamine ( data not shown ) . relatively higher doses of deferiprone ( > 0.5 mm ) were required to inhibit the growths of staphylococci than those were required to inhibit the growth of y. enterocolitica or v. vulnificus ( < 0.2 mm ) . this is attributed to the more efficient iron - uptake systems of staphylococci , which enable them to more actively grow under severely iron - limited conditions than y. enterocolitica or v. vulnificus ; the latter two are considered archetypal ferrophilic or iron - sensitive bacteria that require higher levels of available iron for growth than other bacteria ( 26 , 39 ) . ascorbic acid is known to enhance the efficacy of deferiprone as it does for deferoxamine ( 14 - 18 , 40 ) . accordingly , the deferiprone doses required to inhibit the growths of staphylococci may be reduced by administering it in combination with ascorbic acid . also , more potent iron - chelators capable of efficiently inhibiting the growth of staphylococci , especially s. aureus , are required ( 40 ) . overall , the present study indicates that , in contrast with deferoxamine , deferiprone can inhibit the growths of staphylococci , especially cons , by reducing iron - availability at relatively high doses ; moreover , at least it does not promote the growths of staphylococci , especially s. aureus , at relatively low doses . accordingly , it is thought that deferiprone can be a useful iron - chelator to prevent staphylococcal infections , as well as to improve iron overload , in iron - overloaded patients .

the standard iron - chelator deferoxamine is known to prevent the growth of coagulase - negative staphylococci ( cons ) which are major pathogens in iron - overloaded patients . however , we found that deferoxamine rather promotes the growth of coagulase - positive staphylococcus aureus . accordingly , we tested whether deferiprone , a new clinically - available iron - chelator , can prevent the growth of s. aureus strains as well as cons . deferiprone did not at least promote the growth of all s. aureus strains ( n=26 ) and cons ( n=27 ) at relatively low doses ; moreover , it could significantly inhibit the growth of all staphylococci on non - transferrin - bound - iron and the growth of all cons on transferrin - bound iron at relatively high doses . at the same doses , it did not at least promote the growth of all s. aureus strains on transferrin - bound - iron . these findings indicate that deferiprone can be useful to prevent staphylococcal infections , as well as to improve iron overload , in iron - overloaded patients .

INTRODUCTION MATERIALS AND METHODS Bacterial strains, media and reagents Observations of the effect of deferoxamine on staphylococcal growth Observations of the effect of deferiprone on staphylococcal growths 6 M urea-gel electrophoresis RESULTS DISCUSSION

however , it maintains an iron - restricted condition , as most iron is bound to iron - withholding glycoproteins such as transferrin and lactoferrin , or is sequestered within cells . this low iron - availability provides a non - specific defense mechanism that limits bacterial growth within the human body . among these systems , the siderophore - mediated iron - uptake system is basic and essential in most bacteria ( 1 , 2 ) . bacterial iron - uptake systems are attractive targets for the development of preventive or therapeutic vaccines , especially for multidrug - resistant bacteria . similarly , iron - chelation therapies capable of preventing bacterial iron - uptake have also received attention as attractive novel preventive or therapeutic modalities ( 3 - 7 ) . in vivo iron - availability increases under some pathological conditions . in vivo bacterial growth can be stimulated by increased iron - availability , and conversely , bacterial growth can be suppressed by reducing iron - availability . coagulase - negative staphylococci ( cons ) , especially s. epidermidis , are known to be major causative agents of septicemia in patients who have received anticancer chemotherapy or stem cell transplantation , because serum iron levels are elevated in these patients ( 8 - 10 ) . recently , it was reported that s. epidermidis infections in such patients can be prevented by administering human apotransferrin , which is a major iron - withholding protein ( 11 ) . moreover , it was reported that the growth of coagulase - positive staphylococcus aureus in human serum is suppressed by administrating lactoferrin , another iron - withholding protein ( 12 ) . these findings suggest a possibility that chemical iron - chelating agents can also be used to prevent staphylococcal infections , as well as to improve iron overload in such patients . deferoxamine ( c25h48n6o8ch4o3s ) is the best known hydroxamate siderophore derived from streptomyces pilosus , and is currently being used as the standard parenteral iron - chelator for the treatment of iron - overload ( 13 ) . in addition , deferoxamine is known to have an antimicrobial effect , mainly because it competes with bacteria for available iron ( 14 ) . in vitro studies have demonstrated that deferoxamine has bacteriostatic activity against some pathogenic bacteria including s. epidermidis , especially in the presence of ascorbic acid ( 14 - 18 ) . however , one of the drawbacks of deferoxamine is that some pathogenic bacteria , including yersinia enterocolitica , vibrio vulnificus and s. aureus , are able to utilize the drug for iron - uptake , in the same manner as they use their own siderophores via specific receptors ( 19 - 23 ) . this cautions that deferoxamine therapy in iron - overloaded patients can facilitate fatal infections caused by y. enterocolitica , v. vulnificus and s. aureus . recently , a new synthetic oral iron - chelator deferiprone ( 1,2-dimethyl-3-hydroxypyrid-4-one ) became clinically available ( 24 , 25 ) . moreover , deferiprone has been found to inhibit the growths of y. enterocolitica and v. vulnificus capable of utilizing deferoxamine because structurally it is completely unlike deferoxamine ( 20 , 26 ) . however , whether deferiprone can inhibit or facilitate the growth of staphylococci , especially s. aureus , has not been determined yet . accordingly , in this study , we determined the effect of deferiprone on the in vitro growth of staphylococci . s. aureus kctc1927 ( actc6538 ) , s. epidermidis kctc1917 ( actc12228 ) and s. saprophyticus kctc3345 ( actc15305 ) strains were purchased from the korean collection for type cultures ( http://kctc.kribb.re.kr/ ) . cons ( n=25 ) and s. aureus strains ( n=25 ) isolated from various clinical samples were used in this study . cops included methicillin - susceptible strains ( n=11 ) and methicillin - resistant strains ( n=14 ) . agar was used to subculture all staphylococci . when necessary , 1 m of ferric chloride ( fc ) as a non - transferrin - bound - iron source or 0.5 mg / ml of human holotransferrin ( ht ; 1,200 - 1,600 g of iron per 1 g of protein ) as a transferrin - bound - iron source was added to ssd agars or broths . staphylococci were preconditioned in bhi broth containing 200 m dipyridyl , an iron chelator , at 37 overnight in order to adapt them to iron - restricted conditions and to reduce intracellular iron stores . because s. aureus strains generally grew more rapidly than cons on ssd agars , we observed s. aureus growths after 24 hr but cons growths after 48 hr . response to deferoxamine was determined by the presence of a growth - stimulatory zone or a growth - inhibitory zone around discs containing deferoxamine , as shown in fig . response was arbitrarily expressed using od ratios , as ; less than 0.8 ( growth inhibition ) , 0.8 - 1.2 ( no response ) and more than 1.2 ( growth stimulation ) . the responses of s. aureus kctc1927 , s. epidermidis kctc1917 , and s. saprophyticus kctc3345 strains are presented as growth kinetics over 24 hr . s. aureus kctc1927 ( actc6538 ) , s. epidermidis kctc1917 ( actc12228 ) and s. saprophyticus kctc3345 ( actc15305 ) strains were purchased from the korean collection for type cultures ( http://kctc.kribb.re.kr/ ) . cons ( n=25 ) and s. aureus strains ( n=25 ) isolated from various clinical samples were used in this study . production of coagulase was tested using staphaurex plus kits ( murex biotech limited , dartford , u.k . ) cops included methicillin - susceptible strains ( n=11 ) and methicillin - resistant strains ( n=14 ) . staphylococcal siderophore detection ( ssd ) medium , which was recommended as a minimal medium containing low phosphate and citrate for studying staphylococcal iron - uptake systems ( 27 ) , was used without deferration , because its iron concentration was less than 1 g / dl ( 28 ) . when necessary , 1 m of ferric chloride ( fc ) as a non - transferrin - bound - iron source or 0.5 mg / ml of human holotransferrin ( ht ; 1,200 - 1,600 g of iron per 1 g of protein ) as a transferrin - bound - iron source was added to ssd agars or broths . staphylococci were preconditioned in bhi broth containing 200 m dipyridyl , an iron chelator , at 37 overnight in order to adapt them to iron - restricted conditions and to reduce intracellular iron stores . because s. aureus strains generally grew more rapidly than cons on ssd agars , we observed s. aureus growths after 24 hr but cons growths after 48 hr . response to deferoxamine was determined by the presence of a growth - stimulatory zone or a growth - inhibitory zone around discs containing deferoxamine , as shown in fig . response was arbitrarily expressed using od ratios , as ; less than 0.8 ( growth inhibition ) , 0.8 - 1.2 ( no response ) and more than 1.2 ( growth stimulation ) . the responses of s. aureus kctc1927 , s. epidermidis kctc1917 , and s. saprophyticus kctc3345 strains are presented as growth kinetics over 24 hr . on 6 m urea - gel , transferrin molecules are separated into four forms according to their iron - saturation levels , i.e. the growth of s. aureus kctc1927 was enhanced but those of s. epidermidis kctc1917 and s. saprophyticus kctc3345 were inhibited by deferoxamine . the growths of 15 s. aureus strains were enhanced around discs containing deferoxamine , but the other 10 strains did not respond . moreover , the responses of s. aureus strains to deferoxamine were not associated with their susceptibilities to methicillin ( data not shown ) . 1 , some s. aureus and cons strains appeared to respond to phosphate - buffered saline alone , indicating that they can use phosphate for iron - uptake . to determine whether deferiprone can inhibit the growths of staphylococci on non - transferrin - bound - iron , we cultured s. aureus kctc1927 , s. epidermidis kctc1917 and s. saprophyticus kctc3345 strains in ssd broths only , in ssd broths containing 1 m fc , or in ssd broths containing 1 m fc plus 0.5 - 1.5 mm deferiprone . s. aureus kctc1927 grew more actively than s. epidermidis kctc1917 or s. saprophyticus kctc3345 in iron - limited ssd broths or in ssd broths containing fc . at less than 0.5 mm , deferiprone did not affect the growths of all the three staphylococci ; that is to say , it did not facilitate the growths of all the three staphylococci in contrast with deferoxamine ( data not shown ) . to determine whether deferiprone inhibits the growths of s. aureus ( n=25 ) and cons ( n=25 ) strains on non - transferrin - bound - iron , we cultured all 50 staphylococci in ssd broths containing 1 m fc or in ssd broths containing 1 m fc plus 1.5 mm deferiprone at 37 for 6 hr . deferiprone inhibited the growths of all s. aureus and cons strains on non - transferrin - bound - iron regardless of the ability to utilize deferoxamine ( table 2 ) and methicillin susceptibility ( data not shown ) . to determine whether deferiprone can also inhibit the growths of s. aureus kctc1927 , s. epidermidis kctc1917 and s. saprophyticus kctc3345 strains on transferrin - bound - iron , we cultured these strains in ssd broths only , in ssd broths containing 0.5 mg / ml ht or in ssd broths containing 0.5 mg / ml ht plus 0.5 - 1.5 mm deperiprone for 24 hr . s. aureus kctc1927 grew more actively than s. epidermidis kctc1917 or s. saprophyticus kctc3345 in ssd broths containing ht ( fig . 3 ) , and it also utilized iron from transferrin more efficiently than the other two staphylococci ( fig . the growth stimulations of s. epidermidis kctc1917 and s. saprophyticus kctc3345 by ht were inhibited dose - dependently by deferiprone . in contrast , the growth stimulation of s. aureus kctc1927 by ht was not inhibited by deferiprone . nevertheless , s. aureus kctc1927 still utilized iron from transferrin more efficiently than the other two staphylococci . to determine whether deferiprone inhibits the growths of s. aureus ( n=25 ) and cons ( n=25 ) strains on transferrin - bound - iron , we cultured all 50 staphylococci in ssd broths containing 0.5 mg / ml ht or in ssd broths containing 0.5 mg / ml ht plus 1.5 mm deferiprone at 37 for 6 hr . in contrast , the growths of all s. aureus strains were not inhibited by deferiprone . reported that deferoxamine can inhibit the growths of staphylococci , especially in the presence of ascorbic acid ( 16 - 18 ) . however , according to our results , the responses of staphylococci to deferoxamine appear to vary among staphylococcal species and strains . deferoxamine was found to stimulate the growths of a few cons strains as well as of most s. aureus strains ( table 1 ) . our results are supported by recent reports , in which s. aureus was found to utilize deferoxamine for iron - acquisition and growth via a specific atp - binding cassette ( abc ) transporter system ( 23 , 29 ) . one iron - regulated abc transporter has also been reported in s. epidermidis ( 30 ) . however , it is not determined whether this transporter system is involved in the uptake of deferoxamine - iron complex by s. epidermidis . overall , it appears that deferoxamine therapy in patients with iron - overload can facilitate staphylococcal infections , especially s. aureus infections . moreover , deferoxamine is known to stimulate the growths of other pathogenic bacteria including y. enterocolitica and v. vulnificus ( 19 - 22 , 26 ) . these problems severely limit the use of deferoxamine for the treatment of iron - overload , although the drug has been used for some time as a standard iron - chelator for the treatment of iron - overload ( 13 ) . s. aureus appear to possess more efficient iron - uptake systems than cons . in our previous studies ( 31 - 34 ) , s. aureus was found to acquire iron efficiently from partially iron - saturated transferrin as well as highly iron - saturated transferrin , whereas s. epidermidis and s. saprophyticus were able to acquire iron only from highly iron - saturated transferrin , but less efficiently than s. aureus . found that s. aureus is able to actively grow and produce siderophores in severely iron - limited ssd medium , whereas cons grew poorly and did not produce siderophores ( 27 ) . trivier and courcol reported that s. aureus could grow in severely iron - limited media containing 0.04 m fe whereas other bacteria could not ( 35 ) . also found that the growth initiation of s. epidermidis is dependent on the presence of readily - available iron , i.e. , non - transferrin - bound - iron ( 10 ) . according to the results of the present study , s. aureus grew more actively in ssd broths containing fc or ht and utilized iron from transferrin more efficiently than s. epidermidis or s. saprophyticus ( fig . in addition , deferiprone was found to inhibit the growths of all cons strains , but not of s. aureus strains , on transferrin - bound - iron . overall , all these findings indicate that s. aureus possess more efficient iron - uptake systems that enable them to grow more actively in severely iron - limited conditions and to utilize transferrin - bound - iron more efficiently than cons . cons , but not s. aureus , are known to be major causative agents of septicemia in iron - overloaded patients who have received anticancer chemotherapy or stem cell transplantation . cons growth proved to be stimulated by elevated non - transferrin - bound - iron or transferrin - bound iron in such iron - overloaded patients ( 8 , 9 ) . according to our results , deferiprone can inhibit significantly the growths of staphylococci , especially cons , by reducing iron - availability at high doses ; moreover , at least it does not promote the growths of staphylococci , especially s. aureus , at low doses and on transferrin - bound - iron . accordingly , deferiprone can be useful for the prevention of cons infections along with the treatment of iron - overload by reducing both the level of non - transferrin - bound - iron and the iron - saturation level of transferrin . it has been reported that apotransferrin can prevent s. epidermidis growth in patients with iron - overload ( 11 ) and that lactoferrin or apotransferrin can prevent s. aureus growth in human serum or human peritoneal dialysate ( 12 , 32 ) . however , our results show that s. aureus can still efficiently acquire iron from low iron - saturated transferrin ( fig . it was reported that deferiprone can also inhibit the growth of y. enterocolitica by decreasing iron - availability ( 20 ) . in addition , we recently found that deferiprone can also inhibit the growth of v. vulnificus , which causes fatal septicemia with an associated mortality rate of over 50% ( 26 ) . according to our results , deferiprone did not promote the growths of staphylococci at less than 0.5 mm in contrast with deferoxamine ( data not shown ) . relatively higher doses of deferiprone ( > 0.5 mm ) were required to inhibit the growths of staphylococci than those were required to inhibit the growth of y. enterocolitica or v. vulnificus ( < 0.2 mm ) . this is attributed to the more efficient iron - uptake systems of staphylococci , which enable them to more actively grow under severely iron - limited conditions than y. enterocolitica or v. vulnificus ; the latter two are considered archetypal ferrophilic or iron - sensitive bacteria that require higher levels of available iron for growth than other bacteria ( 26 , 39 ) . ascorbic acid is known to enhance the efficacy of deferiprone as it does for deferoxamine ( 14 - 18 , 40 ) . accordingly , the deferiprone doses required to inhibit the growths of staphylococci may be reduced by administering it in combination with ascorbic acid . also , more potent iron - chelators capable of efficiently inhibiting the growth of staphylococci , especially s. aureus , are required ( 40 ) . overall , the present study indicates that , in contrast with deferoxamine , deferiprone can inhibit the growths of staphylococci , especially cons , by reducing iron - availability at relatively high doses ; moreover , at least it does not promote the growths of staphylococci , especially s. aureus , at relatively low doses . accordingly , it is thought that deferiprone can be a useful iron - chelator to prevent staphylococcal infections , as well as to improve iron overload , in iron - overloaded patients .

epithelial ovarian cancer is a disease with poor prognosis , few useful early diagnostic markers , and limited treatment options. chemotherapeutic agents based on platinum derivatives have been widely used to treat a broad range of cancers including epithelial ovarian cancer with some success. generally , platinum compounds are dna damaging agents , and proliferating cancer cells are more prone to being killed by these dna damaging compounds . currently , a platinum- and taxane - based combination regimen remains the standard front - line chemotherapy for ovarian cancer. in advanced disease , the standard treatment following diagnosis ( in the united states ) is maximal surgical debulking followed by paclitaxel / carboplatin chemotherapy to remove residual cancer . the widely adapted protocol is administration of paclitaxel ( 135 mg / m , 24 h infusion ) plus cisplatin ( 75 mg / m ) or paclitaxel ( 175 mg / m , 3 h infusion ) plus carboplatin . these basic regimens can be tailored to many clinical studies by adjusting the dose and duration , sequence and route of administration , and combination of additional agents to maximize the effectiveness of the therapy . unfortunately , intrinsic and acquired resistance to cisplatin / taxane has greatly limited the efficacy of this therapy , . new second - line agents under development and testing , such as gemcitabine , doxorubicin , and topotecan that convey anti - cancer activities via different mechanisms , are being evaluated in clinical trials for treatment of cisplatin - resistant cancer , and some have been adopted for clinical application. these new agents are often found to have incremental effects on improving survival of ovarian cancer patients . the mechanism of drug resistance has been an intense subject of investigation in ovarian cancer . progressive understanding on this topic has been obtained , and strategies to reverse drug - resistance are being tested . resistance to cytotoxic chemotherapy remains a key problem , and most women ultimately die of ovarian cancer . development of additional chemotherapeutic regimens , biological therapeutic agents , and other unique approaches for treatment of ovarian cancer is a high priority . certainly , the therapeutic approaches are expected to be progressively fine - tuned and improved , but no dramatic advance or alternative approaches that are projected to significantly impact the survival rate of ovarian cancer patients are anticipated on the immediate horizon . the idea to use viruses to kill cancer cells may have been around for many years , and the notion of using a harmful agent to treat a dreadful illness was rooted in the thinking of traditional oriental medicine . the first well - known , truly scientific attempt in modern times was in 1996 , when frank mccormick and colleagues conceived the idea of using a mutant adenovirus that lacks the e1b gene , which encodes a protein that inactivates p53 , to treat cancer. they hypothesized that the onyx-15 mutant adenovirus would only replicate in and kill cancer cells , which generally lack the p53 tumor suppressor , and would be attenuated in p53-containing non - cancer cells. the idea was brilliant , but proven incorrect because the mutant virus killed tumor cells preferentially regardless of p53 mutation status . however , for financial and regulatory reasons , human trials of the onyx-15 mutant adenovirus ( onyx pharmaceuticals ) for cancer therapy were aborted before going into phase iii trial in 2000 . nevertheless , an almost identical virus , h101 , was shown to have efficacy in the first cancer viral therapy in humans and achieved some degree of success in november 2005 in china , . further studies and trials showed that there are substantial limitations of the mutant adenovirus as an oncolytic agent , and continuing development and improvement of mutant adenovirus for cancer treatment persists . following the studies of mutant adenovirus as potential cancer therapy , the idea to use particular types of viruses as agents to selectively kill cancer cells firmly took root , , . these viruses , referred to as oncolytic viruses , are capable of replicating in cancer cells but not in normal cells , . a number of viruses that exhibit oncolytic activity , including retrovirus , measles , newcastle disease virus , mumps , influenza , and vesicular stomatitis virus , are under investigation . various labs and small companies are currently studying biology and testing strategies with genetic engineering to optimize the recombinant viruses for cancer therapy . some of these oncolytic viruses are currently in phase i trials or late preclinical development , . certainly , there are many obstacles , both technical and logistical , in the development of oncolytic viral therapy . the established concept and further understanding of biology , along with the ongoing research effort will likely bring us one or more effective cancer oncolytic therapies . as discussed above , the oncolytic virus that is most considered and invested in development as a cancer therapy in the past decades is adenovirus , , . therapy with engineered conditional , replication - competent , oncolytic adenovirus was regarded with much enthusiasm in preclinical studies . however , clinical trials of oncolytic adenovirus encountered several difficult barriers including the lack of tumor - specific viral targeting / infection and clearance of the virus by the immune system , , . in humans , although adenovirus can efficiently infect most mammalian cells through specific receptors , the infectivity seems to be reduced in neoplastic cells , and the majority of adenovirus delivered is sequestered in the liver . while oncolytic adenovirus still has promise , strategies to modify the basic viral structure will be needed to overcome the existing shortcomings . currently , several other viral vectors that appear to lack the intrinsic faults associated with adenoviral vectors are being studied , . one such virus , the vesicular stomatitis virus ( vsv ) , is emerging as a promising new oncolytic vector . with its specificity for transformed cells and its ability to target tumors without accumulating in other organs , vsv may have great promise as an oncolytic agent for treating drug - resistant ovarian cancer . vsv is a negative - stranded rna virus of the rhabdoviridae family , which has more than one hundred members with hosts that include plants , invertebrates , vertebrates , and mammals . vsv can infect a wide spectrum of cell types through an as - yet - unidentified but likely ubiquitous cell surface receptor(s ) . vsv replicates in the cytoplasm of infected cells , but the viral genome does not integrate into the host genome , nor does it have transforming activity , . additionally , the vsv genome can be manipulated to modify properties and to insert and express transgenes , making it suitable to be studied and engineered in the laboratory setting , , . the 11-kb vsv genome encodes 5 proteins : nucleocapsid ( n ) , phosphoprotein ( p ) , large polymerase ( l ) , matrix ( m ) , and surface glycoprotein ( g ) , ( figure 1 ) . the negative sense , single - stranded vsv rna is bound by multiple copies of n protein , forming a bead - on - a - string helical structure , and is encapsulated in a bullet - shaped viral particle approximately 100400 nm long and 45100 nm in diameter . vsv is an enveloped virus coated with multiple copies of trimeric surface glycoprotein g , which is embedded in a lipid membrane . within the lipid capsule , trace amounts of p , l , and m proteins are packaged along for the initiation of viral replication following entry into cells . the green fluorescence protein ( gfp ) or luciferase transgene can be inserted to monitor viral infection and proliferation . b , the five viral proteins function as follows : the glycoprotein ( g ) catalyzes fusion of viral and cell membranes ; the nucleoprotein ( n ) binds the rna and forms an rna - dependent rna - polymerase complex with the phosphoprotein ( p ) and large polymerase protein ( l ) ; and the matrix protein ( m ) has multiple critical roles in viral replication and pathogenesis . vsv particles enter cells through surface glycoprotein g mediated binding to the cell surface , endocytosis , and membrane fusion , . upon entry into the cytoplasm , packaged p and l proteins form an rna - dependent rna polymerase complex with the n protein - bound vsv rna genome and initiate transcription to produce capped and polyadenylated sub - genomic transcripts . the vsv mrna transcripts depend on the host translation machinery , golgi apparatus , and endoplasmic reticulum to synthesize and process the viral proteins . at some point after sufficient viral proteins are produced , the rna - dependent rna polymerase complex starts to generate full - length viral rnas , which are then used as templates to copy the negative strain viral rna genome . the full - length viral rna genome is then bound by multiple copies of the n protein , and the ribonucleic acid complexes are shuttled to plasma membranes . with additional assembly and packaging , functional vsv virions are budded off and released from the cell surface. the entire process of viral replication takes place in the cytoplasm . the m protein plays multiple regulatory roles in viral assembly and pathogenesis. m protein connects the vsv nucleoprotein capsule and cellular plasma membrane in the assembly of viral particles , , and plays a role in the release of the viral particles by budding from the host cells . mutations in the m gene can also produce spherical extracellular viral particles instead of the bullet shape of native vsv , . m protein is also important for modulation of host gene expression , interaction with host cellular signaling pathways , and altering immunosurveillance , , . by itself , m protein can induce death in mammalian cells , and can also modulate cellular signaling pathways to affect the immune response . m protein is not essential for viral replication because m - mutant vsv can still infect and replicate in mammalian cells , albeit with reduced efficiency . m - mutant vsv can also cause prolonged infection of neuronal cells , instead of inducing rapid cell death . vsv can infect essentially all human cells in culture and undergo robust replication in certain ( often cancerous ) cells ; however , vsv is relatively non - pathologic for humans , likely because of the induction of strong immune response to suppress viral replication and amplification , . while vsv is largely asymptomatic for humans , domestic and farm animals can become non - lethally infected , with symptoms such as lesions in the mucous membranes of the mouth and nose , . vsv has also been reported to be neuropathic in mice , following intranasal inoculation and subsequent infection and replication in the central nervous system. vsv infection can be cleared through activation of both the innate and adaptive immune responses ( figure 2 ) . the interferons ( ifn ) are critically important in antiviral innate immunity and are a family of cytokines produced in response to vsv infection. upon vsv infection , the initial immune response against the virus is the activation of the innate immune system and production of interferon beta ( ifn- ) . the released ifn- protein acts in an autocrine or paracrine manner and leads to the activation of ifn - stimulated genes , up - regulation of antigen processing machinery , and activation / maturation of antigen presenting cells ( i.e. , dendritic cells , nk cells , macrophages). signaling via ifn results in suppression of viral gene expression and clearance of infected cells by leukocytes. extensive studies in mutant mice have identified several key pathways and mechanisms involved in clearing vsv . for example , mouse embryonic fibroblasts ( mefs ) and mice deficient in double - stranded rna dependent rna kinase ( pkr ) , signal transducer and activator of transcritpion 1 ( stat1 ) , nuclear factor associated with soluble stranded - rna ( nfar ) , or interferon alpha receptor ( ifnar ) are highly susceptible to infection by vsv. vsv particles enter host cells by binding to a ubiquitous surface receptor and undergoing endocytosis . upon reaching the cytoplasm , the viral negative - strain rna genome undergoes transcription and replication , and viral proteins are produced and packaged with newly replicated viral genomic rna to form new vsv particles that are released outside cells . the pattern recognition receptors retinoic acid inducible gene i ( rig-1 ) , toll - like receptor ( tlr ) , and melanoma differentiation antigen 5 ( mda-5 ) recognize viral rna and activate interferon ( ifn ) response through stimulator of interferon genes ( sting ) and tank binding kinase 1 ( tbki)mediated signaling pathways . double - stranded rna - dependent rna kinase ( pkr ) is an ifn - induced gene and is also activated by double - stranded rna , forming an amplifying circuit . in a second phase of immune response , mice that with components of the innate immune system knocked out , such as ifnar- , pkr- , or stat1-null animals , show lethality around 45 days after infection because of the virus 's ability to replicate efficiently throughout the mouse. for example , pkr plays an important role in the initial immune response to vsv infection by restricting translation of viral mrna , , . pkr is an ifn - inducible serine / threonine protein kinase that becomes autophosphorylated in response to double - stranded rna species . this is the initial control of infection following induction of ifn and ifn - stimulated genes including pkr . however , activation of ifn and pkr alone is not sufficient to completely clear the vsv infection ; the adapted secondary immune responses also play critical roles in vsv clearance . b cell deficient mice that maintain an otherwise intact innate response often show toxicity around 9 or 10 days post infection because of the lack of endogenous circulating antibodies and the production of neutralizing antibodies primarily against the n and g proteins by plasma cells . further , t cell deficient mice died 30 days after infection due to their inability to provide b cells help and generate memory against the virus . these studies indicate that the innate response is a short - term acute maneuver for the initial suppression of vsv replication , and this allows time for subsequent activation of the adaptive immune response for complete viral clearance . the innate immune system relies on several pattern recognition receptors that detect specific pathogen - associated molecular patterns , including non - self nucleic acid such as positive and negative sense single - stranded rna , double - stranded rna and dna , and cpg dna , and subsequently activates an antiviral response characterized by induction of ifns ( figure 2 ) . three specific cellular dna sensing pathways , namely the retinoic acid inducible gene i ( rig - i ) and rig - i like helicase ( rlh ) , toll - like receptor ( tlr ) , and absent in melanoma 2 ( aim2 ) and stimulator of interferon genes ( sting ) pathways , are responsible for indentifying viral infection and activating an antiviral response. activation of the rlh cascade to produce ifn- and - relies on two dexd / h helicases , rig - i and melanoma differentiation antigen 5 ( mda-5 ) , . rig - i recognizes 5-triphosphates on viral rna , such as double - stranded rna intermediates produced during viral replication and those encoded by negative - stranded viruses like vsv , . mda-5 recognizes longer rnas ( > 1 kb ) that are encoded by positive - stranded viruses such as the picornavirus encephalomyocarditis virus . the recently identified sting and aim2 ( absent in melanoma 2 ) pathway is particularly interesting in that the sting pathway is activated by cellular dna but not rna species. nevertheless , sting is important in response to the single - stranded rna vsv , likely because it mediates the translocation of tank binding kinase 1 ( tbk1 ) and interacts with the rig - i pathways , , . vsv infection occurs through a ubiquitous , undetermined receptor , and thus , vsv has the unique ability to target many different cell types and malignancies and is a promising vector for oncolytic therapy , . in humans or experimental animals , vsv may initially enter and infect cells it encounters , but the activation of both innate and adapted immunity will suppress viral replication and ultimately clear infected cells . thus , the sensitivity of malignant cells to vsv may be caused by their increased sensitivity to apoptosis or , more likely , their intrinsic defects in innate immune signaling pathways that allow the unchecked viral replication . significant progress has been made in understanding the mechanisms of vsv oncolytic activity and selectivity. cancer cells generally have defective immune signaling , , which may be crucial for proliferation and escape from the tumor suppression mechanism of host immunosurveillance . in addition , the defective immune regulatory system , which includes dysregulation of interferon induction and response , is an important factor in regulating vsv replication in cancer cells , . cancer cells have the ability to proliferate continuously because of translation dysregulation , which allows high levels of viral protein production and replication within malignant cells and leads to cell deathi , , . a defective pkr - mediated innate immune signaling pathway , which results in the inability to suppress viral protein translational in cancer cells , is another key factor for vsv replication and oncolytic activity , . other defects in innate immune signaling pathways in cancer cells include alternative splicing of irf3 or myd88 , cpg methylation of irf7 , mutations of cyld , reduced phosphorylation of stat1 , suppressed transcription of ifn - stimulated genes , and mutations in jaks and traf-3. thus , oncolytic viruses can likely kill transformed cells preferentially over normal cells because of defects in both the innate signaling pathways and the translational control systems . the robust production of vsv proteins in cancer cells likely leads to the killing of the malignant cells . m protein produced in cancer cells can bind the nuclear pore , suppress cell cycle progression , and induce apoptosis , . moreover , the released vsv particles can then infect and kill adjacent cancer cells in the tumor , leading to additional activation of host immune response for tumor elimination . vsv 's ability to selectively replicate in and kill malignant but not normal cells has been well established in cultured cells and mouse xenografts of human cancer cells,. vsv has oncolytic activity in a large range of cancer types, , including ovarian cancer cells . mechanistic studies indicate that tumor cells transformed by oncogenic ras , c - myc , or p53 inactivation are susceptible to killing by vsv . the differential susceptibility of normal and ovarian cancer cells to vsv oncolytic activity is unequivocal . when vsv was added to culture cells , only a small fraction of primary ovarian surface epithelial cells were observed to express a low level of vsv - encoded proteins , indicating vsv infection and proliferation . normal ovarian surface epithelial cells maintained a normal growth pattern up to 3 weeks following exposure to vsv . in contrast , all of the ovarian cancer cell lines tested were sensitive to vsv , and the cells died within 3 days of adding vsv to the cultures . while the efficacy of vsv for cancer therapy has been established in preclinical studies , further studies in immune - competent model organisms , rigorous evaluation of safety , and careful documentation of potential toxic side effects may move vsv oncolytic therapy to the next step , a clinical trial in human cancer patients . to that end , targeting of ovarian tumors by vsv was evaluated in an immune - competent wv ( white spotting variant ) mouse model that develops ovarian epithelial tumors spontaneously . wv mice are deficient in ovarian germ cells and initially develop benign ovarian epithelial tumors known as tubular adenomas , which can acquire increasingly neoplastic features in older mice . the in situ ovarian tumor - bearing mice are anatomically correct and more accurately reflect the accessibility of vsv to tumor cells and the potential toxic side effects of oncolytic therapy . this study demonstrated that vsv delivered through various routes in immune - competent mice explicitly targeted ovarian tumors without significantly affecting any other organs or showing observable toxicity . vsv treatment was very effective in eliminating the epithelial component of tumors , leaving only tumor - free ovarian stroma after treatment . likely , expression and replication of vsv proteins induces apoptosis in the infected tumor cells , and the progeny vsv particles released will infect and kill surrounding tumor cells in a manner of amplification . additionally , tumor antigens that are released from cell lysis may be recognized and processed by immune cells . ultimately , the tumor cell antigens are cross - presented to nave t cells , triggering a tumor - specific t cell response that potentially induces immunologic memory against the tumor. indeed , replication - incompetent vsv ( vsv- g ) prompted both antiviral and anti - tumor immune responses , thereby demonstrating the contribution of anti - tumor immune response after vsv exposure . recurrent and drug - resistant ovarian epithelial cancer appears to be a very suitable setting for treatment with oncolytic virus administered into the peritoneal cavity . these ovarian tumors present as numerous small nodules seeded throughout the peritoneum and on the surface of organs . vsv particles infused into the abdomen will likely be able to access , infect , and kill all tumor cells in each small nodule . naturally , vsv is not a human pathogen , hence deliberate inoculation of high dose viral particles to humans in therapy will be a safety concern . one advantage of using vsv for cancer therapy is the widespread tropism to readily infect cells . unlike adenovirus , which is largely sequestered in the liver , vsv can infect most organ and cell types and only proliferate in the susceptible cancer cells upon injection into experimental animals . also , vsv has a low incidence in the human population and is mostly nave for the human adaptive immune system , which is another advantage over adenovirus , a flu - like virus that humans have often encountered and developed immune recognition . thus , injection of vsv for cancer treatment will not cause an immediate immune response to clear the virus , a major limitation of using adenovirus . the oncolytic activity and efficacy of vsv as a cancer therapy are well demonstrated in mouse models , and the important remaining issues are the safety and selectivity . one risk factor associated with vsv is its ability to infect the central nervous system and thereby cause potential neuropathology . vsv infection in neurons may lead to immediate morbidity or even mortality in humans , or it may cause a persistent non - lethal infection as seen in other mammals , presenting as sores on the feet or mucus membranes of the mouth and nose , . one idea for improving the safety of vsv oncolytic therapy is to design recombinant vsv that has reduced virulence in the neuronal system and/or has altered tropism to be selective for tumor cells. strategies include engineering vsv surface protein to bind tumor antigens and including transgenes or manipulating the viral genome to modulate virulence in neuronal cells . upon challenge by high - dose vsv in therapy , the patient may experience a possibly massive immune response and inflammatory reaction that may be very harmful or lethal . thus , host immune and inflammatory responses need to be carefully monitored and modulated during treatment to prevent inflammatory injury . if the immune system kills the virus faster than viral replication and tumor cell killing , one possible solution is to administer drugs to suppress b cells during vsv oncolytic therapy . preclinical studies have established the possibility of using vsv to treat ovarian cancer . while safety is a concern , continuing studies to create and test better vsv strains for safety , efficacy , and selectivity are ongoing . thus , vsv oncolytic therapy may be a very promising approach , especially for recurrent and drug - resistant ovarian cancer for which treatment options have been exhausted . additional studies of the biology of vsv , its oncolytic activity , and its regulation in benign and malignant cells , as well as rigorous testing in additional ovarian cancer animal models for safety and efficacy may bring vsv oncolytic therapy closer for treatment of drug - resistant ovarian cancer patients .

in the last decade , we have gained significant understanding of the mechanism by which vesicular stomatitis virus ( vsv ) specifically kills cancer cells . dysregulation of translation and defective innate immunity are both thought to contribute to vsv oncolysis . safety and efficacy are important objectives to consider in evaluating vsv as a therapy for malignant disease . ongoing efforts may enable vsv virotherapy to be considered in the near future to treat drug - resistant ovarian cancer when other options have been exhausted . in this article , we review the development of vsv as a potential therapeutic approach for recurrent or drug - resistant ovarian cancer .

Current State of Ovarian Cancer Treatment A Brief History of Oncolytic Virus VSV Biology VSV Clearance in Immune-Competent Hosts Mechanistic Basis of VSV Oncolytic Selectivity The Potential of Recombinant VSV in Ovarian Cancer Treatment Barriers to VSV as A Cancer Therapy Remarks

epithelial ovarian cancer is a disease with poor prognosis , few useful early diagnostic markers , and limited treatment options. chemotherapeutic agents based on platinum derivatives have been widely used to treat a broad range of cancers including epithelial ovarian cancer with some success. generally , platinum compounds are dna damaging agents , and proliferating cancer cells are more prone to being killed by these dna damaging compounds . currently , a platinum- and taxane - based combination regimen remains the standard front - line chemotherapy for ovarian cancer. in advanced disease , the standard treatment following diagnosis ( in the united states ) is maximal surgical debulking followed by paclitaxel / carboplatin chemotherapy to remove residual cancer . the widely adapted protocol is administration of paclitaxel ( 135 mg / m , 24 h infusion ) plus cisplatin ( 75 mg / m ) or paclitaxel ( 175 mg / m , 3 h infusion ) plus carboplatin . these basic regimens can be tailored to many clinical studies by adjusting the dose and duration , sequence and route of administration , and combination of additional agents to maximize the effectiveness of the therapy . unfortunately , intrinsic and acquired resistance to cisplatin / taxane has greatly limited the efficacy of this therapy , . new second - line agents under development and testing , such as gemcitabine , doxorubicin , and topotecan that convey anti - cancer activities via different mechanisms , are being evaluated in clinical trials for treatment of cisplatin - resistant cancer , and some have been adopted for clinical application. these new agents are often found to have incremental effects on improving survival of ovarian cancer patients . the mechanism of drug resistance has been an intense subject of investigation in ovarian cancer . progressive understanding on this topic has been obtained , and strategies to reverse drug - resistance are being tested . resistance to cytotoxic chemotherapy remains a key problem , and most women ultimately die of ovarian cancer . development of additional chemotherapeutic regimens , biological therapeutic agents , and other unique approaches for treatment of ovarian cancer is a high priority . certainly , the therapeutic approaches are expected to be progressively fine - tuned and improved , but no dramatic advance or alternative approaches that are projected to significantly impact the survival rate of ovarian cancer patients are anticipated on the immediate horizon . the idea to use viruses to kill cancer cells may have been around for many years , and the notion of using a harmful agent to treat a dreadful illness was rooted in the thinking of traditional oriental medicine . the first well - known , truly scientific attempt in modern times was in 1996 , when frank mccormick and colleagues conceived the idea of using a mutant adenovirus that lacks the e1b gene , which encodes a protein that inactivates p53 , to treat cancer. they hypothesized that the onyx-15 mutant adenovirus would only replicate in and kill cancer cells , which generally lack the p53 tumor suppressor , and would be attenuated in p53-containing non - cancer cells. however , for financial and regulatory reasons , human trials of the onyx-15 mutant adenovirus ( onyx pharmaceuticals ) for cancer therapy were aborted before going into phase iii trial in 2000 . nevertheless , an almost identical virus , h101 , was shown to have efficacy in the first cancer viral therapy in humans and achieved some degree of success in november 2005 in china , . further studies and trials showed that there are substantial limitations of the mutant adenovirus as an oncolytic agent , and continuing development and improvement of mutant adenovirus for cancer treatment persists . following the studies of mutant adenovirus as potential cancer therapy , the idea to use particular types of viruses as agents to selectively kill cancer cells firmly took root , , . these viruses , referred to as oncolytic viruses , are capable of replicating in cancer cells but not in normal cells , . a number of viruses that exhibit oncolytic activity , including retrovirus , measles , newcastle disease virus , mumps , influenza , and vesicular stomatitis virus , are under investigation . various labs and small companies are currently studying biology and testing strategies with genetic engineering to optimize the recombinant viruses for cancer therapy . some of these oncolytic viruses are currently in phase i trials or late preclinical development , . certainly , there are many obstacles , both technical and logistical , in the development of oncolytic viral therapy . the established concept and further understanding of biology , along with the ongoing research effort will likely bring us one or more effective cancer oncolytic therapies . as discussed above , the oncolytic virus that is most considered and invested in development as a cancer therapy in the past decades is adenovirus , , . therapy with engineered conditional , replication - competent , oncolytic adenovirus was regarded with much enthusiasm in preclinical studies . however , clinical trials of oncolytic adenovirus encountered several difficult barriers including the lack of tumor - specific viral targeting / infection and clearance of the virus by the immune system , , . in humans , although adenovirus can efficiently infect most mammalian cells through specific receptors , the infectivity seems to be reduced in neoplastic cells , and the majority of adenovirus delivered is sequestered in the liver . while oncolytic adenovirus still has promise , strategies to modify the basic viral structure will be needed to overcome the existing shortcomings . currently , several other viral vectors that appear to lack the intrinsic faults associated with adenoviral vectors are being studied , . one such virus , the vesicular stomatitis virus ( vsv ) , is emerging as a promising new oncolytic vector . with its specificity for transformed cells and its ability to target tumors without accumulating in other organs , vsv may have great promise as an oncolytic agent for treating drug - resistant ovarian cancer . vsv is a negative - stranded rna virus of the rhabdoviridae family , which has more than one hundred members with hosts that include plants , invertebrates , vertebrates , and mammals . vsv replicates in the cytoplasm of infected cells , but the viral genome does not integrate into the host genome , nor does it have transforming activity , . additionally , the vsv genome can be manipulated to modify properties and to insert and express transgenes , making it suitable to be studied and engineered in the laboratory setting , , . the 11-kb vsv genome encodes 5 proteins : nucleocapsid ( n ) , phosphoprotein ( p ) , large polymerase ( l ) , matrix ( m ) , and surface glycoprotein ( g ) , ( figure 1 ) . the negative sense , single - stranded vsv rna is bound by multiple copies of n protein , forming a bead - on - a - string helical structure , and is encapsulated in a bullet - shaped viral particle approximately 100400 nm long and 45100 nm in diameter . vsv is an enveloped virus coated with multiple copies of trimeric surface glycoprotein g , which is embedded in a lipid membrane . within the lipid capsule , trace amounts of p , l , and m proteins are packaged along for the initiation of viral replication following entry into cells . the green fluorescence protein ( gfp ) or luciferase transgene can be inserted to monitor viral infection and proliferation . b , the five viral proteins function as follows : the glycoprotein ( g ) catalyzes fusion of viral and cell membranes ; the nucleoprotein ( n ) binds the rna and forms an rna - dependent rna - polymerase complex with the phosphoprotein ( p ) and large polymerase protein ( l ) ; and the matrix protein ( m ) has multiple critical roles in viral replication and pathogenesis . upon entry into the cytoplasm , packaged p and l proteins form an rna - dependent rna polymerase complex with the n protein - bound vsv rna genome and initiate transcription to produce capped and polyadenylated sub - genomic transcripts . the full - length viral rna genome is then bound by multiple copies of the n protein , and the ribonucleic acid complexes are shuttled to plasma membranes . with additional assembly and packaging , functional vsv virions are budded off and released from the cell surface. the entire process of viral replication takes place in the cytoplasm . m protein connects the vsv nucleoprotein capsule and cellular plasma membrane in the assembly of viral particles , , and plays a role in the release of the viral particles by budding from the host cells . mutations in the m gene can also produce spherical extracellular viral particles instead of the bullet shape of native vsv , . vsv can infect essentially all human cells in culture and undergo robust replication in certain ( often cancerous ) cells ; however , vsv is relatively non - pathologic for humans , likely because of the induction of strong immune response to suppress viral replication and amplification , . while vsv is largely asymptomatic for humans , domestic and farm animals can become non - lethally infected , with symptoms such as lesions in the mucous membranes of the mouth and nose , . vsv has also been reported to be neuropathic in mice , following intranasal inoculation and subsequent infection and replication in the central nervous system. the interferons ( ifn ) are critically important in antiviral innate immunity and are a family of cytokines produced in response to vsv infection. upon vsv infection , the initial immune response against the virus is the activation of the innate immune system and production of interferon beta ( ifn- ) . the released ifn- protein acts in an autocrine or paracrine manner and leads to the activation of ifn - stimulated genes , up - regulation of antigen processing machinery , and activation / maturation of antigen presenting cells ( i.e. for example , mouse embryonic fibroblasts ( mefs ) and mice deficient in double - stranded rna dependent rna kinase ( pkr ) , signal transducer and activator of transcritpion 1 ( stat1 ) , nuclear factor associated with soluble stranded - rna ( nfar ) , or interferon alpha receptor ( ifnar ) are highly susceptible to infection by vsv. upon reaching the cytoplasm , the viral negative - strain rna genome undergoes transcription and replication , and viral proteins are produced and packaged with newly replicated viral genomic rna to form new vsv particles that are released outside cells . the pattern recognition receptors retinoic acid inducible gene i ( rig-1 ) , toll - like receptor ( tlr ) , and melanoma differentiation antigen 5 ( mda-5 ) recognize viral rna and activate interferon ( ifn ) response through stimulator of interferon genes ( sting ) and tank binding kinase 1 ( tbki)mediated signaling pathways . double - stranded rna - dependent rna kinase ( pkr ) is an ifn - induced gene and is also activated by double - stranded rna , forming an amplifying circuit . in a second phase of immune response , mice that with components of the innate immune system knocked out , such as ifnar- , pkr- , or stat1-null animals , show lethality around 45 days after infection because of the virus 's ability to replicate efficiently throughout the mouse. for example , pkr plays an important role in the initial immune response to vsv infection by restricting translation of viral mrna , , . pkr is an ifn - inducible serine / threonine protein kinase that becomes autophosphorylated in response to double - stranded rna species . however , activation of ifn and pkr alone is not sufficient to completely clear the vsv infection ; the adapted secondary immune responses also play critical roles in vsv clearance . b cell deficient mice that maintain an otherwise intact innate response often show toxicity around 9 or 10 days post infection because of the lack of endogenous circulating antibodies and the production of neutralizing antibodies primarily against the n and g proteins by plasma cells . these studies indicate that the innate response is a short - term acute maneuver for the initial suppression of vsv replication , and this allows time for subsequent activation of the adaptive immune response for complete viral clearance . activation of the rlh cascade to produce ifn- and - relies on two dexd / h helicases , rig - i and melanoma differentiation antigen 5 ( mda-5 ) , . rig - i recognizes 5-triphosphates on viral rna , such as double - stranded rna intermediates produced during viral replication and those encoded by negative - stranded viruses like vsv , . mda-5 recognizes longer rnas ( > 1 kb ) that are encoded by positive - stranded viruses such as the picornavirus encephalomyocarditis virus . nevertheless , sting is important in response to the single - stranded rna vsv , likely because it mediates the translocation of tank binding kinase 1 ( tbk1 ) and interacts with the rig - i pathways , , . in humans or experimental animals , vsv may initially enter and infect cells it encounters , but the activation of both innate and adapted immunity will suppress viral replication and ultimately clear infected cells . thus , the sensitivity of malignant cells to vsv may be caused by their increased sensitivity to apoptosis or , more likely , their intrinsic defects in innate immune signaling pathways that allow the unchecked viral replication . significant progress has been made in understanding the mechanisms of vsv oncolytic activity and selectivity. cancer cells generally have defective immune signaling , , which may be crucial for proliferation and escape from the tumor suppression mechanism of host immunosurveillance . in addition , the defective immune regulatory system , which includes dysregulation of interferon induction and response , is an important factor in regulating vsv replication in cancer cells , . cancer cells have the ability to proliferate continuously because of translation dysregulation , which allows high levels of viral protein production and replication within malignant cells and leads to cell deathi , , . a defective pkr - mediated innate immune signaling pathway , which results in the inability to suppress viral protein translational in cancer cells , is another key factor for vsv replication and oncolytic activity , . other defects in innate immune signaling pathways in cancer cells include alternative splicing of irf3 or myd88 , cpg methylation of irf7 , mutations of cyld , reduced phosphorylation of stat1 , suppressed transcription of ifn - stimulated genes , and mutations in jaks and traf-3. thus , oncolytic viruses can likely kill transformed cells preferentially over normal cells because of defects in both the innate signaling pathways and the translational control systems . the robust production of vsv proteins in cancer cells likely leads to the killing of the malignant cells . m protein produced in cancer cells can bind the nuclear pore , suppress cell cycle progression , and induce apoptosis , . moreover , the released vsv particles can then infect and kill adjacent cancer cells in the tumor , leading to additional activation of host immune response for tumor elimination . vsv has oncolytic activity in a large range of cancer types, , including ovarian cancer cells . mechanistic studies indicate that tumor cells transformed by oncogenic ras , c - myc , or p53 inactivation are susceptible to killing by vsv . the differential susceptibility of normal and ovarian cancer cells to vsv oncolytic activity is unequivocal . when vsv was added to culture cells , only a small fraction of primary ovarian surface epithelial cells were observed to express a low level of vsv - encoded proteins , indicating vsv infection and proliferation . normal ovarian surface epithelial cells maintained a normal growth pattern up to 3 weeks following exposure to vsv . in contrast , all of the ovarian cancer cell lines tested were sensitive to vsv , and the cells died within 3 days of adding vsv to the cultures . while the efficacy of vsv for cancer therapy has been established in preclinical studies , further studies in immune - competent model organisms , rigorous evaluation of safety , and careful documentation of potential toxic side effects may move vsv oncolytic therapy to the next step , a clinical trial in human cancer patients . to that end , targeting of ovarian tumors by vsv was evaluated in an immune - competent wv ( white spotting variant ) mouse model that develops ovarian epithelial tumors spontaneously . the in situ ovarian tumor - bearing mice are anatomically correct and more accurately reflect the accessibility of vsv to tumor cells and the potential toxic side effects of oncolytic therapy . this study demonstrated that vsv delivered through various routes in immune - competent mice explicitly targeted ovarian tumors without significantly affecting any other organs or showing observable toxicity . likely , expression and replication of vsv proteins induces apoptosis in the infected tumor cells , and the progeny vsv particles released will infect and kill surrounding tumor cells in a manner of amplification . additionally , tumor antigens that are released from cell lysis may be recognized and processed by immune cells . indeed , replication - incompetent vsv ( vsv- g ) prompted both antiviral and anti - tumor immune responses , thereby demonstrating the contribution of anti - tumor immune response after vsv exposure . recurrent and drug - resistant ovarian epithelial cancer appears to be a very suitable setting for treatment with oncolytic virus administered into the peritoneal cavity . these ovarian tumors present as numerous small nodules seeded throughout the peritoneum and on the surface of organs . naturally , vsv is not a human pathogen , hence deliberate inoculation of high dose viral particles to humans in therapy will be a safety concern . unlike adenovirus , which is largely sequestered in the liver , vsv can infect most organ and cell types and only proliferate in the susceptible cancer cells upon injection into experimental animals . also , vsv has a low incidence in the human population and is mostly nave for the human adaptive immune system , which is another advantage over adenovirus , a flu - like virus that humans have often encountered and developed immune recognition . thus , injection of vsv for cancer treatment will not cause an immediate immune response to clear the virus , a major limitation of using adenovirus . the oncolytic activity and efficacy of vsv as a cancer therapy are well demonstrated in mouse models , and the important remaining issues are the safety and selectivity . vsv infection in neurons may lead to immediate morbidity or even mortality in humans , or it may cause a persistent non - lethal infection as seen in other mammals , presenting as sores on the feet or mucus membranes of the mouth and nose , . one idea for improving the safety of vsv oncolytic therapy is to design recombinant vsv that has reduced virulence in the neuronal system and/or has altered tropism to be selective for tumor cells. strategies include engineering vsv surface protein to bind tumor antigens and including transgenes or manipulating the viral genome to modulate virulence in neuronal cells . thus , host immune and inflammatory responses need to be carefully monitored and modulated during treatment to prevent inflammatory injury . preclinical studies have established the possibility of using vsv to treat ovarian cancer . while safety is a concern , continuing studies to create and test better vsv strains for safety , efficacy , and selectivity are ongoing . thus , vsv oncolytic therapy may be a very promising approach , especially for recurrent and drug - resistant ovarian cancer for which treatment options have been exhausted . additional studies of the biology of vsv , its oncolytic activity , and its regulation in benign and malignant cells , as well as rigorous testing in additional ovarian cancer animal models for safety and efficacy may bring vsv oncolytic therapy closer for treatment of drug - resistant ovarian cancer patients .

ten subjects with type 2 diabetes and 10 age - matched healthy control subjects participated , all fully informed of the risks and discomforts associated with the experiments . the ethics committee for the copenhagen county approved the study . patients were recruited from steno diabetes center , an outpatient clinic specialized in treating patients with a history of poor glucose control or diabetes complications ; subject characteristics are provided in table 1 . the patients continued their usual medication because antidiabetic , antihypertensive , and lipid - lowering drugs exhibit long - term effects without acute affection of cardiovascular regulation . only insulin ( four participants ) patients with nephropathy or history of angina , acute myocardial infarction , or claudication were excluded . demographic data for diabetic subjects ( n = 10 ) and control subjects ( n = 10 ) data are presented as means se , with the exception of years of diabetes ( median value ) . p - glucose sampled at the beginning of the first intervention because study participants were not fasting . all participants with diabetes were treated with lipid - lowering medication ; one participant was only treated with sulfonyl , nine participants were treated with metformin ( in combination with sulfonyl in four of these ) . all male patients received antihypertensive treatment with ace inhibitors or at - ii antagonist ( in combination with thiazide in three of these and with addition of ca - antagonist in one of these ) . none of the control subjects received medication . as a marker of muscle endothelial function , ach , acetylcholine ; ado , adenosine ; dm , diabetes mellitus ; ex , exercise ; tyr , tyramine . * different from the previous intervention . control subjects were recruited through advertising in local newspapers , had no history of impaired glucose tolerance , and received no medications . on the day of the experiment bmi and leg mass were calculated from whole - body dual energy x - ray absorptiometry scanning ( ge medical systems , fairfield , ct ) . with the participant resting in a supine position , three catheters were placed under local anesthesia in the femoral artery and vein of the right leg and in the femoral artery of the left leg using the seldinger technique . the participants underwent the following protocol : a pretest in which the individual target lbf was determined during 2 min of knee - extensor exercise at 15 w ( 11 ) . lbf was increased in a dose - response manner by infusion of adenosine or atp until lbf matched that obtained during the pre - exercise test . adenosine ( 18.7 mol / ml ; item development ab , stockholm , sweden ) was infused at rates of 0.8 in control subjects and 1.1 mol / min in patients ( p = 0.38 ) , whereas atp ( 1 mol / ml , sigma a7699 ; sigma - aldrich co. , st . louis , mo ) was infused at rates of 0.8 and 0.9 mol / min in control subjects and patients ( p = 0.62 ) , respectively , to increase blood flow to target lbf ( 2.8 l / min ) . this amount of infused atp , sufficient to increase plasma content by an estimated 500 nmol , is within physiologic range ( 12 ) . the vasoconstrictor effect of tyramine ( 5.9 mol / ml , sigma t-2879 ; sigma - aldrich co. ) , which evokes endogenous noradrenaline ( na ) release from sympathetic nerve endings and subsequent postjunctional -adrenergic vasoconstriction , was examined during adenosine ( control ) , atp , and exercise - induced hyperemia ; the latter two were randomized ( 13 ) . tyramine was coinfused during adenosine at rates of 5.4 and 7.4 mol / min in control subjects and patients , respectively ( p = 0.12 ) , to reduce lbf by 50% without affecting arterial blood pressure ( 6 ) . the individual infusion rate of tyramine , resulting in 50% reduction of lbf during adenosine , was used in the following tyramine trials . lbf was calculated from measurements of diameter and blood velocity using the doppler ultrasound method : probe 8c ( vivid 7 ; ge healthcare , little chalfont , buckinghamshire , u.k . ) ( 10,14 ) . lbf represents the average of three measurements obtained at baseline , 4 min after the start of exercise , or 4 min after reaching steady state under infusion of atp , adenosine , or coinfusion of tyramine . pressure transducers ( pressure monitoring kit ; baxter , deerfield , il ) monitored mean arterial pressure ( map ) ; heart rate was determined from an electrocardiogram , with all data continuously recorded using a powerlab system ( adinstruments , sydney , australia ) . -values of all hemodynamic variables were calculated as the difference between baselines immediately before the intervention and steady state during the intervention and analyzed by two - way anova repeated measurements with nucleotides as within - subject factors and control / type 2 diabetes as between - subject factors . the vasodilatory potency of adenosine and atp was similar in control subjects and patients ( 309 54 vs. 250 81 ml/mol atpkg [ p = 0.48 ] and 13.3 1.7 vs. 12.5 4 ml/mol adenosinekg [ p = 0.38 ] ) . during adenosine and atp infusions , lbf increased ninefold in both control subjects and patients to similar levels as during the exercise intervention ( 2.7 0.2 l / min , fig . tyramine infusion reduced lbf during coinfusion with adenosine from 2.6 0.2 to 1.4 0.1 l / min , whereas infusion of the same amount of tyramine during exercise did not reduce lbf in either group ( 2.6 0.25 l / min ) . coinfusion with tyramine during atp infusion reduced lbf ( from 2.9 0.2 to 2.0 0.2 l / min in control subjects and from 2.7 0.3 to 2.2 0.2 l / min in patients ; p = 0.55 for control subjects vs. patients ) ( fig . leg vascular conductance ( lvc ) increased similarly from baseline values of 3 0.4 to 27 2 ml / minmmhg during adenosine and 30 4 ml / minmmhg during atp and decreased similarly during coinfusion of tyramine ( 12 2 during adenosine and 22 3 ml / minmmhg during atp ) . during exercise , lvc increased in both groups to 21 4 ml / minmmhg and was not affected by tyramine coinfusion . map at baseline was slightly higher in the control group , yet not significant and potentially reflecting antihypertensive treatment in the group with diabetes . map increased from baseline ( 101 4 in control subjects vs. 94 4 mmhg in patients ) to similar values during exercise , 120 4 mmhg in both groups . during adenosine infusion , map was unaltered , but during coinfusion with tyramine , map increased in both groups to 107 3 mmhg . atp infusion lowered map to 94 3 in control subjects and 86 4 mmhg in patients . although coinfusion of atp and tyramine did not affect map in control subjects ( 95 4 mmhg ) , map increased ( to 94 4 mmhg ) in patients ( p < 0.05 ) . the arteriovenous differences of o2 content did not differ at baseline ( 92 7 in control subjects vs. 77 9 ml / l in patients , p = 0.24 ) or during the different interventions . during exercise , the arteriovenous difference increased similarly ( 130 6 ml / l ) and coinfusion with tyramine did not alter the values . reflecting the alterations in lbf , with combined infusion of adenosine and tyramine , leg arteriovenous o2 difference increased from 11 2 ml / l with adenosine infusion to 19 3 ml / l with combined adenosine and tyramine and from 14 3 to 18 4 ml / l during atp and atp - tyramine infusion , respectively , with no differences in the groups . o2 delivery was proportional to lbf , and a small difference in baseline lbf was reflected in a baseline difference of o2 delivery ( p = 0.14 ) . however , there were no differences in o2 delivery or uptake during the infusions of adenosine , atp , exercise , or coinfusion of tyramine . heart rate at baseline tended to be higher in patients ( 68 4 in control subjects vs. 79 4 bpm in patients , p = 0.06 ) , but during the interventions , values were similar . in addition , cardiac output differed at baseline ( 4.0 0.4 in control subjects vs. 6.0 0.5 l / min in patients , p = 0.005 ) , but during atp infusion and exercise , values were similar . in contrast , cardiac output increased more in the group with diabetes during adenosine infusion , both with and without tyramine , p = 0.03 ( control subjects vs. patients , both adenosine and coinfusion of adenosine and tyramine ) . at baseline , femoral venous na was different ( 2.6 0.2 in control subjects vs. 1.8 0.2 nmol / l in patients , p = 0.001 ) , but na increased similarly during exercise ( 1.3 0.5 nmol / l , p = 0.98 , fig . the increases during tyramine coinfusion also were similar in the two groups ( 2.2 0.4 for adenosine , 1.5 0.3 for atp , and 3.2 0.5 nmol / l during exercise p = 0.4 to 0.6 ) , as were the increases in venous na adjusted for the individual infusion rates of tyramine ( 0.38 0.08 during adenosine infusion , 0.25 0.04 during atp infusion , and 0.56 0.11 nmol / l during exercise per micromole of tyramine ) . during adenosine infusions , venous na did not change in the two groups , whereas during atp infusion , na increased in the control group ( p = 0.003 ) . this difference was also reflected during combined atp and tyramine infusions , where venous na in the group with diabetes tended to be lower compared with adenosine ( p = 0.053 ) . the flow reduction per micromole of tyramine was similar in the two groups ( 245 40 ml/mol tyramine ) , whereas during atp infusion , the sensitivity to tyramine in terms of flow reduction was lower in the group with diabetes ( 182 27 vs. 92 34 ml/mol tyramine , p = 0.042 ) . the vasodilatory potency of adenosine and atp was similar in control subjects and patients ( 309 54 vs. 250 81 ml/mol atpkg [ p = 0.48 ] and 13.3 1.7 vs. 12.5 4 ml/mol adenosinekg [ p = 0.38 ] ) . during adenosine and atp infusions , lbf increased ninefold in both control subjects and patients to similar levels as during the exercise intervention ( 2.7 0.2 l / min , fig . tyramine infusion reduced lbf during coinfusion with adenosine from 2.6 0.2 to 1.4 0.1 l / min , whereas infusion of the same amount of tyramine during exercise did not reduce lbf in either group ( 2.6 0.25 l / min ) . coinfusion with tyramine during atp infusion reduced lbf ( from 2.9 0.2 to 2.0 0.2 l / min in control subjects and from 2.7 0.3 to 2.2 0.2 l / min in patients ; p = 0.55 for control subjects vs. patients ) ( fig . leg vascular conductance ( lvc ) increased similarly from baseline values of 3 0.4 to 27 2 ml / minmmhg during adenosine and 30 4 ml / minmmhg during atp and decreased similarly during coinfusion of tyramine ( 12 2 during adenosine and 22 3 ml / minmmhg during atp ) . during exercise , lvc increased in both groups to 21 4 ml / minmmhg and was not affected by tyramine coinfusion . map at baseline was slightly higher in the control group , yet not significant and potentially reflecting antihypertensive treatment in the group with diabetes . map increased from baseline ( 101 4 in control subjects vs. 94 4 mmhg in patients ) to similar values during exercise , 120 4 mmhg in both groups . during adenosine infusion , map was unaltered , but during coinfusion with tyramine , map increased in both groups to 107 3 mmhg . atp infusion lowered map to 94 3 in control subjects and 86 4 mmhg in patients . although coinfusion of atp and tyramine did not affect map in control subjects ( 95 4 mmhg ) , map increased ( to 94 4 mmhg ) in patients ( p < 0.05 ) . the arteriovenous differences of o2 content did not differ at baseline ( 92 7 in control subjects vs. 77 9 ml / l in patients , p = 0.24 ) or during the different interventions . during exercise , the arteriovenous difference increased similarly ( 130 6 ml / l ) and coinfusion with tyramine did not alter the values . reflecting the alterations in lbf , with combined infusion of adenosine and tyramine , leg arteriovenous o2 difference increased from 11 2 ml / l with adenosine infusion to 19 3 ml / l with combined adenosine and tyramine and from 14 3 to 18 4 ml / l during atp and atp - tyramine infusion , respectively , with no differences in the groups . o2 delivery was proportional to lbf , and a small difference in baseline lbf was reflected in a baseline difference of o2 delivery ( p = 0.14 ) . however , there were no differences in o2 delivery or uptake during the infusions of adenosine , atp , exercise , or coinfusion of tyramine . heart rate at baseline tended to be higher in patients ( 68 4 in control subjects vs. 79 4 bpm in patients , p = 0.06 ) , but during the interventions , values were similar . in addition , cardiac output differed at baseline ( 4.0 0.4 in control subjects vs. 6.0 0.5 l / min in patients , p = 0.005 ) , but during atp infusion and exercise , values were similar . in contrast , cardiac output increased more in the group with diabetes during adenosine infusion , both with and without tyramine , p = 0.03 ( control subjects vs. patients , both adenosine and coinfusion of adenosine and tyramine ) . at baseline , femoral venous na was different ( 2.6 0.2 in control subjects vs. 1.8 0.2 nmol / l in patients , p = 0.001 ) , but na increased similarly during exercise ( 1.3 0.5 nmol / l , p = 0.98 , fig . the increases during tyramine coinfusion also were similar in the two groups ( 2.2 0.4 for adenosine , 1.5 0.3 for atp , and 3.2 0.5 nmol / l during exercise p = 0.4 to 0.6 ) , as were the increases in venous na adjusted for the individual infusion rates of tyramine ( 0.38 0.08 during adenosine infusion , 0.25 0.04 during atp infusion , and 0.56 0.11 nmol / l during exercise per micromole of tyramine ) . during adenosine infusions , venous na did not change in the two groups , whereas during atp infusion , na increased in the control group ( p = 0.003 ) . this difference was also reflected during combined atp and tyramine infusions , where venous na in the group with diabetes tended to be lower compared with adenosine ( p = 0.053 ) . the flow reduction per micromole of tyramine was similar in the two groups ( 245 40 ml/mol tyramine ) , whereas during atp infusion , the sensitivity to tyramine in terms of flow reduction was lower in the group with diabetes ( 182 27 vs. 92 34 ml/mol tyramine , p = 0.042 ) . the main findings of the current study are that patients with type 2 diabetes and intact endothelial function have a similar capacity to blunt sympathetic vasoconstriction during moderate exercise as healthy age- and bmi - matched control subjects . however , both groups were only partially capable of blunting -adrenergic vasoconstriction during atp - induced hyperemia . in addition , the vasodilatory potency of adenosine and atp did not differ between patients and control subjects . finally , the subjects with diabetes had a lower venous content of na but a similar elevation of venous na during the adenosine infusions , exercise , and tyramine interventions . consistent with a study on young healthy subjects ( 6 ) , exercise fully blunted sympathetic vasoconstriction in both groups , indicating that the ability of functional sympatholysis during moderate exercise was not reduced in the group with diabetes . this may be clinically relevant given that pathology , affecting the vasodilatory function , could be expected to limit skeletal muscle blood flow because of enhanced sympathetic vasoconstriction in the active muscles , thereby potentially reducing exercise capacity . a study on elderly healthy humans ( > 65 years ) demonstrated that aging is associated with a greater vasoconstrictor tone in active muscles during exercise compared with young adults ( 15 ) . the magnitude of functional sympatholysis was significantly lower in older persons compared with young persons in the presence of tyramine , leading the authors to conclude that aging is associated with impaired functional sympatholysis in the vascular beds of contracting forearm muscle . the present finding of intact functional sympatholysis in middle - aged healthy subjects and patients with diabetes could demonstrate that the phenomenon may be age - dependent ; however , limb differences should also be kept in mind ( 16 ) . moreover , a component of systemic limitation to peripheral blood flow during exercise may explain the observed low blood flows in some studies of patients with type 2 diabetes ; the knee - extensor model eliminates this risk and therefore allows studies of the local microcirculation and may not be translated to all skeletal muscles or applied to high - intensity exercise with a large muscle mass but is likely to represent the leg muscles , which accounts for the largest part of vascular resistance in the body and is of particular interest because insulin resistance is primarily present in leg muscles ( 17 ) . in young subjects , luminal atp was shown to abolish tyramine - induced sympathetic vasoconstriction , indicating that atp is contributing to functional sympatholysis via activation of the p2y2 receptor ( 6 ) . in contrast with this finding , both groups in the current study had a reduction in lbf during combined atp and tyramine infusion . the sympatholytic effect of atp is graded and dose - dependent ; during very modest atp infusions , atp had no sympatholytic effect ( 18 ) . however , the present infusion rates of atp increased lbf ninefold , most likely increasing arterial atp to levels sufficient to limit -adrenergic vasoconstriction in young subjects . therefore , the sympatholytic effect of atp may be affected by age . the amount of tyramine leading to 50% reduction in lvc and lbf during adenosine infusion resulted in a 30% reduction during atp ; thus , atp has a role in functional sympatholysis in the elderly , but other factors must be of importance to offset sympathetic vasoconstriction , because there were no changes in lvc or lbf during exercise with coinfusion of tyramine . functional sympatholysis in middle - aged persons could gradually be more dependent on factors other than atp , such as katp channel activation , which opposes -adrenergic vasoconstriction during muscle contraction ( 19 ) . regardless of the compounds responsible for functional sympatholysis , endothelial function also could be of importance . the present findings do not exclude that endothelial dysfunction affects the ability of sympatholysis during exercise . the amount of atp and adenosine needed to increase lbf to levels matching exercise was similar in the two groups . in contrast , in a previous study on patients with diabetes and age - matched control subjects , we found a 50% reduction in the lbf response to atp and adenosine in the group with diabetes ( 10 ) . the lbf response in control groups was identical , whereas the response in the present group with diabetes was higher in regard to both atp and adenosine . when comparing the two groups of patients with diabetes , there were no differences in demographic variables or medication . only the lbf response to acetylcholine infusion was significantly different , indicating that the reduction in effect of purinergic agonist may be proportional to the extent of endothelial dysfunction . a comparison of data from our two studies of middle - aged subjects , both control subjects and patients with diabetes , and previous studies of young subjects ( 20,21 ) demonstrates a decline in lbf response to adenosine in particular , which was reduced fourfold . in regard to atp , there was a clear decline in sensitivity only in the group with more developed endothelial dysfunction . still , it is a possibility that the response to atp also is diminished in an elderly population , possibly to a minor extent , because the dose - response in young healthy subjects may not be linear for the dose interval under study , hampering data extrapolation and direct comparison ( 6 ) . the decline in lbf responses to atp and adenosine may reflect age - related changes of the signaling pathways ( 20,21 ) . however , physical inactivity and obesity also affect endothelial function and cardiovascular regulation and should be of note , because both groups in the current study had moderately elevated bmi and the majority of the subjects had a sedentary lifestyle . the interpretation of plasma levels of na and its correlation to sympathetic activity is complex ; plasma na elevation may not be a precise measure of the effect on msna but merely a reflection of direction of change . it should be noted that during infusion with adenosine alone , there was no increase in venous na , despite a ninefold increase in lbf , indicating that the increase in na was indeed due to tyramine infusion and not to hyperemia . -values of venous na between the two groups were similar during all three tyramine interventions . furthermore , during moderate exercise , both with and without tyramine , the venous na increase in the groups was similar and additive , indicating that the stimulus of exercise to the sympathetic system also may have been uniform . these findings are consistent with previous studies in young subjects , showing that na spillover is a function of active muscle mass and exercise intensity ( 22 ) . in young subjects ( 6 ) , plasma na increased from 1.7 nmol l at baseline to 3 nmol l during tyramine infusions with comparable infusion rates , thus lower than in the present middle - aged control subjects . however , the levels of na in the present group of patients with diabetes were similar to those of young subjects . despite increases in sympathetic nerve activity in patients with diabetes , plasma na level has been found to be reduced compared with control subjects ( 23 ) . therefore , it is of interest to investigate whether the presented differences are a result of antidiabetic treatment or a physiologic response to elevation in sympathetic nerve activity , leading to enhanced reuptake and thus a reduced spillover . circulating atp mimics exercise hyperemia by its vasodilatory potency and by increasing msna and circulating na ( 6 ) . measurements of interstitial na in skeletal muscle during atp infusion with the microdialysis technique showed a dose - dependent increase in interstitial na , whereas interstitial na increased to a larger extent during exercise , despite similar lbf , consistent with the results in the present control group ( 24 ) . the lack of increase in venous na during atp infusion in the group with diabetes may be to the result of an impaired baroreceptor function ( 25 ) or altered function of the atp inducement of na exocytosis because the changes in map during atp infusions were similar in the two groups . -values of venous na during tyramine infusion and the changes in lvc were proportional and similar in the two groups , suggesting that the subjects with diabetes were equally sensitive to na as the control subjects . therefore , the present measurements of venous na may reflect interstitial conditions , and the preserved functional sympatholysis in the group with diabetes could be partly attributed to a reduced level of na , perhaps in combination with or attributed to a reduction in atp - induced sympathetic activity . the primary limitation is that no truly selective human antagonists and ligands of p2 receptors are currently available , which hinders confirmatory studies of the role of the purinergic system . power calculations ahead were impeded because the current study is the first to investigate this particular area in patients with diabetes . in our previous study on 10 control subjects and 10 patients with diabetes and endothelial dysfunction , we demonstrated clear and significant differences in the relevant variables ( 10 ) . however , patients with type 2 diabetes are a heterogenic group , and sample size for the current study does not necessarily reflect such heterogeneity . antidiabetic and antihypertensive treatment has been shown to improve endothelial function through several pathways , and studies of patients with diabetes have been carried out primarily during withdrawal of medications ; however , in most studies analyzing risk profiles of different chronic diseases , the patients are usually taking medication . it may be more accurate to investigate patients in their normal functional status if the drugs do not affect the measurements directly , because little is known of the time course after withdrawal . consistent with a study on young healthy subjects ( 6 ) , exercise fully blunted sympathetic vasoconstriction in both groups , indicating that the ability of functional sympatholysis during moderate exercise was not reduced in the group with diabetes . this may be clinically relevant given that pathology , affecting the vasodilatory function , could be expected to limit skeletal muscle blood flow because of enhanced sympathetic vasoconstriction in the active muscles , thereby potentially reducing exercise capacity . a study on elderly healthy humans ( > 65 years ) demonstrated that aging is associated with a greater vasoconstrictor tone in active muscles during exercise compared with young adults ( 15 ) . the magnitude of functional sympatholysis was significantly lower in older persons compared with young persons in the presence of tyramine , leading the authors to conclude that aging is associated with impaired functional sympatholysis in the vascular beds of contracting forearm muscle . the present finding of intact functional sympatholysis in middle - aged healthy subjects and patients with diabetes could demonstrate that the phenomenon may be age - dependent ; however , limb differences should also be kept in mind ( 16 ) . moreover , a component of systemic limitation to peripheral blood flow during exercise may explain the observed low blood flows in some studies of patients with type 2 diabetes ; the knee - extensor model eliminates this risk and therefore allows studies of the local microcirculation and may not be translated to all skeletal muscles or applied to high - intensity exercise with a large muscle mass but is likely to represent the leg muscles , which accounts for the largest part of vascular resistance in the body and is of particular interest because insulin resistance is primarily present in leg muscles ( 17 ) . in young subjects , luminal atp was shown to abolish tyramine - induced sympathetic vasoconstriction , indicating that atp is contributing to functional sympatholysis via activation of the p2y2 receptor ( 6 ) . in contrast with this finding , both groups in the current study had a reduction in lbf during combined atp and tyramine infusion . the sympatholytic effect of atp is graded and dose - dependent ; during very modest atp infusions , atp had no sympatholytic effect ( 18 ) . however , the present infusion rates of atp increased lbf ninefold , most likely increasing arterial atp to levels sufficient to limit -adrenergic vasoconstriction in young subjects . therefore , the sympatholytic effect of atp may be affected by age . the amount of tyramine leading to 50% reduction in lvc and lbf during adenosine infusion resulted in a 30% reduction during atp ; thus , atp has a role in functional sympatholysis in the elderly , but other factors must be of importance to offset sympathetic vasoconstriction , because there were no changes in lvc or lbf during exercise with coinfusion of tyramine . functional sympatholysis in middle - aged persons could gradually be more dependent on factors other than atp , such as katp channel activation , which opposes -adrenergic vasoconstriction during muscle contraction ( 19 ) . regardless of the compounds responsible for functional sympatholysis , endothelial function also could be of importance . the present findings do not exclude that endothelial dysfunction affects the ability of sympatholysis during exercise . the amount of atp and adenosine needed to increase lbf to levels matching exercise was similar in the two groups . in contrast , in a previous study on patients with diabetes and age - matched control subjects , we found a 50% reduction in the lbf response to atp and adenosine in the group with diabetes ( 10 ) . the lbf response in control groups was identical , whereas the response in the present group with diabetes was higher in regard to both atp and adenosine . when comparing the two groups of patients with diabetes , there were no differences in demographic variables or medication . only the lbf response to acetylcholine infusion was significantly different , indicating that the reduction in effect of purinergic agonist may be proportional to the extent of endothelial dysfunction . a comparison of data from our two studies of middle - aged subjects , both control subjects and patients with diabetes , and previous studies of young subjects ( 20,21 ) demonstrates a decline in lbf response to adenosine in particular , which was reduced fourfold . in regard to atp , there was a clear decline in sensitivity only in the group with more developed endothelial dysfunction . still , it is a possibility that the response to atp also is diminished in an elderly population , possibly to a minor extent , because the dose - response in young healthy subjects may not be linear for the dose interval under study , hampering data extrapolation and direct comparison ( 6 ) . the decline in lbf responses to atp and adenosine may reflect age - related changes of the signaling pathways ( 20,21 ) . however , physical inactivity and obesity also affect endothelial function and cardiovascular regulation and should be of note , because both groups in the current study had moderately elevated bmi and the majority of the subjects had a sedentary lifestyle . the interpretation of plasma levels of na and its correlation to sympathetic activity is complex ; plasma na elevation may not be a precise measure of the effect on msna but merely a reflection of direction of change . it should be noted that during infusion with adenosine alone , there was no increase in venous na , despite a ninefold increase in lbf , indicating that the increase in na was indeed due to tyramine infusion and not to hyperemia . -values of venous na between the two groups were similar during all three tyramine interventions . furthermore , during moderate exercise , both with and without tyramine , the venous na increase in the groups was similar and additive , indicating that the stimulus of exercise to the sympathetic system also may have been uniform . these findings are consistent with previous studies in young subjects , showing that na spillover is a function of active muscle mass and exercise intensity ( 22 ) . in young subjects ( 6 ) , plasma na increased from 1.7 nmol l at baseline to 3 nmol l during tyramine infusions with comparable infusion rates , thus lower than in the present middle - aged control subjects . however , the levels of na in the present group of patients with diabetes were similar to those of young subjects . despite increases in sympathetic nerve activity in patients with diabetes , plasma na level has been found to be reduced compared with control subjects ( 23 ) . therefore , it is of interest to investigate whether the presented differences are a result of antidiabetic treatment or a physiologic response to elevation in sympathetic nerve activity , leading to enhanced reuptake and thus a reduced spillover . circulating atp mimics exercise hyperemia by its vasodilatory potency and by increasing msna and circulating na ( 6 ) . measurements of interstitial na in skeletal muscle during atp infusion with the microdialysis technique showed a dose - dependent increase in interstitial na , whereas interstitial na increased to a larger extent during exercise , despite similar lbf , consistent with the results in the present control group ( 24 ) . the lack of increase in venous na during atp infusion in the group with diabetes may be to the result of an impaired baroreceptor function ( 25 ) or altered function of the atp inducement of na exocytosis because the changes in map during atp infusions were similar in the two groups . -values of venous na during tyramine infusion and the changes in lvc were proportional and similar in the two groups , suggesting that the subjects with diabetes were equally sensitive to na as the control subjects . therefore , the present measurements of venous na may reflect interstitial conditions , and the preserved functional sympatholysis in the group with diabetes could be partly attributed to a reduced level of na , perhaps in combination with or attributed to a reduction in atp - induced sympathetic activity . the primary limitation is that no truly selective human antagonists and ligands of p2 receptors are currently available , which hinders confirmatory studies of the role of the purinergic system . power calculations ahead were impeded because the current study is the first to investigate this particular area in patients with diabetes . in our previous study on 10 control subjects and 10 patients with diabetes and endothelial dysfunction , we demonstrated clear and significant differences in the relevant variables ( 10 ) . however , patients with type 2 diabetes are a heterogenic group , and sample size for the current study does not necessarily reflect such heterogeneity . antidiabetic and antihypertensive treatment has been shown to improve endothelial function through several pathways , and studies of patients with diabetes have been carried out primarily during withdrawal of medications ; however , in most studies analyzing risk profiles of different chronic diseases , the patients are usually taking medication . it may be more accurate to investigate patients in their normal functional status if the drugs do not affect the measurements directly , because little is known of the time course after withdrawal . patients with well - diagnosed type 2 diabetes and only minor or no endothelial affection have an intact capacity of functional sympatholysis during moderate exercise . however , both the patients and the aging control subjects have a lower sympatholytic effect of atp . because this does not compromise functional sympatholysis , atp is not mandatory for an adequate hyperemic response during exercise . the lbf response to atp and adenosine was similar in the two groups ; thus , purinergic - induced lbf may not be affected by diabetes per se ; attenuation of purinergic vasodilation in patients with diabetes could be a result of endothelial dysfunction . when comparing the present findings with those of young subjects , the vasodilatory potency of adenosine in particular may be markedly reduced by aging and further aggravated with diabetes , in correlation to the grade of endothelial affection .

objectivesympathetic vasoconstriction is blunted in contracting human skeletal muscles ( functional sympatholysis ) . in young subjects , infusion of adenosine and atp increases blood flow , and the latter compound also attenuates -adrenergic vasoconstriction . in patients with type 2 diabetes and age - matched healthy subjects , we tested 1 ) the sympatholytic capacity during one - legged exercise , 2 ) the vasodilatory capacity of adenosine and atp , and 3 ) the ability to blunt -adrenergic vasoconstriction during atp infusion.research design and methodsin 10 control subjects and 10 patients with diabetes and normal endothelial function , determined by leg blood flow ( lbf ) response to acetylcholine infusion , we measured lbf and venous na , with and without tyramine - induced sympathetic vasoconstriction , during adenosine- , atp- , and exercise - induced hyperemia.resultslbf during acetylcholine did not differ significantly . lbf increased ninefold during exercise and during adenosine- and atp - induced hyperemia . infusion of tyramine during exercise did not reduce lbf in either the control or the patient group . during combined atp and tyramine infusions , lbf decreased by 30% in both groups . adenosine had no sympatholytic effect.conclusionsin patients with type 2 diabetes and normal endothelial function , functional sympatholysis was intact during moderate exercise . the vasodilatory response for adenosine and atp did not differ between the patients with diabetes and the control subjects ; however , the vasodilatory effect of adenosine and atp and the sympatholytic effect of atp seem to decline with age .

RESEARCH DESIGN AND METHODS RESULTS Hemodynamic variables Venous NA CONCLUSIONS Functional sympatholysis and sympatholytic effect of ATP Vasodilatory action of adenosine and ATP Venous NA and sensitivity Limitations CONCLUSIONS

ten subjects with type 2 diabetes and 10 age - matched healthy control subjects participated , all fully informed of the risks and discomforts associated with the experiments . the vasoconstrictor effect of tyramine ( 5.9 mol / ml , sigma t-2879 ; sigma - aldrich co. ) , which evokes endogenous noradrenaline ( na ) release from sympathetic nerve endings and subsequent postjunctional -adrenergic vasoconstriction , was examined during adenosine ( control ) , atp , and exercise - induced hyperemia ; the latter two were randomized ( 13 ) . lbf represents the average of three measurements obtained at baseline , 4 min after the start of exercise , or 4 min after reaching steady state under infusion of atp , adenosine , or coinfusion of tyramine . the vasodilatory potency of adenosine and atp was similar in control subjects and patients ( 309 54 vs. 250 81 ml/mol atpkg [ p = 0.48 ] and 13.3 1.7 vs. 12.5 4 ml/mol adenosinekg [ p = 0.38 ] ) . during adenosine and atp infusions , lbf increased ninefold in both control subjects and patients to similar levels as during the exercise intervention ( 2.7 0.2 l / min , fig . tyramine infusion reduced lbf during coinfusion with adenosine from 2.6 0.2 to 1.4 0.1 l / min , whereas infusion of the same amount of tyramine during exercise did not reduce lbf in either group ( 2.6 0.25 l / min ) . coinfusion with tyramine during atp infusion reduced lbf ( from 2.9 0.2 to 2.0 0.2 l / min in control subjects and from 2.7 0.3 to 2.2 0.2 l / min in patients ; p = 0.55 for control subjects vs. patients ) ( fig . map increased from baseline ( 101 4 in control subjects vs. 94 4 mmhg in patients ) to similar values during exercise , 120 4 mmhg in both groups . although coinfusion of atp and tyramine did not affect map in control subjects ( 95 4 mmhg ) , map increased ( to 94 4 mmhg ) in patients ( p < 0.05 ) . reflecting the alterations in lbf , with combined infusion of adenosine and tyramine , leg arteriovenous o2 difference increased from 11 2 ml / l with adenosine infusion to 19 3 ml / l with combined adenosine and tyramine and from 14 3 to 18 4 ml / l during atp and atp - tyramine infusion , respectively , with no differences in the groups . however , there were no differences in o2 delivery or uptake during the infusions of adenosine , atp , exercise , or coinfusion of tyramine . in addition , cardiac output differed at baseline ( 4.0 0.4 in control subjects vs. 6.0 0.5 l / min in patients , p = 0.005 ) , but during atp infusion and exercise , values were similar . in contrast , cardiac output increased more in the group with diabetes during adenosine infusion , both with and without tyramine , p = 0.03 ( control subjects vs. patients , both adenosine and coinfusion of adenosine and tyramine ) . the increases during tyramine coinfusion also were similar in the two groups ( 2.2 0.4 for adenosine , 1.5 0.3 for atp , and 3.2 0.5 nmol / l during exercise p = 0.4 to 0.6 ) , as were the increases in venous na adjusted for the individual infusion rates of tyramine ( 0.38 0.08 during adenosine infusion , 0.25 0.04 during atp infusion , and 0.56 0.11 nmol / l during exercise per micromole of tyramine ) . during adenosine infusions , venous na did not change in the two groups , whereas during atp infusion , na increased in the control group ( p = 0.003 ) . this difference was also reflected during combined atp and tyramine infusions , where venous na in the group with diabetes tended to be lower compared with adenosine ( p = 0.053 ) . the flow reduction per micromole of tyramine was similar in the two groups ( 245 40 ml/mol tyramine ) , whereas during atp infusion , the sensitivity to tyramine in terms of flow reduction was lower in the group with diabetes ( 182 27 vs. 92 34 ml/mol tyramine , p = 0.042 ) . the vasodilatory potency of adenosine and atp was similar in control subjects and patients ( 309 54 vs. 250 81 ml/mol atpkg [ p = 0.48 ] and 13.3 1.7 vs. 12.5 4 ml/mol adenosinekg [ p = 0.38 ] ) . during adenosine and atp infusions , lbf increased ninefold in both control subjects and patients to similar levels as during the exercise intervention ( 2.7 0.2 l / min , fig . tyramine infusion reduced lbf during coinfusion with adenosine from 2.6 0.2 to 1.4 0.1 l / min , whereas infusion of the same amount of tyramine during exercise did not reduce lbf in either group ( 2.6 0.25 l / min ) . coinfusion with tyramine during atp infusion reduced lbf ( from 2.9 0.2 to 2.0 0.2 l / min in control subjects and from 2.7 0.3 to 2.2 0.2 l / min in patients ; p = 0.55 for control subjects vs. patients ) ( fig . map increased from baseline ( 101 4 in control subjects vs. 94 4 mmhg in patients ) to similar values during exercise , 120 4 mmhg in both groups . although coinfusion of atp and tyramine did not affect map in control subjects ( 95 4 mmhg ) , map increased ( to 94 4 mmhg ) in patients ( p < 0.05 ) . reflecting the alterations in lbf , with combined infusion of adenosine and tyramine , leg arteriovenous o2 difference increased from 11 2 ml / l with adenosine infusion to 19 3 ml / l with combined adenosine and tyramine and from 14 3 to 18 4 ml / l during atp and atp - tyramine infusion , respectively , with no differences in the groups . however , there were no differences in o2 delivery or uptake during the infusions of adenosine , atp , exercise , or coinfusion of tyramine . in addition , cardiac output differed at baseline ( 4.0 0.4 in control subjects vs. 6.0 0.5 l / min in patients , p = 0.005 ) , but during atp infusion and exercise , values were similar . in contrast , cardiac output increased more in the group with diabetes during adenosine infusion , both with and without tyramine , p = 0.03 ( control subjects vs. patients , both adenosine and coinfusion of adenosine and tyramine ) . the increases during tyramine coinfusion also were similar in the two groups ( 2.2 0.4 for adenosine , 1.5 0.3 for atp , and 3.2 0.5 nmol / l during exercise p = 0.4 to 0.6 ) , as were the increases in venous na adjusted for the individual infusion rates of tyramine ( 0.38 0.08 during adenosine infusion , 0.25 0.04 during atp infusion , and 0.56 0.11 nmol / l during exercise per micromole of tyramine ) . during adenosine infusions , venous na did not change in the two groups , whereas during atp infusion , na increased in the control group ( p = 0.003 ) . this difference was also reflected during combined atp and tyramine infusions , where venous na in the group with diabetes tended to be lower compared with adenosine ( p = 0.053 ) . the flow reduction per micromole of tyramine was similar in the two groups ( 245 40 ml/mol tyramine ) , whereas during atp infusion , the sensitivity to tyramine in terms of flow reduction was lower in the group with diabetes ( 182 27 vs. 92 34 ml/mol tyramine , p = 0.042 ) . the main findings of the current study are that patients with type 2 diabetes and intact endothelial function have a similar capacity to blunt sympathetic vasoconstriction during moderate exercise as healthy age- and bmi - matched control subjects . however , both groups were only partially capable of blunting -adrenergic vasoconstriction during atp - induced hyperemia . in addition , the vasodilatory potency of adenosine and atp did not differ between patients and control subjects . finally , the subjects with diabetes had a lower venous content of na but a similar elevation of venous na during the adenosine infusions , exercise , and tyramine interventions . consistent with a study on young healthy subjects ( 6 ) , exercise fully blunted sympathetic vasoconstriction in both groups , indicating that the ability of functional sympatholysis during moderate exercise was not reduced in the group with diabetes . this may be clinically relevant given that pathology , affecting the vasodilatory function , could be expected to limit skeletal muscle blood flow because of enhanced sympathetic vasoconstriction in the active muscles , thereby potentially reducing exercise capacity . the present finding of intact functional sympatholysis in middle - aged healthy subjects and patients with diabetes could demonstrate that the phenomenon may be age - dependent ; however , limb differences should also be kept in mind ( 16 ) . moreover , a component of systemic limitation to peripheral blood flow during exercise may explain the observed low blood flows in some studies of patients with type 2 diabetes ; the knee - extensor model eliminates this risk and therefore allows studies of the local microcirculation and may not be translated to all skeletal muscles or applied to high - intensity exercise with a large muscle mass but is likely to represent the leg muscles , which accounts for the largest part of vascular resistance in the body and is of particular interest because insulin resistance is primarily present in leg muscles ( 17 ) . in young subjects , luminal atp was shown to abolish tyramine - induced sympathetic vasoconstriction , indicating that atp is contributing to functional sympatholysis via activation of the p2y2 receptor ( 6 ) . in contrast with this finding , both groups in the current study had a reduction in lbf during combined atp and tyramine infusion . the sympatholytic effect of atp is graded and dose - dependent ; during very modest atp infusions , atp had no sympatholytic effect ( 18 ) . however , the present infusion rates of atp increased lbf ninefold , most likely increasing arterial atp to levels sufficient to limit -adrenergic vasoconstriction in young subjects . therefore , the sympatholytic effect of atp may be affected by age . the amount of tyramine leading to 50% reduction in lvc and lbf during adenosine infusion resulted in a 30% reduction during atp ; thus , atp has a role in functional sympatholysis in the elderly , but other factors must be of importance to offset sympathetic vasoconstriction , because there were no changes in lvc or lbf during exercise with coinfusion of tyramine . in contrast , in a previous study on patients with diabetes and age - matched control subjects , we found a 50% reduction in the lbf response to atp and adenosine in the group with diabetes ( 10 ) . a comparison of data from our two studies of middle - aged subjects , both control subjects and patients with diabetes , and previous studies of young subjects ( 20,21 ) demonstrates a decline in lbf response to adenosine in particular , which was reduced fourfold . furthermore , during moderate exercise , both with and without tyramine , the venous na increase in the groups was similar and additive , indicating that the stimulus of exercise to the sympathetic system also may have been uniform . however , the levels of na in the present group of patients with diabetes were similar to those of young subjects . measurements of interstitial na in skeletal muscle during atp infusion with the microdialysis technique showed a dose - dependent increase in interstitial na , whereas interstitial na increased to a larger extent during exercise , despite similar lbf , consistent with the results in the present control group ( 24 ) . -values of venous na during tyramine infusion and the changes in lvc were proportional and similar in the two groups , suggesting that the subjects with diabetes were equally sensitive to na as the control subjects . therefore , the present measurements of venous na may reflect interstitial conditions , and the preserved functional sympatholysis in the group with diabetes could be partly attributed to a reduced level of na , perhaps in combination with or attributed to a reduction in atp - induced sympathetic activity . in our previous study on 10 control subjects and 10 patients with diabetes and endothelial dysfunction , we demonstrated clear and significant differences in the relevant variables ( 10 ) . however , patients with type 2 diabetes are a heterogenic group , and sample size for the current study does not necessarily reflect such heterogeneity . antidiabetic and antihypertensive treatment has been shown to improve endothelial function through several pathways , and studies of patients with diabetes have been carried out primarily during withdrawal of medications ; however , in most studies analyzing risk profiles of different chronic diseases , the patients are usually taking medication . consistent with a study on young healthy subjects ( 6 ) , exercise fully blunted sympathetic vasoconstriction in both groups , indicating that the ability of functional sympatholysis during moderate exercise was not reduced in the group with diabetes . this may be clinically relevant given that pathology , affecting the vasodilatory function , could be expected to limit skeletal muscle blood flow because of enhanced sympathetic vasoconstriction in the active muscles , thereby potentially reducing exercise capacity . the present finding of intact functional sympatholysis in middle - aged healthy subjects and patients with diabetes could demonstrate that the phenomenon may be age - dependent ; however , limb differences should also be kept in mind ( 16 ) . moreover , a component of systemic limitation to peripheral blood flow during exercise may explain the observed low blood flows in some studies of patients with type 2 diabetes ; the knee - extensor model eliminates this risk and therefore allows studies of the local microcirculation and may not be translated to all skeletal muscles or applied to high - intensity exercise with a large muscle mass but is likely to represent the leg muscles , which accounts for the largest part of vascular resistance in the body and is of particular interest because insulin resistance is primarily present in leg muscles ( 17 ) . in young subjects , luminal atp was shown to abolish tyramine - induced sympathetic vasoconstriction , indicating that atp is contributing to functional sympatholysis via activation of the p2y2 receptor ( 6 ) . in contrast with this finding , both groups in the current study had a reduction in lbf during combined atp and tyramine infusion . the sympatholytic effect of atp is graded and dose - dependent ; during very modest atp infusions , atp had no sympatholytic effect ( 18 ) . however , the present infusion rates of atp increased lbf ninefold , most likely increasing arterial atp to levels sufficient to limit -adrenergic vasoconstriction in young subjects . therefore , the sympatholytic effect of atp may be affected by age . the amount of tyramine leading to 50% reduction in lvc and lbf during adenosine infusion resulted in a 30% reduction during atp ; thus , atp has a role in functional sympatholysis in the elderly , but other factors must be of importance to offset sympathetic vasoconstriction , because there were no changes in lvc or lbf during exercise with coinfusion of tyramine . in contrast , in a previous study on patients with diabetes and age - matched control subjects , we found a 50% reduction in the lbf response to atp and adenosine in the group with diabetes ( 10 ) . a comparison of data from our two studies of middle - aged subjects , both control subjects and patients with diabetes , and previous studies of young subjects ( 20,21 ) demonstrates a decline in lbf response to adenosine in particular , which was reduced fourfold . furthermore , during moderate exercise , both with and without tyramine , the venous na increase in the groups was similar and additive , indicating that the stimulus of exercise to the sympathetic system also may have been uniform . however , the levels of na in the present group of patients with diabetes were similar to those of young subjects . measurements of interstitial na in skeletal muscle during atp infusion with the microdialysis technique showed a dose - dependent increase in interstitial na , whereas interstitial na increased to a larger extent during exercise , despite similar lbf , consistent with the results in the present control group ( 24 ) . -values of venous na during tyramine infusion and the changes in lvc were proportional and similar in the two groups , suggesting that the subjects with diabetes were equally sensitive to na as the control subjects . therefore , the present measurements of venous na may reflect interstitial conditions , and the preserved functional sympatholysis in the group with diabetes could be partly attributed to a reduced level of na , perhaps in combination with or attributed to a reduction in atp - induced sympathetic activity . in our previous study on 10 control subjects and 10 patients with diabetes and endothelial dysfunction , we demonstrated clear and significant differences in the relevant variables ( 10 ) . however , patients with type 2 diabetes are a heterogenic group , and sample size for the current study does not necessarily reflect such heterogeneity . antidiabetic and antihypertensive treatment has been shown to improve endothelial function through several pathways , and studies of patients with diabetes have been carried out primarily during withdrawal of medications ; however , in most studies analyzing risk profiles of different chronic diseases , the patients are usually taking medication . patients with well - diagnosed type 2 diabetes and only minor or no endothelial affection have an intact capacity of functional sympatholysis during moderate exercise . however , both the patients and the aging control subjects have a lower sympatholytic effect of atp . the lbf response to atp and adenosine was similar in the two groups ; thus , purinergic - induced lbf may not be affected by diabetes per se ; attenuation of purinergic vasodilation in patients with diabetes could be a result of endothelial dysfunction . when comparing the present findings with those of young subjects , the vasodilatory potency of adenosine in particular may be markedly reduced by aging and further aggravated with diabetes , in correlation to the grade of endothelial affection .

adolescence is a critical period for normal growth and development where there is a gradual transition between childhood and adulthood , and a period which marks puberty and physical maturation . sleep , in a complex association with many other processes , plays an important role in adolescents as dramatic maturational changes in sleep and its neurobiological regulation occurs in this period . the sleep patterns undergo substantial changes from childhood to adolescence characterized by a progressive delay in the sleep phase at the onset of puberty , as well as an endogenous preference for later bedtimes than children and adults . at the same time , societal demands such as school schedules or external factors frequently require earlier wake times and lead to shorter total sleep time in adolescents . studies done by carskadon shows that older adolescents may actually have a physiological need for more sleep than preadolescents . national sleep foundation , united states of america , 2006b , recommends 8.59.5 h of sleep per day for adolescents ages 13 to adult and 911 h of sleep for children from 5 to 12 years . longitudinal studies of sleep needs through puberty have demonstrated that adolescents need more than 9 h of sleep at night with some adolescents requiring additional sleep during the day , whereas laboratory studies showed that they require 9.25 h of sleep per night , with no difference in sleep duration of adolescents across different pubertal stages . but unfortunately , the typical adolescent sleeps one to one and a half hours less than the optimal amount per night . research shows that approximately 45% and 85% of 612 grade students report sleeping less than the recommended amount during school nights . many studies have shown that adolescents on an average obtains 7.58.5 h per night sleep with 26.6% getting less than 6.5 h of sleep per night and only 15% of them gets 8.5 h or more . a study reported 6.3 h of sleep for 10 through 12 graders in japan , and 5.4 h of school nights sleep among 11 and 12 graders and 6.6 h of sleep among 9 and 10 graders in korea . sleep deprivation is sleep less than the average and about 9 h of sleep per night for adolescents . sleep patterns refers to variations in sleep and waking patterns ( bed times and sleep duration ) measured on school and weekend nights . the sleep patterns of adolescents , in contrast to their childhood show a considerable delay on weekends when compared to weekdays , with a later sleep onset and offset during weekends . a cyclical sleep deprivation or sleep debt on school days , mitigated by a sleep on weekends and holidays is often shown by some children and adolescents , which makes them more difficult to fall asleep on subsequent school nights , producing an ongoing cycle of delayed sleep timing with impaired daytime functioning or causing impairment in quality of life . psychosocial factors like academic and social demands , physiological concomitants of puberty , and environmental factors like reduced parental influence on bedtimes , extracurricular activities , watching television , using internet and computer , and playing videogames causes a reduced sleep time . an average of 1 and 2 h of difference between weekend and school - night 's bedtimes is observed in older than younger adolescents . gender - based differences in sleep had been reported in many studies , but often showed inconsistent results . a hong kong study reported more sleep disturbances among female adolescent students . in some studies , girls found to wake up earlier than boys on weekdays , but accumulated higher sleep debt resulting in delayed wake up times on weekends than boys , without a change in their sleep length . these variations could be due to the differences in the measurement of sleep time or due to differences in the age group of participants in the study . sleep hygiene refers to a variety of different practices that are necessary to have normal , quality nighttime sleep and full daytime alertness . poor sleep hygiene practices impair cognitive and behavioral ability in children , and sleep - deprived habits established in adolescence can often lead to problems during subsequent college years . monitoring all adolescents and college - aged students for sleep insufficiency is imperative to improve both academic and emotional well - being . the adolescents in bahrain , especially those who are in higher grades have been observed to have late evening tuition classes that subsequently delay their sleep timing , unless they complete their routine academic sessions and home works earlier . the schools earlier start time at 7.30 am in most of the schools also tends to have an effect of inadequate sleep times among adolescents . however , no published studies regarding sleep duration or sleep hygiene behaviors among indian adolescents in bahrain are so far available to determine the extent of their sleep problems . thus , the aim of the present study is to examine the sleep patterns of adolescents on school days and weekends , to identify the sleep hygiene practices and the extent of parental monitoring among indian adolescents in bahrain and also to determine age and gender differences in sleep duration and sleep hygiene practices . indian adolescents studying in various schools of bahrain from grade 6 to 12 between 11 and 17 years were recruited to participate in the study using convenience sampling , by an informal method of approaching the churches and tuition centers . a written parental consent was taken for all participating adolescents . a letter to parents requesting permission for their adolescent children to participate in the study in addition , adolescent 's willingness to be a part of the study was obtained through the child assent form , as they have not attained the legal age for consent involved in research , which is 18 years . information was collected using a structured questionnaire , and was given to adolescents in the churches during sunday school classes to complete , and , to take it home to be filled for those from the tuition centers . out of 145 questionnaires distributed , the questionnaire consisted of two parts : the first part contained 28 items and included three questions related to demographic profile such as age , sex , and grade level . the remaining questions were related to sleep duration on school days and weekends , reasons for going to bed on school days and weekends at that time , the method of getting up from sleep on school days and weekends , questions related to exercises , and afternoon napping and parental influence while going to sleep and getting up from sleep . questions to assess sleep duration ( given on a 12-h format ) on school days included at what time do you go to bed on a typical school day ? and at what time do you get up from sleep on a typical school day ? the same were asked for sleep duration on weekends . the total sleep duration ( on school days and weekends ) = ( sleep duration on school days 5 ) + ( sleep duration on weekends 2)/7 . napping on school days and weekends and doing exercises were asked by yes or no questions . in addition , for those who answered yes were asked to indicate the duration of napping and the frequency of performing exercises . parental influence on sleep was assessed by questions like , does your parent influence the time of going to bed on school days ? and does your parent influence the time of getting up from bed on school days ? the same were asked to know the parental influence for going to bed and getting up from bed on weekends . the second part assessed the sleep hygiene behaviors through a 13-item questionnaire developed by mastin , bryson and corwyn , 2006 . the scale consisted of items to assess the presence of behavior comprising of sleep hygiene , and participants were asked to indicate how frequently they engage in specific behavior through a 5-point likert scale . the items summed up provided a global assessment of sleep hygiene , ranging from 13 to 65 , with higher scores indicative of more maladaptive sleep hygiene status . the scale has an internal consistency of cronbach 's 0.66 , and good test - retest reliability , 0.71 , p < 0.01 . among the total of 140 adolescents , 90 ( 64% ) were females and 50 ( 36% ) were males . majority ( 66% ) were middle adolescents in the age group of 1517 years and belonged to upper grade levels and the remaining ( 34% ) were younger adolescents from 11 to 14 years ( classified according to adolescent classification by american academy of pediatrics ) . when grouped according to grade levels , 32 adolescents ( 22.86% ) belonged to grade 12 , 25 ( 17.86% ) in grade 11 , 39 ( 27.9% ) in grade 10 , 22 ( 15.7% ) in grade 9 , 12 ( 8.57% ) in grade 8 and five each ( 3.57% ) in grade 6 and 7 , respectively . the time of going to bed and getting up from sleep for adolescents during a typical school day was 10.9 0.9 h and 5.5 0.5 h , respectively . the adolescents average sleep duration during school days was 06.5 1.0 h , much less than the average recommended sleep for this age group . the sleep debt on school days was 2.4 h , compared to the standard 9 h of sleep for this age group . the adolescents time of going to bed and wake times were delayed during weekends . on weekends , the time of going to bed was 12.2 1.5 h , 1.3 h later than school days . the wake time on weekends was 8.6 2.1 h. the sleep duration on a weekend was 8.4 2.7 h , i.e. 1.8 h more than during a school day indicating a more pronounced weekend oversleep among adolescents . the duration of sleep including both school days and weekends was 7.07 1.13 h. sleep duration by gender showed that male adolescents obtained slightly longer sleep duration on school days than female adolescents ( 6.7 h vs. 6.5 h , respectively ) . on weekends , female adolescents slept few minutes longer than male adolescents ( 8.4 h vs. 8.3 h , respectively ) . however , gender difference assessed using t - test for both school days and weekends for sleep duration showed that there is no statistically significant difference in sleep duration between male and female adolescents on school days and weekends since the p values 0.299 and 0.776 were at > 0.05 significance level [ tables 1 and 2 ] . influence of gender and grade level on sleep duration on school days influence of gender and grade level on sleep duration on weekends sleep duration of adolescents when grouped according to grade level on school days and weekends assessed using the kruskal wallis test showed a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels since the p values were < 0.001 and 0.001 , respectively [ tables 1 and 2 ] . the sleep duration of adolescents on higher grade levels were less when compared to sleep duration of adolescents on lower grade levels . majority of adolescents ( 52.1% ) reported that they take daytime naps during school days . twenty - eight of them ( 38.4% ) took daytime naps lasting for 12 h [ table 3 ] . during weekends , daytime napping with duration on school days and weekends only 62 adolescents ( 44.3% ) were involved in doing exercises regularly and the remaining 78 ( 55.7% ) were not involved in any kind of exercises . eighty - four adolescents ( 60.0% ) reported that their parents do not influence their bedtimes on school days and weekends and only 56 ( 40.0% ) adolescents parents influenced their bedtimes on school day . however , 60.7% adolescents parents influenced their time of getting up from bed on school days . during weekends , only 44 adolescents ( 31.4% ) reported that their parents influenced their rise times and the remaining 96 ( 68.6% ) reported that their parents did not monitor their rise times . a significant difference between parental monitoring at the time of getting up on school days and sleep duration was obtained ( p value 0.026 at 0.05 level of significance ) . whereas parental monitoring at the time of going to bed on school days , and that at the time of getting up and sleep duration on weekends was not statistically significant [ table 4 ] . relationship between parental monitoring and sleep duration the mean sleep hygiene score of adolescents was 31.0 5.9 . when grouped according to gender , the male adolescents obtained slightly higher sleep hygiene scores than female adolescents ( mean 31.3 6.2 vs. 30.8 5.8 , respectively ) . the highest score was obtained among grade 10 adolescents , followed by 7 graders and 12 graders , indicating poorer sleep hygiene practices among these adolescents ( mean 32.5 5.4 , 32.2 7.2 and 31.4 5.6 , respectively ) . however , the t - test and kruskal wallis test to find the influence of gender and grade level on sleep hygiene practices showed no significant difference as the p values 0.664 and 0.282 were > 0.05 level of significance . also , taking naps during school days and weekends and doing exercise do not show any significance with sleep hygiene practices as the p values 0.068 , 0.253 and 0.231 respectively were at > 0.05 level of significance [ table 5 ] . among the total of 140 adolescents , 90 ( 64% ) were females and 50 ( 36% ) were males . majority ( 66% ) were middle adolescents in the age group of 1517 years and belonged to upper grade levels and the remaining ( 34% ) were younger adolescents from 11 to 14 years ( classified according to adolescent classification by american academy of pediatrics ) . when grouped according to grade levels , 32 adolescents ( 22.86% ) belonged to grade 12 , 25 ( 17.86% ) in grade 11 , 39 ( 27.9% ) in grade 10 , 22 ( 15.7% ) in grade 9 , 12 ( 8.57% ) in grade 8 and five each ( 3.57% ) in grade 6 and 7 , respectively . the time of going to bed and getting up from sleep for adolescents during a typical school day was 10.9 0.9 h and 5.5 0.5 h , respectively . the adolescents average sleep duration during school days was 06.5 1.0 h , much less than the average recommended sleep for this age group . the sleep debt on school days was 2.4 h , compared to the standard 9 h of sleep for this age group . the adolescents time of going to bed and wake times were delayed during weekends . on weekends , the time of going to bed was 12.2 1.5 h , 1.3 h later than school days . the wake time on weekends was 8.6 2.1 h. the sleep duration on a weekend was 8.4 2.7 h , i.e. 1.8 h more than during a school day indicating a more pronounced weekend oversleep among adolescents . the duration of sleep including both school days and weekends was 7.07 1.13 h. sleep duration by gender showed that male adolescents obtained slightly longer sleep duration on school days than female adolescents ( 6.7 h vs. 6.5 h , respectively ) . on weekends , female adolescents slept few minutes longer than male adolescents ( 8.4 h vs. 8.3 h , respectively ) . however , gender difference assessed using t - test for both school days and weekends for sleep duration showed that there is no statistically significant difference in sleep duration between male and female adolescents on school days and weekends since the p values 0.299 and 0.776 were at > 0.05 significance level [ tables 1 and 2 ] . influence of gender and grade level on sleep duration on school days influence of gender and grade level on sleep duration on weekends sleep duration of adolescents when grouped according to grade level on school days and weekends assessed using the kruskal wallis test showed a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels since the p values were < 0.001 and 0.001 , respectively [ tables 1 and 2 ] . the sleep duration of adolescents on higher grade levels were less when compared to sleep duration of adolescents on lower grade levels . the time of going to bed and getting up from sleep for adolescents during a typical school day was 10.9 0.9 h and 5.5 0.5 h , respectively . the adolescents average sleep duration during school days was 06.5 1.0 h , much less than the average recommended sleep for this age group . the sleep debt on school days was 2.4 h , compared to the standard 9 h of sleep for this age group . the adolescents time of going to bed and wake times were delayed during weekends . on weekends , the time of going to bed was 12.2 1.5 h , 1.3 h later than school days . the wake time on weekends was 8.6 2.1 h. the sleep duration on a weekend was 8.4 2.7 h , i.e. 1.8 h more than during a school day indicating a more pronounced weekend oversleep among adolescents . sleep duration by gender showed that male adolescents obtained slightly longer sleep duration on school days than female adolescents ( 6.7 h vs. 6.5 h , respectively ) . on weekends , female adolescents slept few minutes longer than male adolescents ( 8.4 h vs. 8.3 h , respectively ) . however , gender difference assessed using t - test for both school days and weekends for sleep duration showed that there is no statistically significant difference in sleep duration between male and female adolescents on school days and weekends since the p values 0.299 and 0.776 were at > 0.05 significance level [ tables 1 and 2 ] . influence of gender and grade level on sleep duration on school days influence of gender and grade level on sleep duration on weekends sleep duration of adolescents when grouped according to grade level on school days and weekends assessed using the kruskal wallis test showed a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels since the p values were < 0.001 and 0.001 , respectively [ tables 1 and 2 ] . the sleep duration of adolescents on higher grade levels were less when compared to sleep duration of adolescents on lower grade levels . majority of adolescents ( 52.1% ) reported that they take daytime naps during school days . twenty - eight of them ( 38.4% ) took daytime naps lasting for 12 h [ table 3 ] . during weekends , daytime napping with duration on school days and weekends only 62 adolescents ( 44.3% ) were involved in doing exercises regularly and the remaining 78 ( 55.7% ) were not involved in any kind of exercises . eighty - four adolescents ( 60.0% ) reported that their parents do not influence their bedtimes on school days and weekends and only 56 ( 40.0% ) adolescents parents influenced their bedtimes on school day . however , 60.7% adolescents parents influenced their time of getting up from bed on school days . during weekends , only 44 adolescents ( 31.4% ) reported that their parents influenced their rise times and the remaining 96 ( 68.6% ) reported that their parents did not monitor their rise times . a significant difference between parental monitoring at the time of getting up on school days and sleep duration was obtained ( p value 0.026 at 0.05 level of significance ) . whereas parental monitoring at the time of going to bed on school days , and that at the time of getting up and sleep duration on weekends was not statistically significant [ table 4 ] . when grouped according to gender , the male adolescents obtained slightly higher sleep hygiene scores than female adolescents ( mean 31.3 6.2 vs. 30.8 5.8 , respectively ) . the highest score was obtained among grade 10 adolescents , followed by 7 graders and 12 graders , indicating poorer sleep hygiene practices among these adolescents ( mean 32.5 5.4 , 32.2 7.2 and 31.4 5.6 , respectively ) . however , the t - test and kruskal wallis test to find the influence of gender and grade level on sleep hygiene practices showed no significant difference as the p values 0.664 and 0.282 were > 0.05 level of significance . also , taking naps during school days and weekends and doing exercise do not show any significance with sleep hygiene practices as the p values 0.068 , 0.253 and 0.231 respectively were at > 0.05 level of significance [ table 5 ] . the present study investigated the sleep - wake patterns of indian adolescents studying in bahrain on school days and weekends , sleep duration , and the associations between sleep duration , sleep hygiene behaviors and parental monitoring . the adolescents in this study obtained less than the average recommended 9 h of sleep for their age group , i.e. 6.5 h on school days , 8.4 h on weekends and an average of 7.07 h total sleep including school days and weekends . the bedtime on school days was 10.9 h and on weekends it was delayed to 12.2 h. these findings were similar to those obtained in taiwan , where 54% of middle school and 74% of high school adolescents slept less than 68 h on school days , and in greece , where total nocturnal sleep duration among the high school adolescents were 6.58 h and 7.28 h with afternoon napping . also , 41.3% of south australian adolescents obtained less than 8 h of sleep and 78% reported discrepant school versus weekend morning out of bed timings . the adolescents in bahrain obtained less sleep than hong kong chinese adolescents on school nights , and a much shorter sleep than indian adolescents on school days and weekends . whereas a u.s . study showed that 23% of the adolescent respondents went to bed at 11.15 pm or later and slept fewer than the recommended 9 h of sleep . female adolescents in this study slept slightly less than male adolescents during school days ( 6.5 h vs. 6.7 h ) and the reverse was observed during weekends for girls and boys ( 8.4 h vs. 8.3 h , respectively ) . this weekday and weekend sleep were consistent with studies in spain and in usa . and in contrast with sleep reported among indian adolescents . a 36 min later rise time of females , with a sleep length of 9 h 45 min and an average rise time of 10:31 on weekends than boys is reported in another . however , gender differences were not significant in this study for sleep duration similar to studies in greece , florida , and india . whereas a significant difference between boys and girls in sleep duration was observed among german adolescents . the findings of this study indicated that a highly significant difference exists in total sleep duration between adolescents of various grade levels on both school days and weekends , where adolescents on higher grade slept less compared to adolescents on lower grade levels . adolescents in grade 12 obtained only 6.1 1.1 h of sleep in comparison to adolescents in grade 6 , who slept for 7.7 0.9 h. similar trends were observed in korea , where 1012 graders slept 5.78 1.3 h in comparison with 56 graders who slept for 7.95 1.05 h , and in taiwan . since 9 h is considered as the general requirement of adolescent sleep , all adolescents in this study were obtaining less than the required amount of sleep and thus are sleep - deprived . this could be explained by the fact that as the indian adolescents enter into higher grade levels , they spend considerable time in completing school works , assignments , and projects and in addition , go for private tutoring and thus reach late at home between 7 pm and 10 pm . they also prepare for the entrance exams for getting admission to professional courses after finishing the 12 grade to further pursue their career . these make them getting reduced hours of sleep and sleeping at late hours at night . however , majority of adolescents in bahrain took a daytime nap regularly on school days lasting 12 h as a method to compensate for their lost sleep similar to 60% of students in greece . but on weekends , the tendency of daytime napping was comparatively less among the adolescents and could be due to the long hours of sleep they obtain during weekends . majority of the adolescents in this study reported that parental monitoring was absent during school days and weekends for their bedtimes . but , most of the adolescents rise times on school days were influenced by parents , even though majority of the adolescents rise times on weekends were not monitored by them . parental monitoring showed significant difference with sleep duration on rise times of adolescents on school days . randler reports significant age differences in the setting of bedtimes of adolescents by parents , whereas 35% of adolescent bedtimes were set by parents on weekdays in contrast to 9% of parental setting of bedtimes on weekends . parental monitoring was absent by the age of 17 years and in those adolescents whose sleep was not monitored by parents went to bed later than those with parental monitoring . the sleep hygiene practices were worse among 10 graders followed by 7 and 12 graders , even though gender and grade levels were not statistically significant . it could be due to the influence of puberty , higher amounts of stress and poor sleep hygiene practices . this study also supports previous research findings about poor sleep hygiene practices : an irregular bed / stimulating mental activity before bedtime , staying up late to study or academic commitments and earlier wake up times are associated with sleep problems in adolescents . whereas better sleep hygiene practices were observed among italian adolescents than american adolescents . this study indicates that there is a high prevalence of short sleep in adolescents , which poses a risk by making them sleep deprived . accumulated sleep deprivation causes impairment in cognitive , vigilance and memory and mood disturbances among adolescents , which can be prevented by promoting good sleep hygiene and increasing their total sleep time . in addition , the biological , behavioral and social and environmental factors have to be addressed to develop regular sleep schedules and to entrain the circadian system since poor sleep seems to be a universal and prevalent problem in modern society . there were some limitations in this study . the study used a non - probability sampling method and the sample collection from informal centers might not be representative of the more general population of indian adolescents in bahrain . the use of a random sampling and inclusion of an equal proportion of female and male adolescents would have been valuable . also , the study used a cross - sectional method and the data was collected using a self - rated questionnaire . using longitudinal studies and more objective measures for sleep like actigraphy could provide better measures about variations in sleep across adolescent population . there is also evidence that sleep quality is a better measure for adequate sleep than sleep times which has to be investigated further . this will need to be addressed in subsequent research using interventional programs and strategies for better sleep practices in adolescents . even though these limitations are present , the findings of this research may be valuable in exploring the sleep patterns and could contribute to the sleep practices among indian adolescents in bahrain . the study investigated adolescents sleep patterns and its association with sleep hygiene behaviors and parental monitoring . the results suggest that there is a high prevalence of insufficient sleep and irregular bedtime schedule in adolescent students in bahrain which give an insight into promoting good sleep practices in adolescents . parents , teachers and researchers should be also taught about the importance of sleep in adolescents and the health consequences with lack of sleep . interventions to help adolescents with the sleep issues , promoting good sleep hygiene strategies along with time management skills to facilitate healthy sleep are required . future studies could consider the relationship of daytime sleepiness and the influence of sleep quality with sleep behaviors as well as the pubertal changes associated with sleep to further understand the sleep problems in adolescents .

introduction : sleep plays an important role in adolescent 's health and undergoes substantial changes with puberty and physical maturation with a preference for later bed times . evidence shows that many adolescents are not obtaining the required amounts of sleep which is 9.25 h , due to inadequate sleep practices , academic and societal demands . this study aims at describing the ( 1 ) sleep patterns of adolescents on school days and weekends , ( 2 ) sleep hygiene practices and the extent of parental monitoring and ( 3 ) gender and grade level differences in sleep duration and sleep hygiene practices among indian adolescents in bahrain.materials and methods : study used a descriptive correlational design . a total of 145 adolescents from 11 to 17 years from grade 6 to 12 were selected using convenience sampling . data was collected from november 2012 to march 2013 . a structured questionnaire for sleep patterns and mastin et al . 's sleep hygiene index for assessing sleep hygiene practices were used.results:the adolescents total sleep duration was 7.07 1.13 hours . a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels ( p < 0.001 and 0.001 , respectively ) and between parental monitoring at the time of getting up on school days and sleep duration ( p value 0.026 at 0.05 level of significance ) was found . gender was not significant with the sleep duration , and also with sleep hygiene index scores.conclusion:the results suggest that there is a high prevalence of insufficient sleep and irregular bed - time schedule among indian adolescents in bahrain . interventions directed toward improving sleep and promoting good sleep hygiene strategies are required to improve the physical and emotional health of adolescents .

Introduction Materials and Methods Results Profile of the respondents Sleep wake patterns and sleep duration of the respondents Sleep duration on school days and weekends Sleep duration by gender Sleep duration by grade levels Afternoon napping and exercises Parental monitoring and sleep Part B: Sleep hygiene index scores of respondents Discussion Conclusion

adolescence is a critical period for normal growth and development where there is a gradual transition between childhood and adulthood , and a period which marks puberty and physical maturation . sleep , in a complex association with many other processes , plays an important role in adolescents as dramatic maturational changes in sleep and its neurobiological regulation occurs in this period . the sleep patterns undergo substantial changes from childhood to adolescence characterized by a progressive delay in the sleep phase at the onset of puberty , as well as an endogenous preference for later bedtimes than children and adults . longitudinal studies of sleep needs through puberty have demonstrated that adolescents need more than 9 h of sleep at night with some adolescents requiring additional sleep during the day , whereas laboratory studies showed that they require 9.25 h of sleep per night , with no difference in sleep duration of adolescents across different pubertal stages . sleep patterns refers to variations in sleep and waking patterns ( bed times and sleep duration ) measured on school and weekend nights . the sleep patterns of adolescents , in contrast to their childhood show a considerable delay on weekends when compared to weekdays , with a later sleep onset and offset during weekends . these variations could be due to the differences in the measurement of sleep time or due to differences in the age group of participants in the study . poor sleep hygiene practices impair cognitive and behavioral ability in children , and sleep - deprived habits established in adolescence can often lead to problems during subsequent college years . monitoring all adolescents and college - aged students for sleep insufficiency is imperative to improve both academic and emotional well - being . however , no published studies regarding sleep duration or sleep hygiene behaviors among indian adolescents in bahrain are so far available to determine the extent of their sleep problems . thus , the aim of the present study is to examine the sleep patterns of adolescents on school days and weekends , to identify the sleep hygiene practices and the extent of parental monitoring among indian adolescents in bahrain and also to determine age and gender differences in sleep duration and sleep hygiene practices . indian adolescents studying in various schools of bahrain from grade 6 to 12 between 11 and 17 years were recruited to participate in the study using convenience sampling , by an informal method of approaching the churches and tuition centers . information was collected using a structured questionnaire , and was given to adolescents in the churches during sunday school classes to complete , and , to take it home to be filled for those from the tuition centers . out of 145 questionnaires distributed , the questionnaire consisted of two parts : the first part contained 28 items and included three questions related to demographic profile such as age , sex , and grade level . the remaining questions were related to sleep duration on school days and weekends , reasons for going to bed on school days and weekends at that time , the method of getting up from sleep on school days and weekends , questions related to exercises , and afternoon napping and parental influence while going to sleep and getting up from sleep . questions to assess sleep duration ( given on a 12-h format ) on school days included at what time do you go to bed on a typical school day ? the same were asked for sleep duration on weekends . the total sleep duration ( on school days and weekends ) = ( sleep duration on school days 5 ) + ( sleep duration on weekends 2)/7 . napping on school days and weekends and doing exercises were asked by yes or no questions . parental influence on sleep was assessed by questions like , does your parent influence the time of going to bed on school days ? and does your parent influence the time of getting up from bed on school days ? the scale has an internal consistency of cronbach 's 0.66 , and good test - retest reliability , 0.71 , p < 0.01 . majority ( 66% ) were middle adolescents in the age group of 1517 years and belonged to upper grade levels and the remaining ( 34% ) were younger adolescents from 11 to 14 years ( classified according to adolescent classification by american academy of pediatrics ) . when grouped according to grade levels , 32 adolescents ( 22.86% ) belonged to grade 12 , 25 ( 17.86% ) in grade 11 , 39 ( 27.9% ) in grade 10 , 22 ( 15.7% ) in grade 9 , 12 ( 8.57% ) in grade 8 and five each ( 3.57% ) in grade 6 and 7 , respectively . the time of going to bed and getting up from sleep for adolescents during a typical school day was 10.9 0.9 h and 5.5 0.5 h , respectively . the adolescents average sleep duration during school days was 06.5 1.0 h , much less than the average recommended sleep for this age group . the sleep debt on school days was 2.4 h , compared to the standard 9 h of sleep for this age group . on weekends , the time of going to bed was 12.2 1.5 h , 1.3 h later than school days . the wake time on weekends was 8.6 2.1 h. the sleep duration on a weekend was 8.4 2.7 h , i.e. the duration of sleep including both school days and weekends was 7.07 1.13 h. sleep duration by gender showed that male adolescents obtained slightly longer sleep duration on school days than female adolescents ( 6.7 h vs. 6.5 h , respectively ) . on weekends , female adolescents slept few minutes longer than male adolescents ( 8.4 h vs. 8.3 h , respectively ) . however , gender difference assessed using t - test for both school days and weekends for sleep duration showed that there is no statistically significant difference in sleep duration between male and female adolescents on school days and weekends since the p values 0.299 and 0.776 were at > 0.05 significance level [ tables 1 and 2 ] . influence of gender and grade level on sleep duration on school days influence of gender and grade level on sleep duration on weekends sleep duration of adolescents when grouped according to grade level on school days and weekends assessed using the kruskal wallis test showed a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels since the p values were < 0.001 and 0.001 , respectively [ tables 1 and 2 ] . the sleep duration of adolescents on higher grade levels were less when compared to sleep duration of adolescents on lower grade levels . during weekends , daytime napping with duration on school days and weekends only 62 adolescents ( 44.3% ) were involved in doing exercises regularly and the remaining 78 ( 55.7% ) were not involved in any kind of exercises . eighty - four adolescents ( 60.0% ) reported that their parents do not influence their bedtimes on school days and weekends and only 56 ( 40.0% ) adolescents parents influenced their bedtimes on school day . however , 60.7% adolescents parents influenced their time of getting up from bed on school days . a significant difference between parental monitoring at the time of getting up on school days and sleep duration was obtained ( p value 0.026 at 0.05 level of significance ) . whereas parental monitoring at the time of going to bed on school days , and that at the time of getting up and sleep duration on weekends was not statistically significant [ table 4 ] . relationship between parental monitoring and sleep duration the mean sleep hygiene score of adolescents was 31.0 5.9 . when grouped according to gender , the male adolescents obtained slightly higher sleep hygiene scores than female adolescents ( mean 31.3 6.2 vs. 30.8 5.8 , respectively ) . the highest score was obtained among grade 10 adolescents , followed by 7 graders and 12 graders , indicating poorer sleep hygiene practices among these adolescents ( mean 32.5 5.4 , 32.2 7.2 and 31.4 5.6 , respectively ) . however , the t - test and kruskal wallis test to find the influence of gender and grade level on sleep hygiene practices showed no significant difference as the p values 0.664 and 0.282 were > 0.05 level of significance . also , taking naps during school days and weekends and doing exercise do not show any significance with sleep hygiene practices as the p values 0.068 , 0.253 and 0.231 respectively were at > 0.05 level of significance [ table 5 ] . majority ( 66% ) were middle adolescents in the age group of 1517 years and belonged to upper grade levels and the remaining ( 34% ) were younger adolescents from 11 to 14 years ( classified according to adolescent classification by american academy of pediatrics ) . when grouped according to grade levels , 32 adolescents ( 22.86% ) belonged to grade 12 , 25 ( 17.86% ) in grade 11 , 39 ( 27.9% ) in grade 10 , 22 ( 15.7% ) in grade 9 , 12 ( 8.57% ) in grade 8 and five each ( 3.57% ) in grade 6 and 7 , respectively . the time of going to bed and getting up from sleep for adolescents during a typical school day was 10.9 0.9 h and 5.5 0.5 h , respectively . the sleep debt on school days was 2.4 h , compared to the standard 9 h of sleep for this age group . on weekends , the time of going to bed was 12.2 1.5 h , 1.3 h later than school days . the wake time on weekends was 8.6 2.1 h. the sleep duration on a weekend was 8.4 2.7 h , i.e. the duration of sleep including both school days and weekends was 7.07 1.13 h. sleep duration by gender showed that male adolescents obtained slightly longer sleep duration on school days than female adolescents ( 6.7 h vs. 6.5 h , respectively ) . on weekends , female adolescents slept few minutes longer than male adolescents ( 8.4 h vs. 8.3 h , respectively ) . however , gender difference assessed using t - test for both school days and weekends for sleep duration showed that there is no statistically significant difference in sleep duration between male and female adolescents on school days and weekends since the p values 0.299 and 0.776 were at > 0.05 significance level [ tables 1 and 2 ] . influence of gender and grade level on sleep duration on school days influence of gender and grade level on sleep duration on weekends sleep duration of adolescents when grouped according to grade level on school days and weekends assessed using the kruskal wallis test showed a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels since the p values were < 0.001 and 0.001 , respectively [ tables 1 and 2 ] . the sleep duration of adolescents on higher grade levels were less when compared to sleep duration of adolescents on lower grade levels . the time of going to bed and getting up from sleep for adolescents during a typical school day was 10.9 0.9 h and 5.5 0.5 h , respectively . the adolescents average sleep duration during school days was 06.5 1.0 h , much less than the average recommended sleep for this age group . the sleep debt on school days was 2.4 h , compared to the standard 9 h of sleep for this age group . on weekends , the time of going to bed was 12.2 1.5 h , 1.3 h later than school days . the wake time on weekends was 8.6 2.1 h. the sleep duration on a weekend was 8.4 2.7 h , i.e. sleep duration by gender showed that male adolescents obtained slightly longer sleep duration on school days than female adolescents ( 6.7 h vs. 6.5 h , respectively ) . on weekends , female adolescents slept few minutes longer than male adolescents ( 8.4 h vs. 8.3 h , respectively ) . however , gender difference assessed using t - test for both school days and weekends for sleep duration showed that there is no statistically significant difference in sleep duration between male and female adolescents on school days and weekends since the p values 0.299 and 0.776 were at > 0.05 significance level [ tables 1 and 2 ] . influence of gender and grade level on sleep duration on school days influence of gender and grade level on sleep duration on weekends sleep duration of adolescents when grouped according to grade level on school days and weekends assessed using the kruskal wallis test showed a highly significant difference in sleep duration on school days and weekends between adolescents of various grade levels since the p values were < 0.001 and 0.001 , respectively [ tables 1 and 2 ] . the sleep duration of adolescents on higher grade levels were less when compared to sleep duration of adolescents on lower grade levels . during weekends , daytime napping with duration on school days and weekends only 62 adolescents ( 44.3% ) were involved in doing exercises regularly and the remaining 78 ( 55.7% ) were not involved in any kind of exercises . eighty - four adolescents ( 60.0% ) reported that their parents do not influence their bedtimes on school days and weekends and only 56 ( 40.0% ) adolescents parents influenced their bedtimes on school day . however , 60.7% adolescents parents influenced their time of getting up from bed on school days . a significant difference between parental monitoring at the time of getting up on school days and sleep duration was obtained ( p value 0.026 at 0.05 level of significance ) . whereas parental monitoring at the time of going to bed on school days , and that at the time of getting up and sleep duration on weekends was not statistically significant [ table 4 ] . when grouped according to gender , the male adolescents obtained slightly higher sleep hygiene scores than female adolescents ( mean 31.3 6.2 vs. 30.8 5.8 , respectively ) . the highest score was obtained among grade 10 adolescents , followed by 7 graders and 12 graders , indicating poorer sleep hygiene practices among these adolescents ( mean 32.5 5.4 , 32.2 7.2 and 31.4 5.6 , respectively ) . however , the t - test and kruskal wallis test to find the influence of gender and grade level on sleep hygiene practices showed no significant difference as the p values 0.664 and 0.282 were > 0.05 level of significance . also , taking naps during school days and weekends and doing exercise do not show any significance with sleep hygiene practices as the p values 0.068 , 0.253 and 0.231 respectively were at > 0.05 level of significance [ table 5 ] . the present study investigated the sleep - wake patterns of indian adolescents studying in bahrain on school days and weekends , sleep duration , and the associations between sleep duration , sleep hygiene behaviors and parental monitoring . the adolescents in this study obtained less than the average recommended 9 h of sleep for their age group , i.e. 6.5 h on school days , 8.4 h on weekends and an average of 7.07 h total sleep including school days and weekends . the bedtime on school days was 10.9 h and on weekends it was delayed to 12.2 h. these findings were similar to those obtained in taiwan , where 54% of middle school and 74% of high school adolescents slept less than 68 h on school days , and in greece , where total nocturnal sleep duration among the high school adolescents were 6.58 h and 7.28 h with afternoon napping . the adolescents in bahrain obtained less sleep than hong kong chinese adolescents on school nights , and a much shorter sleep than indian adolescents on school days and weekends . female adolescents in this study slept slightly less than male adolescents during school days ( 6.5 h vs. 6.7 h ) and the reverse was observed during weekends for girls and boys ( 8.4 h vs. 8.3 h , respectively ) . however , gender differences were not significant in this study for sleep duration similar to studies in greece , florida , and india . whereas a significant difference between boys and girls in sleep duration was observed among german adolescents . the findings of this study indicated that a highly significant difference exists in total sleep duration between adolescents of various grade levels on both school days and weekends , where adolescents on higher grade slept less compared to adolescents on lower grade levels . adolescents in grade 12 obtained only 6.1 1.1 h of sleep in comparison to adolescents in grade 6 , who slept for 7.7 0.9 h. similar trends were observed in korea , where 1012 graders slept 5.78 1.3 h in comparison with 56 graders who slept for 7.95 1.05 h , and in taiwan . since 9 h is considered as the general requirement of adolescent sleep , all adolescents in this study were obtaining less than the required amount of sleep and thus are sleep - deprived . this could be explained by the fact that as the indian adolescents enter into higher grade levels , they spend considerable time in completing school works , assignments , and projects and in addition , go for private tutoring and thus reach late at home between 7 pm and 10 pm . however , majority of adolescents in bahrain took a daytime nap regularly on school days lasting 12 h as a method to compensate for their lost sleep similar to 60% of students in greece . but on weekends , the tendency of daytime napping was comparatively less among the adolescents and could be due to the long hours of sleep they obtain during weekends . majority of the adolescents in this study reported that parental monitoring was absent during school days and weekends for their bedtimes . parental monitoring showed significant difference with sleep duration on rise times of adolescents on school days . parental monitoring was absent by the age of 17 years and in those adolescents whose sleep was not monitored by parents went to bed later than those with parental monitoring . the sleep hygiene practices were worse among 10 graders followed by 7 and 12 graders , even though gender and grade levels were not statistically significant . it could be due to the influence of puberty , higher amounts of stress and poor sleep hygiene practices . this study also supports previous research findings about poor sleep hygiene practices : an irregular bed / stimulating mental activity before bedtime , staying up late to study or academic commitments and earlier wake up times are associated with sleep problems in adolescents . whereas better sleep hygiene practices were observed among italian adolescents than american adolescents . this study indicates that there is a high prevalence of short sleep in adolescents , which poses a risk by making them sleep deprived . accumulated sleep deprivation causes impairment in cognitive , vigilance and memory and mood disturbances among adolescents , which can be prevented by promoting good sleep hygiene and increasing their total sleep time . the study used a non - probability sampling method and the sample collection from informal centers might not be representative of the more general population of indian adolescents in bahrain . also , the study used a cross - sectional method and the data was collected using a self - rated questionnaire . even though these limitations are present , the findings of this research may be valuable in exploring the sleep patterns and could contribute to the sleep practices among indian adolescents in bahrain . the study investigated adolescents sleep patterns and its association with sleep hygiene behaviors and parental monitoring . the results suggest that there is a high prevalence of insufficient sleep and irregular bedtime schedule in adolescent students in bahrain which give an insight into promoting good sleep practices in adolescents . interventions to help adolescents with the sleep issues , promoting good sleep hygiene strategies along with time management skills to facilitate healthy sleep are required . future studies could consider the relationship of daytime sleepiness and the influence of sleep quality with sleep behaviors as well as the pubertal changes associated with sleep to further understand the sleep problems in adolescents .

much research has focused on mechanisms such as competition ( e.g. levine et al . , 2003 ) and predation ( e.g. short et al . , 2002 ) but disruption of host - parasite dynamics may be an important additional route of impact ( thieltges et al . , 2009 ; hartigan et al . , invaders often exhibit reduced parasite levels ( the enemy release hypothesis : marr et al . , 2008 ) , but novel pathogens brought by invaders can still devastate native taxa by directly reducing survival or by mediating the outcome of competition between native and invasive species ( e.g. settle and wilson , 1990 ; hudson and greenman , 1998 ) . the reverse scenario ( the transfer of native pathogens to the invader ) has similar effects ( dunn , 2009 ; hartigan et al . , 2011 ; pizzatto and shine , 2012 ) . past studies have investigated cases where invaders act to increase parasitism of native fauna by introducing a new parasite or by acting as a reservoir for native parasites ( dobson and foufopoulos , 2001 ; mastitsky and veres , 2010 ; pizzatto and shine , 2011 ; hartigan et al . , 2011 ) . however in this instance , native parasites are taken up by the invader but fail to complete their life cycle due to a lack of co - evolutionary history . when the parasite enters a foreign host it becomes disoriented or attacked by the immune system . resistant targets , reducing the density of parasites in the environment and thus , lowering the risk of infection for native hosts ( heimpel et al . , 2003 ; kelly et al . , 2009a ) . because free - living stages of parasites are time - limited and exposed to threats such as predation and desiccation ( johnson and thieltges , 2010 ) they are under heavy selective pressure to rapidly infect an appropriate host . finding a host becomes more of a challenge in assemblages with a number of host species that differ in susceptibility to the parasite , such as invasive systems ( keesing et al . , 2006 ) . sink mechanism has been investigated in only a few invasive systems ( trejo , 1992 ; telfer et al . , 2005 ; kopp and jokela , 2007 ; thieltges et al . , 2009 ; paterson et al . , 2011 , 2013a , b ) but these studies are geographically and taxonomically diverse , meaning the crossover of native parasites to invaders is likely to be common ( dunn , 2009 ) . sink mechanisms in other systems , especially involving parasite and host taxa from lineages that have not been the subjects of previous research in this respect . here , we examine the potential disruption of host - parasite interactions caused by the invasive cane toad , rhinella marina , in australia . recent studies have shown that cane toads and native frogs have separate nematode lungworm fauna ( pizzatto et al . , 2012 ) . the cane toad lungworm , rhabdias pseudosphaerocephala , arrived in australia with the invasive species in 1935 when it was introduced into queensland as a biological control agent for sugar cane pests ( dubey and shine , 2008 ) . these two parasite species are virtually indistinguishable in morphology , but genetic analysis shows that they are indeed separate species ( dubey and shine , 2008 ) , and experimental infections demonstrate that the lungworms perform differently in cane toad and native frog hosts ( pizzatto et al . , 2010 ; nelson et al . r. hylae can penetrate cane toads but is killed by a strong immune response ; it becomes lost inside the novel host 's body and never reaches the lungs , where it would normally mature , produce eggs and complete its life cycle ( nelson , 2014 ; nelson et al . , 2015b ) . r. pseudosphaerocephala readily penetrates native frogs but only reaches the lungs in a small number of species , for similar reasons ( pizzatto et al . , 2010 ; pizzatto and shine , 2011 ) . the only field data to support this scenario come from a recent study conducted in northeastern new south wales . ( 2013 ) found lower rates of infection with lungworms in native frogs living in areas with cane toads , than in the same frog species living in nearby areas that lack cane toads . firstly , the cane toads could be acting as sinks ( removing parasite larvae from the environment , and dooming those parasites to an early death ) . secondly , native frogs that are exposed to the cane toad parasite might thereby develop acquired immunity to their own parasite species ( i.e. , an initial exposure to toad rhabdias spp . may instigate production of antibodies that are also effective targeting native rhabdias spp . ) . a similar priming of the cane toad 's immune system by frog rhabdias spp . against its more virulent native parasite would have substantial benefits for cane toads , and might enhance their invasion success . priming is the principle behind many vaccines , which exploit the capacity of the adaptive immune system to form memories in response to inert parts of pathogens ( brunham and coombs , 1998 ; oettinger et al . , 1999 specific antibodies are generated to defend the body against attack . upon re - infection , memory of previously encountered pathogens and the acquired immune response has been shown to play a role in the improved response of amphibians towards infections ( richmond et al . , 2009 ; tinsley et al . , sink mechanism as it applies to cane toads soaking up native frog parasites , and the possibility that prior exposure to the native frog lungworm primes the cane toad 's immune system such that it is less vulnerable to infection by its own lungworm species . descriptions and details of methods for breeding and husbandry of anurans , and collection and identification of lungworm larvae used in the following experiments , appear in the supplementary material . to measure rates of r. hylae uptake by anurans we exposed each of 69 native frogs ( 30 cyclorana australis and 39 limnodynastes convexiusculus ) and 31 cane toads ( r.marina ) to infective lungworm larvae . feces containing free - living adult worms were collected from adult frogs between 4 and 18 days prior to infection and stored in petri dishes with untreated bore water . after 24 days in these petri dishes , the free - living adult worms had produced infective third stage larvae ( l3 ) that we used for experimental infections . 30 larvae ( l3 ) were collected using a glass pipette under a dissecting microscope and placed in a 3.5 cm - diameter petri dish with 2 ml of water . an anuran was then placed in each dish and held with infective larvae for 1 h. we then removed the anuran and placed the dish under a dissecting microscope to count the larvae remaining . a second anuran was then added to the petri dish for 1 h. after this second 1-h infection period , the second anuran was removed and the number of remaining larvae counted once more . the combination of anurans in each petri dish was as follows : ( 1 ) cane toad ( n = 13 ) followed by l. convexiusculus ( n = 13 ) , ( 2 ) l. convexiusculus ( n = 13 ) followed by l. convexiusculus ( n = 13 ) , ( 3 ) cane toad ( n = 10 ) followed by c. australis ( n = 10 ) , ( 4 ) c. australis ( n = 10 ) followed by c. australis ( n = 10 ) and , ( 5 ) cane toad ( n = 4 ) followed by cane toad ( n = 4 ) . we measured parasite uptake as the difference in number of larvae between the beginning and end of each 1 h trial . this assumes that any missing larvae had crawled onto the anuran host and been removed along with it at the end of the trial . metamorphs varied by a maximum of only 1.62 g , but anuran body mass was still used as a covariate in the analyses . type ( the precedence combination of species 1/species 2 ) , order of exposure of each anuran ( first vs second ) , body mass and the order*type interaction term as independent variables and the number of larvae taken up as the dependent variable . we carried out histological examinations to verify that a 1 h exposure to 30 l3 was sufficient to allow successful larval penetration . five days after the exposure trials a subsample of 17 anurans ( 5 cane toads , 6 c. australis , 6 l. convexiusculus ) were euthanised by immersion in a solution of buffered tricaine methanesulfonate ( ms-222 ) . for histological examination , five to six 5-m serial transverse sections were made encompassing the tissue from the head to the pelvis of each anuran and stained with hematoxylin and eosin ( see pizzatto et al . slides were examined for the presence of larvae and characteristic inflammatory foci associated with degenerating larvae ( nelson et al . , 2015b ) . as part of another study , we exposed 32 metamorph cane toads to 30 infective larvae of r. hylae for 24 h and then measured correlates of fitness over 45 days ( nelson et al . after this experiment had concluded 45 days post - treatment ( dpt ) , we exposed 7 of the cane toads that had been previously exposed to r. hylae as part of this experiment , and 7 control toads ( with no prior exposure to r. hylae , but otherwise identical husbandry conditions ) to 30 r. pseudosphaerocephala larvae . this was done by placing each metamorph separately in a 3.5 cm diameter petri dish with 2 ml of water ( plus the parasite larvae ) for 24 h. toads were housed and fed for a subsequent 20 days and then euthanised by immersion in ms-222 as above . these toads were then dissected on day 65 to determine the number of lungworms in each lung . no cane toads out of the 25 that were dissected contained r. hylae in their lungs after 45 days ( due to a severe immune response by toads and aberrant migration of larvae rather than a lack of penetration by larvae : nelson et al . thus , any lungworms found in the re - exposed toad 's lungs after 65 days were assumed to be r. pseudosphaerocephala , rather than r. hylae . we could therefore determine whether prior exposure to r. hylae influenced the number of r. pseudosphaerocephala that reached the lungs of the experimental toads . we compared the presence versus absence of lungworms at the time of euthanasia between treatments ( prior exposure to r. hylae , versus no prior exposure to r. hylae ) using a nominal logistic regression with treatment as the independent variable . we compared the number of lungworms between treatments using nonparametric statistics ( wilcoxon test ) . this research was approved by the university of sydney animal ethics committee ( aec protocol number : 6042 ) . the number of r. hylae larvae taken up by an anuran was reduced by the prior presence of another anuran in the exposure chamber ( the first anuran took up some of the available larvae , thus reducing the number available to infect the subsequently - available host ) . secondarily - exposed anurans on average took up 42.4% fewer larvae than those exposed first ( f1,90 = 7.37 , p = 0.008 ) . this effect was not dependent on body mass ( f1,90 = 0.05 , p = 0.83 ) or on which anuran species was exposed first versus second ( f4,90 = 0.60 , p = 0.66 ; fig . 1a ) : that is , the second anuran's reduction in parasite uptake was just as high if the anurans exposed first took up 8.68 larvae on average , whereas anurans exposed second took up 3.68 larvae on average . this pattern remained when all native frogs were pooled into one group and compared to toads ( order : f1,92 = 8.11 , p = 0.005 ; mass : f1,92 = 0.04 , p = 0.84 , anuran combination : f2,92 = 1.16 , p = 0.32 ; fig . two of the 17 anurans examined histologically 5 days after infection , showed evidence of successful penetration by larvae . one l. convexiusculus had nematode cross - sections subcutaneously and in its coelom and one c. australis had nematode cross - sections in a lung . we found relatively few lungworms ( mean = 0.34 per toad ) in the lungs of metamorphs when these animals were dissected after 65 days , with no significant difference in the absence or presence of lungworms between treatments ( prior exposure to 30 r. hylae larvae vs. control : 42.9% vs 28.6% with larvae ; = 0.32 , n = 14 , p = 0.58 ) or the number of lungworms between treatments ( mean 0.7 larvae vs mean 0.6 larvae ; wilcoxon : = 0.14 , n = 14 , p = 0.71 ; fig . 2 ) . the average body mass was 0.72 g ( range = 0.3 g ) . body mass did not differ significantly between treatment groups ( wilcoxon : = 0.50 , n = 14 , p = 0.48 ) . our experiments support the plausibility of one putative mechanism by which toad invasion might reduce parasite numbers in native frogs . as predicted by the sink hypothesis , the presence of a toad ( or frog ) in the experimental arena for an hour was enough to remove many of the parasite larvae in a defined area ; and as a result , an anuran that was later placed in the same arena was infected at a lower rate than would otherwise have been the case . however , our data falsified the main prediction from the other hypothesis that we tested ( immunological priming ) : prior infection with the lungworm from native frogs ( r. hylae ) did not render a cane toad more ( or less ) resistant to infection by the cane toad 's own lungworm species ( r. pseudosphaerocephala ) . sink mechanism was tested here with a simplistic experimental design ; the first anuran was forced into close contact with larvae in a small container and the subsequent anuran was placed in precisely the same location . in nature , the impact of prior residency of a frog or a cane toad will depend on infection dynamics , anuran densities and habitat overlap as well as a wealth of other variables ( prenter et al . , 2004 ; the three anuran species used in this experiment have very similar sizes as metamorphs but adult cane toads and c. australis are much larger than l. convexiusculus . thus , the results of this experiment do not reflect the precise magnitude of any sink effect in nature , as larger animals are likely to be penetrated by larvae at a higher rate . however , this experiment is a necessary first step in determining whether the sink is a viable mechanism , as it confirms that a toad can have as much effect as a frog in soaking up infective larvae of the frog parasite. plausibly , toads might have had less effect than frogs for example , toads might be less attractive to larvae , or the individual larvae that infect a toad might not be the same ones as are most likely to infect a frog . that ( at least some ) r. hylae recognise cane toads as a potential host , and are capable of entering their bodies ( nelson et al . , 2015b ) , is somewhat surprising . this nematode species would never have encountered a bufonid before 1935 ( or , in the study area where we worked , before 2005 ) . that the parasite recognises and manages to enter toads suggests that r. hylae may not be very discriminating in their choice of host . the same appears to be true for the toad 's lungworm , r. pseudosphaerocephala , which readily enters australian frogs ( pizzatto and shine , 2011 ) . currently little is known about the specificity of signals used by rhabidas spp . to locate hosts , although it is evident that these nematodes use a combination of chemotaxis and vibrations ( langford , 2010 ) . the lack of discrimination by r. hylae may be a disadvantage to this nematode when invasive cane toads arrive . unless the nematode can quickly adapt to exploit the new host species , its natural life cycle might be interrupted by the abundance of decoy toad hosts in which it can not reproduce ( paterson et al . , 2013b ) . cane toads often attain very high densities , sometimes outnumbering native frogs ( e.g. , freeland and kerin , 1988 ) , so that ( all else being equal ) a high proportion of r. hylae larvae may locate the accidental toad host , where they are eliminated by the toad 's immune defences ( nelson et al . , 2015b ) . whether or not this uptake by toads has a large - scale impact on prevalence and intensity of r. hylae infection in native frog populations in the wild ( as suggested by the data of lettoof et al . , 2013 ) if larval output is high , then the larvae that cane toads extract from the system may have little impact in reducing the numbers still available to infect frogs . have a high output ( between 10 and 100 eggs a day per lungworm ; personal observation ) , suggesting that the environment might be saturated by larvae ( many of which will never reach a frog host , even without toads to contend with ) . sink mechanism in this system ( e.g. laracuente et al . , 1979 ) . sink mechanisms can operate under a wide array of circumstances , such as livestock decoys reducing the spread of human diseases by vectors ( van buskirk and ostfeld , 1995 ; miller and huppert , 2013 ) , invasive snails in new zealand decreasing the transmission of a native trematode to native snails ( kopp and jokela , 2007 ) , and invasive molluscs decreasing parasite ( trematode ) burdens of native european mussels ( thieltges et al . , 2009 ) . field surveys , experiments and population modelling have indicated that invasive salmonids in new zealand act as a , 2011 , 2013a , b ) . in the only other amphibian host - parasite system in which such interactions have been studied , the presence of grey tree frogs reduces the infection rates of toads ( bufo americanus ) to ribeiroia ondatrae , a trematode that causes limb deformities ( johnson et al . , sink effects have been documented not only in the laboratory , but also with field experiments in intertidal zones , outdoor mesocosms and even entire wetlands ( e.g. upatham , 1972 ; upatham and sturrock , 1973 ; laracuente et al . it may often be true that increased biodiversity decreases disease risk in this way ( ostfeld and keesing , 2000 ; johnson et al . , 2008 ; ostfeld and keesing , 2012 ; vourc'h et al . , 2012 ; johnson et al . , these studies suggest that the sink hypothesis may be important if we are to understand wildlife disease ecology in invasive systems . it may have been ignored because it is counter - intuitive ; researchers may expect a detrimental impact from an invader , and hence focus their work on detecting such effects . the toad - frog - lungworm interaction in australia may offer an excellent model for further research on the for example , it would be logistically feasible to repeat our studies in small containers with different densities of the two hosts or in large outdoor enclosures under more natural conditions . it would also be interesting to test the reciprocal scenario ( native frogs infected with the cane toad lungworm , r. pseudosphaerocephala ) to see if native species act as a sink for cane toad lungworms , or tend to spillback the parasite to cane toad populations . the absence of histological evidence of larval penetration in all the anurans examined may be attributable to several factors . some larvae taken up from the infection arena may have been unable to penetrate the skin , being weakened or killed by antimicrobial peptides in the host 's skin secretions ( bowie and tyler , 2006 ) . larvae that reach the host are able to penetrate the skin ( gendron et al . , 2003 ; kelehear et al . , 2012 ) . the serial sections cut from each anuran were 2 mm apart and could have missed intersecting the larvae , which are only 1000 m long and 50 m in diameter . our purpose in conducting histological examinations was not to quantify rates of larval penetration in each individual , but only to verify that the experimental conditions enabled successful infection . in contrast to our experiments on the sink hypothesis , our attempt to test the there are many cases in immunology where initial exposure to a pathogen enables an animal to later recognise and reject a similar but not identical pathogen ( this is the principle behind vaccines : brunham and coombs , 1998 ; oettinger et al . , 1999 ; hooper et al . , 2004 ) . however , our experiments suggest that prior exposure to r. hylae has no effect on cane toad resistance towards r. pseudosphaerocephala . histological studies ( nelson et al . , 2015b ) demonstrate that cane toads do mount an immune response to r. hylae larvae but the current experiment indicates that this activation of the toad 's immune system does not immunise toads against subsequent attack by larvae of its co - evolved parasite , r. pseudosphaerocephala . although amphibians possess adaptive immune systems broadly similar to those seen in avian / mammalian species , some components are lacking ( fournier et al . , 2005 ) and therefore the efficacy of the system is in question ( hsu , 1998 ) . however , acquired immunity in amphibians can reduce the intensity and impact of subsequent re - infections ; frogs with previous exposure to chytridiomycosis are better able to survive re - infection than are immunologically nave frogs ( richmond et al . , 2009 ) . similarly , acquired immunity in xenopus laevis due to prior infection with a monogenean ( protopolystoma xenopodis ) increased resistance to re - infection ( tinsley et al . , 2012 ) . given the long time frame in our experiment ( 45 days ) , we expected the first exposure to prime the toad 's adaptive immune system to rhabdias spp chemicals . this immunological memory of the parasite might then translate into efficacy against a similar invader . contrary to this expectation , the initial exposure to r. hylae had no impact on the cane toad 's resistance to secondary infection by a congeneric lungworm . in summary , sinks for r. hylae and do not acquire immunity to their native lungworms through prior exposure to frog lungworms . this would be good news for native frogs if the advantage of reduced parasitism outweighed the stresses imposed by toads . however , the advantages of reduced parasitism may be low : nelson et al . ( 2015a ) showed that native frogs suffer few ill effects from infection by r. hylae . also , cane toad invasion typically does not have major overall effects on the abundance or viability of frog populations ( shine , 2014 ) . thus , the influence of cane toads on native frogs populations via the disruption of host - parasite interactions ( via sink mechanism ) may be minimal .

many invading species have brought devastating parasites and diseases to their new homes , thereby imperiling native taxa . potentially , though , invaders might have the opposite effect . if they take up parasites that otherwise would infect native taxa , but those parasites fail to develop in the invader , the introduced species might reduce parasite burdens of the native fauna . similarly , earlier exposure to the other taxon 's parasites might prime an anuran 's immune system such that it is then able to reject subsequent infection by its own parasite species . field surveys suggest that lungworm counts in native australian frogs decrease after the arrival of invasive cane toads ( rhinella marina ) , and laboratory studies confirm that native lungworm larvae enter , but do not survive in , the toads . in laboratory trials , we confirmed that the presence of anurans ( either frogs or toads ) in an experimental arena reduced uptake rates of lungworm larvae by anurans that were later added to the same arena . however , experimental exposure to lungworms from native frogs did not enhance a toad 's ability to reject subsequent infection by its own lungworm species .

Introduction Materials and methods Results Discussion Conflict of interest

, invaders often exhibit reduced parasite levels ( the enemy release hypothesis : marr et al . , 2008 ) , but novel pathogens brought by invaders can still devastate native taxa by directly reducing survival or by mediating the outcome of competition between native and invasive species ( e.g. the reverse scenario ( the transfer of native pathogens to the invader ) has similar effects ( dunn , 2009 ; hartigan et al . past studies have investigated cases where invaders act to increase parasitism of native fauna by introducing a new parasite or by acting as a reservoir for native parasites ( dobson and foufopoulos , 2001 ; mastitsky and veres , 2010 ; pizzatto and shine , 2011 ; hartigan et al . however in this instance , native parasites are taken up by the invader but fail to complete their life cycle due to a lack of co - evolutionary history . when the parasite enters a foreign host it becomes disoriented or attacked by the immune system . resistant targets , reducing the density of parasites in the environment and thus , lowering the risk of infection for native hosts ( heimpel et al . finding a host becomes more of a challenge in assemblages with a number of host species that differ in susceptibility to the parasite , such as invasive systems ( keesing et al . here , we examine the potential disruption of host - parasite interactions caused by the invasive cane toad , rhinella marina , in australia . recent studies have shown that cane toads and native frogs have separate nematode lungworm fauna ( pizzatto et al . these two parasite species are virtually indistinguishable in morphology , but genetic analysis shows that they are indeed separate species ( dubey and shine , 2008 ) , and experimental infections demonstrate that the lungworms perform differently in cane toad and native frog hosts ( pizzatto et al . r. hylae can penetrate cane toads but is killed by a strong immune response ; it becomes lost inside the novel host 's body and never reaches the lungs , where it would normally mature , produce eggs and complete its life cycle ( nelson , 2014 ; nelson et al . r. pseudosphaerocephala readily penetrates native frogs but only reaches the lungs in a small number of species , for similar reasons ( pizzatto et al . ( 2013 ) found lower rates of infection with lungworms in native frogs living in areas with cane toads , than in the same frog species living in nearby areas that lack cane toads . firstly , the cane toads could be acting as sinks ( removing parasite larvae from the environment , and dooming those parasites to an early death ) . secondly , native frogs that are exposed to the cane toad parasite might thereby develop acquired immunity to their own parasite species ( i.e. , an initial exposure to toad rhabdias spp . a similar priming of the cane toad 's immune system by frog rhabdias spp . against its more virulent native parasite would have substantial benefits for cane toads , and might enhance their invasion success . priming is the principle behind many vaccines , which exploit the capacity of the adaptive immune system to form memories in response to inert parts of pathogens ( brunham and coombs , 1998 ; oettinger et al . upon re - infection , memory of previously encountered pathogens and the acquired immune response has been shown to play a role in the improved response of amphibians towards infections ( richmond et al . , sink mechanism as it applies to cane toads soaking up native frog parasites , and the possibility that prior exposure to the native frog lungworm primes the cane toad 's immune system such that it is less vulnerable to infection by its own lungworm species . descriptions and details of methods for breeding and husbandry of anurans , and collection and identification of lungworm larvae used in the following experiments , appear in the supplementary material . to measure rates of r. hylae uptake by anurans we exposed each of 69 native frogs ( 30 cyclorana australis and 39 limnodynastes convexiusculus ) and 31 cane toads ( r.marina ) to infective lungworm larvae . after 24 days in these petri dishes , the free - living adult worms had produced infective third stage larvae ( l3 ) that we used for experimental infections . 30 larvae ( l3 ) were collected using a glass pipette under a dissecting microscope and placed in a 3.5 cm - diameter petri dish with 2 ml of water . an anuran was then placed in each dish and held with infective larvae for 1 h. we then removed the anuran and placed the dish under a dissecting microscope to count the larvae remaining . a second anuran was then added to the petri dish for 1 h. after this second 1-h infection period , the second anuran was removed and the number of remaining larvae counted once more . the combination of anurans in each petri dish was as follows : ( 1 ) cane toad ( n = 13 ) followed by l. convexiusculus ( n = 13 ) , ( 2 ) l. convexiusculus ( n = 13 ) followed by l. convexiusculus ( n = 13 ) , ( 3 ) cane toad ( n = 10 ) followed by c. australis ( n = 10 ) , ( 4 ) c. australis ( n = 10 ) followed by c. australis ( n = 10 ) and , ( 5 ) cane toad ( n = 4 ) followed by cane toad ( n = 4 ) . this assumes that any missing larvae had crawled onto the anuran host and been removed along with it at the end of the trial . metamorphs varied by a maximum of only 1.62 g , but anuran body mass was still used as a covariate in the analyses . type ( the precedence combination of species 1/species 2 ) , order of exposure of each anuran ( first vs second ) , body mass and the order*type interaction term as independent variables and the number of larvae taken up as the dependent variable . we carried out histological examinations to verify that a 1 h exposure to 30 l3 was sufficient to allow successful larval penetration . five days after the exposure trials a subsample of 17 anurans ( 5 cane toads , 6 c. australis , 6 l. convexiusculus ) were euthanised by immersion in a solution of buffered tricaine methanesulfonate ( ms-222 ) . for histological examination , five to six 5-m serial transverse sections were made encompassing the tissue from the head to the pelvis of each anuran and stained with hematoxylin and eosin ( see pizzatto et al . slides were examined for the presence of larvae and characteristic inflammatory foci associated with degenerating larvae ( nelson et al . as part of another study , we exposed 32 metamorph cane toads to 30 infective larvae of r. hylae for 24 h and then measured correlates of fitness over 45 days ( nelson et al . after this experiment had concluded 45 days post - treatment ( dpt ) , we exposed 7 of the cane toads that had been previously exposed to r. hylae as part of this experiment , and 7 control toads ( with no prior exposure to r. hylae , but otherwise identical husbandry conditions ) to 30 r. pseudosphaerocephala larvae . no cane toads out of the 25 that were dissected contained r. hylae in their lungs after 45 days ( due to a severe immune response by toads and aberrant migration of larvae rather than a lack of penetration by larvae : nelson et al . thus , any lungworms found in the re - exposed toad 's lungs after 65 days were assumed to be r. pseudosphaerocephala , rather than r. hylae . we could therefore determine whether prior exposure to r. hylae influenced the number of r. pseudosphaerocephala that reached the lungs of the experimental toads . we compared the presence versus absence of lungworms at the time of euthanasia between treatments ( prior exposure to r. hylae , versus no prior exposure to r. hylae ) using a nominal logistic regression with treatment as the independent variable . the number of r. hylae larvae taken up by an anuran was reduced by the prior presence of another anuran in the exposure chamber ( the first anuran took up some of the available larvae , thus reducing the number available to infect the subsequently - available host ) . secondarily - exposed anurans on average took up 42.4% fewer larvae than those exposed first ( f1,90 = 7.37 , p = 0.008 ) . 1a ) : that is , the second anuran's reduction in parasite uptake was just as high if the anurans exposed first took up 8.68 larvae on average , whereas anurans exposed second took up 3.68 larvae on average . this pattern remained when all native frogs were pooled into one group and compared to toads ( order : f1,92 = 8.11 , p = 0.005 ; mass : f1,92 = 0.04 , p = 0.84 , anuran combination : f2,92 = 1.16 , p = 0.32 ; fig . two of the 17 anurans examined histologically 5 days after infection , showed evidence of successful penetration by larvae . we found relatively few lungworms ( mean = 0.34 per toad ) in the lungs of metamorphs when these animals were dissected after 65 days , with no significant difference in the absence or presence of lungworms between treatments ( prior exposure to 30 r. hylae larvae vs. control : 42.9% vs 28.6% with larvae ; = 0.32 , n = 14 , p = 0.58 ) or the number of lungworms between treatments ( mean 0.7 larvae vs mean 0.6 larvae ; wilcoxon : = 0.14 , n = 14 , p = 0.71 ; fig . body mass did not differ significantly between treatment groups ( wilcoxon : = 0.50 , n = 14 , p = 0.48 ) . our experiments support the plausibility of one putative mechanism by which toad invasion might reduce parasite numbers in native frogs . as predicted by the sink hypothesis , the presence of a toad ( or frog ) in the experimental arena for an hour was enough to remove many of the parasite larvae in a defined area ; and as a result , an anuran that was later placed in the same arena was infected at a lower rate than would otherwise have been the case . however , our data falsified the main prediction from the other hypothesis that we tested ( immunological priming ) : prior infection with the lungworm from native frogs ( r. hylae ) did not render a cane toad more ( or less ) resistant to infection by the cane toad 's own lungworm species ( r. pseudosphaerocephala ) . sink mechanism was tested here with a simplistic experimental design ; the first anuran was forced into close contact with larvae in a small container and the subsequent anuran was placed in precisely the same location . in nature , the impact of prior residency of a frog or a cane toad will depend on infection dynamics , anuran densities and habitat overlap as well as a wealth of other variables ( prenter et al . , 2004 ; the three anuran species used in this experiment have very similar sizes as metamorphs but adult cane toads and c. australis are much larger than l. convexiusculus . thus , the results of this experiment do not reflect the precise magnitude of any sink effect in nature , as larger animals are likely to be penetrated by larvae at a higher rate . however , this experiment is a necessary first step in determining whether the sink is a viable mechanism , as it confirms that a toad can have as much effect as a frog in soaking up infective larvae of the frog parasite. plausibly , toads might have had less effect than frogs for example , toads might be less attractive to larvae , or the individual larvae that infect a toad might not be the same ones as are most likely to infect a frog . that ( at least some ) r. hylae recognise cane toads as a potential host , and are capable of entering their bodies ( nelson et al . , 2015b ) , is somewhat surprising . this nematode species would never have encountered a bufonid before 1935 ( or , in the study area where we worked , before 2005 ) . that the parasite recognises and manages to enter toads suggests that r. hylae may not be very discriminating in their choice of host . the same appears to be true for the toad 's lungworm , r. pseudosphaerocephala , which readily enters australian frogs ( pizzatto and shine , 2011 ) . to locate hosts , although it is evident that these nematodes use a combination of chemotaxis and vibrations ( langford , 2010 ) . the lack of discrimination by r. hylae may be a disadvantage to this nematode when invasive cane toads arrive . cane toads often attain very high densities , sometimes outnumbering native frogs ( e.g. , freeland and kerin , 1988 ) , so that ( all else being equal ) a high proportion of r. hylae larvae may locate the accidental toad host , where they are eliminated by the toad 's immune defences ( nelson et al . whether or not this uptake by toads has a large - scale impact on prevalence and intensity of r. hylae infection in native frog populations in the wild ( as suggested by the data of lettoof et al . , 2013 ) if larval output is high , then the larvae that cane toads extract from the system may have little impact in reducing the numbers still available to infect frogs . have a high output ( between 10 and 100 eggs a day per lungworm ; personal observation ) , suggesting that the environment might be saturated by larvae ( many of which will never reach a frog host , even without toads to contend with ) . sink mechanisms can operate under a wide array of circumstances , such as livestock decoys reducing the spread of human diseases by vectors ( van buskirk and ostfeld , 1995 ; miller and huppert , 2013 ) , invasive snails in new zealand decreasing the transmission of a native trematode to native snails ( kopp and jokela , 2007 ) , and invasive molluscs decreasing parasite ( trematode ) burdens of native european mussels ( thieltges et al . field surveys , experiments and population modelling have indicated that invasive salmonids in new zealand act as a , 2011 , 2013a , b ) . in the only other amphibian host - parasite system in which such interactions have been studied , the presence of grey tree frogs reduces the infection rates of toads ( bufo americanus ) to ribeiroia ondatrae , a trematode that causes limb deformities ( johnson et al . , sink effects have been documented not only in the laboratory , but also with field experiments in intertidal zones , outdoor mesocosms and even entire wetlands ( e.g. , these studies suggest that the sink hypothesis may be important if we are to understand wildlife disease ecology in invasive systems . it may have been ignored because it is counter - intuitive ; researchers may expect a detrimental impact from an invader , and hence focus their work on detecting such effects . the toad - frog - lungworm interaction in australia may offer an excellent model for further research on the for example , it would be logistically feasible to repeat our studies in small containers with different densities of the two hosts or in large outdoor enclosures under more natural conditions . it would also be interesting to test the reciprocal scenario ( native frogs infected with the cane toad lungworm , r. pseudosphaerocephala ) to see if native species act as a sink for cane toad lungworms , or tend to spillback the parasite to cane toad populations . some larvae taken up from the infection arena may have been unable to penetrate the skin , being weakened or killed by antimicrobial peptides in the host 's skin secretions ( bowie and tyler , 2006 ) . larvae that reach the host are able to penetrate the skin ( gendron et al . our purpose in conducting histological examinations was not to quantify rates of larval penetration in each individual , but only to verify that the experimental conditions enabled successful infection . in contrast to our experiments on the sink hypothesis , our attempt to test the there are many cases in immunology where initial exposure to a pathogen enables an animal to later recognise and reject a similar but not identical pathogen ( this is the principle behind vaccines : brunham and coombs , 1998 ; oettinger et al . however , our experiments suggest that prior exposure to r. hylae has no effect on cane toad resistance towards r. pseudosphaerocephala . , 2015b ) demonstrate that cane toads do mount an immune response to r. hylae larvae but the current experiment indicates that this activation of the toad 's immune system does not immunise toads against subsequent attack by larvae of its co - evolved parasite , r. pseudosphaerocephala . , 2005 ) and therefore the efficacy of the system is in question ( hsu , 1998 ) . however , acquired immunity in amphibians can reduce the intensity and impact of subsequent re - infections ; frogs with previous exposure to chytridiomycosis are better able to survive re - infection than are immunologically nave frogs ( richmond et al . similarly , acquired immunity in xenopus laevis due to prior infection with a monogenean ( protopolystoma xenopodis ) increased resistance to re - infection ( tinsley et al . , 2012 ) . given the long time frame in our experiment ( 45 days ) , we expected the first exposure to prime the toad 's adaptive immune system to rhabdias spp chemicals . this immunological memory of the parasite might then translate into efficacy against a similar invader . contrary to this expectation , the initial exposure to r. hylae had no impact on the cane toad 's resistance to secondary infection by a congeneric lungworm . in summary , sinks for r. hylae and do not acquire immunity to their native lungworms through prior exposure to frog lungworms . this would be good news for native frogs if the advantage of reduced parasitism outweighed the stresses imposed by toads . however , the advantages of reduced parasitism may be low : nelson et al . ( 2015a ) showed that native frogs suffer few ill effects from infection by r. hylae . thus , the influence of cane toads on native frogs populations via the disruption of host - parasite interactions ( via sink mechanism ) may be minimal .

leishmaniases are a set of vector - borne diseases caused by a flagellate protozoan transmitted by the bite of an insect vector , the phlebotomine sandfly . this group of diseases affects 98 countries with three distinct entities : cutaneous , mucocutaneous , and visceral leishmaniasis . various clinical outcomes are described from a simple skin lesion that may heal spontaneously to a multi - organ failure , fatal if untreated . there is still no human vaccine against this disease and therapy takes a major place in the control strategies . the use of the liposomal form of amphotericin b , a highly active molecule with reduced side effects , is still restricted to the treatment of visceral leishmaniasis in countries that can afford its cost , such as european countries . however europe accounts for less than 1% of the approximately 500,000 cases per year which occur mainly in the indian subcontinent , sudan , and brazil . other molecules such as pentamidine , miltefosine , or fluconazole are available but their use is restricted owing to side effects , cost , or effectiveness . this accounts for the still predominant place of antimony derivatives , sodium stibiogluconate ( pentostam ) , and meglumine antimoniate ( glucantime ) which have been used in the treatment of the majority of cases of leishmaniases for more than 60 years worldwide . currently , these molecules have two major limitations : first , side effects are frequent and can be serious ; second , parasite resistance is emerging in some endemic areas , causing an increase in treatment failure [ 5 , 6 ] . resistance to antimonials has emerged over 20 years in the region of bihar in india . low dosage or insufficient duration of treatment led to the selection of resistant mutants that were transmitted more easily in this anthroponotic form than in areas where anthropozoonotic cycle occurs . currently , in the most heavily affected areas of india , resistance to antimonials may reach more than 60% of the cases , thus representing a public health problem . it imposed the use of other drugs such as amphotericine b or , more recently , miltefosine . the selection occurs in dogs which , unlike men , are still being treated with antimonials . in other mediterranean areas such as maghreb , albania , and middle east except israel , most infected dogs are left untreated ; antimonials are still the first line treatment and display high efficiency [ 11 , 12 ] . the issue of antileishmanial therapy depends on several factors such as the immune status of the host , the molecule , its preparation , its administration , and the susceptibility of the strain to the drug [ 13 , 14 ] . drug resistance of leishmania may be natural , acquired when the parasite is exposed to suboptimal doses of the drug , or induced in vitro after selection of mutants by exposure to gradually increasing concentrations of the drug . first , we relate methodology , general limitations , and tools used for resistance surveys . in a second step , finally , we focus on molecular resistance pathways and already identified targets within in vitro selected mutants and field isolates . they address the phenotypic , genomic , or proteomic levels . before discussing the different ways of investigation , we present some general considerations related to leishmania isolates , the evolution of their characteristics during maintenance in vitro , selection of mutants , and postulates about cross - resistance mechanisms . ( 1 ) leishmania speciesseveral in vitro experiments were performed on l. tarentolae , a non - infective species for laboratory personnel [ 1520 ] . by using this species , different resistance mechanisms and molecular targets were identified and their presence confirmed in other species selected in vitro or from clinical isolates . however , intrinsic susceptibility to heavy metals and thiols levels found in l. tarentolae can be very different from those encountered in leishmania species of clinical interest . several in vitro experiments were performed on l. tarentolae , a non - infective species for laboratory personnel [ 1520 ] . by using this species , different resistance mechanisms and molecular targets were identified and their presence confirmed in other species selected in vitro or from clinical isolates . however , intrinsic susceptibility to heavy metals and thiols levels found in l. tarentolae can be very different from those encountered in leishmania species of clinical interest . ( 2 ) parasitic stage and resistancemost of in vitro resistance experiments are performed on promastigotes , easier to cultivate and to quantify than amastigotes . however promastigotes are usually not sensitive to sbv which has a stage - specific activity unless they are exposed to pentostam and several experiments indicate that this susceptibility is not due to sodium stibiogluconate but rather to chlorocresol included in pentostam formulation [ 22 , 23 ] . interestingly , chlorocresol concentration for pentostam ic50 was eight - fold lower than chlorocresol ic50 in axenic amastigotes . thus increased susceptibility of axenic amastigotes to pentostam is mainly due to sbv . in another study , l. donovani amastigotes were either sbiii or sbv ( glucantime or pentostam chlorocresol free ) sensitive unlike promastigotes which displayed a very high ic50 . susceptibility to sbv follows stage transformation from promastigotes to axenic amastigotes while resistance to sbv is acquired while amastigotes differentiate into promastigotes . carriers belonging to the abc system and involved in the efflux or sequestration of antimonials have indeed been identified in promastigotes resistant mutants and few studies on the role of these proteins have been conducted on amastigotes . most of in vitro resistance experiments are performed on promastigotes , easier to cultivate and to quantify than amastigotes . however , they do not represent the parasitic stage on which antimonials act in vivo . promastigotes are usually not sensitive to sbv which has a stage - specific activity unless they are exposed to pentostam and several experiments indicate that this susceptibility is not due to sodium stibiogluconate but rather to chlorocresol included in pentostam formulation [ 22 , 23 ] . . showed that l. donovani axenic amastigotes were considerably more susceptible to pentostam than promastigotes . interestingly , chlorocresol concentration for pentostam ic50 was eight - fold lower than chlorocresol ic50 in axenic amastigotes . thus increased susceptibility of axenic amastigotes to pentostam is mainly due to sbv . in another study , l. donovani amastigotes were either sbiii or sbv ( glucantime or pentostam chlorocresol free ) sensitive unlike promastigotes which displayed a very high ic50 . susceptibility to sbv follows stage transformation from promastigotes to axenic amastigotes while resistance to sbv is acquired while amastigotes differentiate into promastigotes . carriers belonging to the abc system and involved in the efflux or sequestration of antimonials have indeed been identified in promastigotes resistant mutants and few studies on the role of these proteins have been conducted on amastigotes . ( 3 ) selection during treatment of the hostthe in vitro sensitivity of strains isolated from untreated hosts did not change despite the successive passages in culture or after animal infection . however , the sensitivity was reduced in strains isolated from either patients or dogs treated with antimonials [ 9 , 10 , 27 ] . the growth and proliferation of cultured strains isolated after treatment were lowered compared to the strains isolated before treatment . the in vitro sensitivity of strains isolated from untreated hosts did not change despite the successive passages in culture or after animal infection . however , the sensitivity was reduced in strains isolated from either patients or dogs treated with antimonials [ 9 , 10 , 27 ] . the growth and proliferation of cultured strains isolated after treatment were lowered compared to the strains isolated before treatment . ( 1 ) culture mediumthe growth rate of l. infantum promastigotes influences their ic50 for sbv chlorocresol - free formulations as reported by carrio et al . in fact , different ic50 values can be obtained for the same strain by varying heat - inactivated fetal calf serum concentration in culture medium . variation of ph can also influence promastigotes susceptibility to sbv mostly for schneider 's medium but also for m199 and rpmi media . the growth rate of l. infantum promastigotes influences their ic50 for sbv chlorocresol - free formulations as reported by carrio et al . in fact , different ic50 values can be obtained for the same strain by varying heat - inactivated fetal calf serum concentration in culture medium . variation of ph can also influence promastigotes susceptibility to sbv mostly for schneider 's medium but also for m199 and rpmi media . ( 2 ) impact of in vitro culture on primary phenotype leishmania parasites isolated from an infected host may present a polyclonal population with a varied expression of different phenotypes such as growth capacity in vitro , infectivity , and drug resistance [ 29 , 30 ] . obtaining a sufficient number of promastigotes to conduct experiments often requires numerous passages and thus can induce changes in the expression pattern of the parasite . the possible selection of one or some clones that were not dominant at the time of parasite isolation may complicate the interpretation of a resistance survey . indeed , the virulence of a strain and its capacity for growth and adaptation in culture can alter the clonal composition of strain over successive passages in the medium and so modify the expression of resistance phenotype . however , experiments have shown that during induction of in vitro resistance by discontinuous exposition of parasites to the drug , the resistance phenotype does not vary either during successive passages in culture in the absence of the drug or after in vivo infection [ 29 , 30 ] . in addition , antimonial resistant l. donovani indian strains cultured with several passages before infection of macrophages or hamsters maintained their resistance profile . leishmania parasites isolated from an infected host may present a polyclonal population with a varied expression of different phenotypes such as growth capacity in vitro , infectivity , and drug resistance [ 29 , 30 ] . obtaining a sufficient number of promastigotes to conduct experiments often requires numerous passages and thus can induce changes in the expression pattern of the parasite . the possible selection of one or some clones that were not dominant at the time of parasite isolation may complicate the interpretation of a resistance survey . indeed , the virulence of a strain and its capacity for growth and adaptation in culture can alter the clonal composition of strain over successive passages in the medium and so modify the expression of resistance phenotype . however , experiments have shown that during induction of in vitro resistance by discontinuous exposition of parasites to the drug , the resistance phenotype does not vary either during successive passages in culture in the absence of the drug or after in vivo infection [ 29 , 30 ] . in addition , antimonial resistant l. donovani indian strains cultured with several passages before infection of macrophages or hamsters maintained their resistance profile . ( 3 ) use of arsenicals for in vitro selectionseveral experiments on leishmania resistance were performed with a progressive selection of mutant strains by exposing promastigotes to increasing concentrations of arsenicals [ 17 , 20 , 32 , 33 ] . these studies suffer the selection bias of arsenic derivatives not used in antileishmanial therapy but whose use in vitro showed that they confer cross - resistance to antimonials . arsenicals have undubitably common characteristics with antimonials in their mode of action such as increased reactive oxygen species ( ros ) , loss of mitochondrial membrane potential and collapse of the available quantity of atp followed by a cell death that occurs by cell shrinkage and dna fragmentation . however , arsenicals induce an increase in intracellular calcium that does not occur with antimonials . conversely , the level of intracellular glutathione which is not affected during treatment with arsenicals is lowered when antimonials are used . the inhibition of calcium influx in case of arsenicals and glutathione supplementation in presence of antimony decreased cell death in both cases . it is therefore conceivable that the mutant strains selected in vitro against arsenic derivatives and then tested against antimonials may have developed resistance mechanisms specific to arsenite along with other common mechanisms , thus explaining the phenomenon of cross - resistance . several experiments on leishmania resistance were performed with a progressive selection of mutant strains by exposing promastigotes to increasing concentrations of arsenicals [ 17 , 20 , 32 , 33 ] . these studies suffer the selection bias of arsenic derivatives not used in antileishmanial therapy but whose use in vitro showed that they confer cross - resistance to antimonials . arsenicals have undubitably common characteristics with antimonials in their mode of action such as increased reactive oxygen species ( ros ) , loss of mitochondrial membrane potential and collapse of the available quantity of atp followed by a cell death that occurs by cell shrinkage and dna fragmentation . however , arsenicals induce an increase in intracellular calcium that does not occur with antimonials . conversely , the level of intracellular glutathione which is not affected during treatment with arsenicals is lowered when antimonials are used . the inhibition of calcium influx in case of arsenicals and glutathione supplementation in presence of antimony decreased cell death in both cases . it is therefore conceivable that the mutant strains selected in vitro against arsenic derivatives and then tested against antimonials may have developed resistance mechanisms specific to arsenite along with other common mechanisms , thus explaining the phenomenon of cross - resistance . ( 4 ) sbiii , and sbv - selected mutantsthe use of sbiii that is not the prescribed form in clinical practice but represents the biologically active form of the drug would lead to the selection of resistant strains with different mechanisms and targets that are not necessarily involved in clinical isolates resistance since these are first exposed to sbv . however , cross - resistance between sbv , sbiii , and asiii that was observed in some leishmania species such as l. tarentolae , l. major , l. mexicana , and l. infantum [ 15 , 35 , 36 ] allowed performing resistance experiments with sbiii . however , in some l. donovani strains , no cross - resistance was found . experiments on l. panamensis strains from clinical relapse showed that while amastigotes were sbv resistant , and promastigotes were sensitive to sbiii . the use of sbiii that is not the prescribed form in clinical practice but represents the biologically active form of the drug would lead to the selection of resistant strains with different mechanisms and targets that are not necessarily involved in clinical isolates resistance since these are first exposed to sbv . however , cross - resistance between sbv , sbiii , and asiii that was observed in some leishmania species such as l. tarentolae , l. major , l. mexicana , and l. infantum [ 15 , 35 , 36 ] allowed performing resistance experiments with sbiii . however , in some l. donovani strains , no cross - resistance was found . experiments on l. panamensis strains from clinical relapse showed that while amastigotes were sbv resistant , and promastigotes were sensitive to sbiii . the infection of mice or hamsters , which was among the first approaches for studying the sensitivity to antimonials in vivo , still faces the problem of variability related to the host and genetic background of these vertebrate hosts may have a major influence upon evolution after infection . they are currently mainly performed for immunological or genetic studies [ 31 , 38 , 39 ] . macrophages cultivation and their infection with promastigotes are useful tools for analysing the sensitivity of amastigotes . this technique is time consuming and it suffers from the variability of infectivity due to the growth characteristics of the strain and to in vitro metacyclogenesis level . however , it remains a reference technique for studying the resitance phenotype of clinical isolates . different cell lines can be used such as murine peritoneal exudate macrophages , murine bone marrow - derived macrophages , human monocyte - derived macrophages , and the human monocytic thp-1 cell line . recently , it has been shown that either the infection kinetics or the activity of the drug especially antimonials depends on the host cell type . consequently , macrophage population must be taken into account before any comparison between studies and there is a need for standardization . the difficulty of sensitivity tests on infected macrophages is partially overcome by the axenic amastigotes approach , also called amastigotes - like . these stages are obtained by gradual changes of temperature and ph of cultured promastigotes until the parasites resemble amastigotes and express specific proteins and morphology of this stage . nevertheless , amastigote - like cells are heterogeneous and do not acquire all the characteristics of in vivo amastigotes . moreover , long - term culture may lead to reappearance of promastigote - like pattern . while several studies have used this model and highlighted axenic amastigotes susceptibility to sbv [ 25 , 35 ] , other reports showed that pentavalent antimony is inactive on these forms and recommend intracellular amastigotes as the gold standard for in vitro sensitivity studies . it is important to assess whether in vitro tests used to determine the sensitivity of a clinical isolate reflect the response to treatment and clinical resistance . evidenced that in clinical isolates , among both hiv and immunocompetent patients , the ic50 of amastigotes within macrophages was significantly higher in patients who have not improved , compared to those who have responded to treatment . similarly , strains from patients previously treated exhibited higher ic50 than strains from untreated patients . there was no difference in the ic50 for patients cured compared to those who developed a relapse after an initial improvement but iterative treatment with antimonials induced an increase in ic50 in strains isolated successively from the same patient . thus , in vitro susceptibility of clinical isolates correlated well with clinical outcome in immediate treatment . in contrast , the long - term treatment did not appear correlated with in vitro tests since relapses occurred even in patients harboring susceptible strains . correlation between the clinical outcome and in vitro susceptibility tests was also observed in indian field isolates . a correlation was also found between treatment failure in patients , the ic50 of l. donovani promastigotes and the lack of response in the animal model suggesting the use of the ic50 study as a reliable test for resistance . in an area highly endemic for visceral leishmaniasis in india , a double - blinded survey was performed in which intracellular amastigotes ic50 was determined for the first group of patients , whereas in the second group , patients were treated without testing the parasite sensitivity . results showed a statistically significant improvement of cure rate reaching 86% in the first group compared to 35% in patients treated with sbv without in vitro tests . nevertheless , clinical outcome and in vitro susceptibility tests can be contradictory as shown for l. donovani clinical isolates tested for their sensitivity to sbv and sbiii . indeed , rijal et al . identified three profiles of resistance : strains sensitive to both forms of antimony , sbv resistant strains but sbiii sensitive and finally strains refractory to the two compounds . all patients unresponsive to treatment were infected with strains resistant to pentavalent antimony but this phenotype was also found in patients who responded to treatment . some strains harvested from cured patients displayed in vitro resistance to sbiii . finally , out of 3 patients whose strains were resistant to both sbv and sbiii , only one experienced treatment failure . these surveys suffer most from a lack of standardization regarding the criteria for treatment failure , the difference of resistance threshold , and the patients ' population under study . as mentioned above , these tests are carried out on selected in vitro mutants , on clinical human isolates , or on strains affecting other vertebrates such as l. tarentolae , a species widely used in vitro . revertant strains are obtained by successive passages in culture of the resistant strain without drug exposure . gene amplification was the first molecular resistance mechanism identified in l. major resistant to methotrexate ( mtx ) and displayed by the presence of h - circle . the presence of circular amplicons in leishmania is often the consequence of homologous recombination between repeated sequences . these events seem to be frequent in leishmania and early associated to resistance phenotype . the presence of these elements has also been found in strains selected for other molecules unrelated to mtx as not only primaquine , and terbinafine , vinblastine but also arsenicals [ 50 , 51 ] . this amplification may concern preexistant or novel h - circles [ 52 , 53 ] . in addition to multidrug - related protein a ( mrpa ) , two other genes have been identified within the h - circles : pteridine reductase 1 ( ptr1 ) and h region terbinafine - associated resistance gene ( htbf ) [ 54 , 55 ] . historical techniques for dna and rna analysis ( southern and northern blotting ) are now replaced by quantitative real time pcr ( qpcr ) or reverse transcription qpcr ( rt - qpcr ) . qpcr is performed in order to compare amplification rate of a target gene between susceptible and resistant leishmania strains . results are expressed as threshold cycle ( ct ) and need to be standardized by using a housekeeping gene in the studied strains hence revealing possible gene amplification in resistant strain compared with wild type one . gene amplification can also be established by southern blot by hybridization of gene of interest with its complementary probe [ 57 , 58 ] . quantitative reverse transcription pcr ( qrt - pcr ) is an approach used in this context to highlight gene overexpression of rna which can be independent of amplification . recent advances in genomic approaches of leishmania resistance were brought by dna microarray technique concomitant with whole genome sequencing of l. infantum and l. major enabling an exhaustive prospection of differences between resistant and susceptible strains at gene level . differential expression among the two strains is easily recognized by disposing of cdna genomic bank and different genetic markers . dna microarray technology allows testing a large set of genes at the dna or rna levels in a unique experiment and so is useful as screening test . functional cloning of cosmid bank is a widely used technique for determination of genes functions . there are two approaches for functional cloning depending on expected effect , dominant positive one and dominant negative one . in case of dominant positive cloning , the cosmid is transfected into a wild - type strain , and acquisition of a new function such as drug resistance is analyzed . by contrast in dominant negative selection resistant strains are transfected with the cosmid in order to obtain a reversal of resistance . once identified , cosmid of interest is retransfected in the strain under study to confirm the phenotype . subsequently , the cosmid is sequenced and blasted with genomic bank in order to identify the resistance gene . in addition , this gene could be transfected into wild type strain to ascertain its role in resistance . gene transfection is used to identify a new phenotype or a new functionality such as resistance . the strain transfected with gene of interest is compared to the same strain transfected with vector only , so keeping the original phenotype . protein localization can also be determined by cotransfection experiments with green fluorescent protein ( gfp ) . phenotype deletion at genomic scale is carried out by gene knockout ( ko ) approach . as leishmania is a diplod organism , two strategies are possible : a single allele ko resulting in a decrease in targeted functionality and double knockout leading to phenotype suppression . isolates of l. donovani have been studied on the basis of their genetic diversities , variations , and molecular phylogenetic structure using fingerprinting approach - amplified fragment length polymorphism ( aflp ) and identified a polymorphism percentage of 55% . genetic studies , analyzing the whole genome , would present the advantage of searching gene of interest without any previous choice concerning their function . pulse field gel electrophoresis ( pfge ) can highlight differences at chromosomal scale between two strains in addition to the possibility of differentiation between linear and supercoiled dna by varying pulse time . proteomic approach was initially reductive but last years global techniques as two - dimensional electrophoresis and more recently mass spectrometry offer a global analysis of cell proteome . proteomic approach can be performed by immuno - enzymatic assays such as western blot ( wb ) , a semiquantitative technique that can reveal the presence of a protein and compare its relative abundance to other strains based on the band intensity . necessity of renaturation of protein during tranfert and availability of specific antibodies constitute the main limiting factors . flow cytometry was used in two aims : as a tools for leishmania quantification in the last stage of sensitivity testing of infected cells and more interestingly for functional analysis of drugs transporter by using fluorescent drug analogs and specific inhibitors . specific inhibitors are commonly used for two purposes : the first is in vitro study of inhibiting an enzyme supposed to participate in resistance phenotype ; the second is withdrawal of resistance profile in animal model using such inhibitor in combination with antimonials . its field of interest can range from screening of proteins giving an overall assessment of the differences that may exist between two strains to the sequencing of a protein . this approach is usually preceded by a two - dimensional electrophoresis or by liquid chromatography . high - performance liquid chromaography ( hplc ) was mainly used for isolation , identification , and quantification of drug and/or thiols metabolites [ 57 , 63 ] . injection - coupled plasma mass spectrometry ( icpms ) is used in leishmania resistance surveys to measure differences in uptake and accumulation of sbv or sbiii depending on strain sensitivity or its stage . all these techniques allowed to discover and investigate one or several mechanisms related to leishmania drug resistance . they involve drug entry , thiol metabolism pathway , drug transport and sequestration , programmed cell death and other minor mechanisms or cofactors as illustrated in figure 1 . resistance pathways in leishmania share some common features with other microorganisms such as drug entry , drug metabolism , efflux and/or sequestration , programmed cell death along with action on host cell . the route of entry of antimonials is still unclear and does not appear to be the same for sbv and sbiii . accumulation of sbv and sbiii by antimonials sensitive and resistant strains of different leishmania species showed that both axenic amastigotes and promastigotes accumulated both forms of antimony using different carriers [ 67 , 68 ] . sbiii entry is in part dependent on a parasitic plasma membrane protein , aquaglyceroporine ( aqp1 ) , also involved in the entry of other metbolites . aqp1 cloning and transfection in l. infantum , l. major , and l. tarentolae promastigotes expressing luciferase gene and infecting thp1 cell line showed increment of their susceptibility to sbiii and asiii . a regain in susceptibility was noticed after transfection of aqp1 to in vitro selected mutants for sbiii resistance . these strains showed faster incorporation of sbiii and asiii with 20- to 50-fold higher steady state accumulation amount compared to those transfected with the vector only . single allele knock - out in a l. major strain had led to a 10-fold decrease in its susceptibility , highlighting the significance of aqp1 amount in resistance phenotype . there was no difference in aqp1 gene copy number between resistant and susceptible strains . in strains exhibiting decreased susceptibility to sbiii , this protein was underexpressed and a correlation was found between accumulation rate of sbiii and rna expression level of aqp1 . the aqp1 gene was found to be underexpressed in antimony resistant clinical isolates from nepal in comparison with susceptible ones . the composition of the amino acid sequence of aqp1 appears to influence the incorporation rate of sbiii in leishmania . in fact , the replacement of alanine at position 163 by serine or threonine induced the decrease of the entry of the active form of antimony . other mutations in the sequence of this protein such as an alteration at glu152 or arg230 were associated with reducing sbiii entry . these mutations were not found in resistant indian clinical isolates with an underexpression of aqp1 . consequently , this mechanism is probably the first barrier that leishmania involves in order to counteract the action of antimonials and induce resistance . it is generally accepted that to be active on the parasite , the antimonial pentavalent form ( sbv ) has to be reduced to its highly active trivalent one ( sbiii ) [ 22 , 35 , 7375 ] . the site of this reduction and its exact mechanism remain somewhat controversial but thought to take place in both macrophage and parasite . evidence of macrophage involvement in reduction is that axenic amastigotes are less susceptible to pentostam than intramacrophagic ones . furthermore , glutathione ( gsh ) which is the major thiol of the host cell can reduce sbv at 37c . in fact , at ph5 , a condition encountered in phagolysosome , this reaction is more important than at ph7.2 ( cytosol ) and a little rate of sbv conversion is found although the highest amount of gsh is cytosolic . hence , it appears from these in vitro experiments that reduction does occur in macrophage but mostly in its acidic compartments such as endosomes , lysosomes , and phagolysosome . other macrophage cytosolic thiols such as cystein and cysteinyl - glycin can also reduce sbv at rates even higher than gsh either in acidic or neutral ph conditions . in a recent study , involvement of macrophages in sbv reduction it has also demonstrated that cellular amount of sbiii is related to that of sbv . the sensitivity of leishmania depends on its ability to reduce sbv since antimonial reduction activity of resistant l. donovani amastigotes was impaired . ( 1 ) enzymatic reductiontwo parasitic enzymes have been identified as being involved in sbv reduction , a thiol dependent reductase 1 ( tdr1 ) , and another one , called lmacr2.the sequence of tdr1 was identified in l. major by in silico analysis , cloned , and the recombinant protein showed high conversion rate of sbv to sbiii using gsh as reducing agent . this enzyme was isolated from both stages of l. major and 10 times higher level of tdr1 was recovered from amastigotes than from promastigotes . furthermore its activity is noticeably greater than nonenzymatic reduction . lmacr2 , an enzyme identified within l. major genomic sequence was cloned and transfected into l. infantum promastigotes : the derived amastigotes ( infecting thp1 macrophages ) were more susceptible to pentostam compared to non transfected strain . transfection of this protein in an indian clinical isolate ( l. donovani ) induced sensitization of this strain to a lower dose of pentostam compared to untransfected one . two parasitic enzymes have been identified as being involved in sbv reduction , a thiol dependent reductase 1 ( tdr1 ) , and another one , called lmacr2 . the sequence of tdr1 was identified in l. major by in silico analysis , cloned , and the recombinant protein showed high conversion rate of sbv to sbiii using gsh as reducing agent . this enzyme was isolated from both stages of l. major and 10 times higher level of tdr1 was recovered from amastigotes than from promastigotes . lmacr2 , an enzyme identified within l. major genomic sequence was cloned and transfected into l. infantum promastigotes : the derived amastigotes ( infecting thp1 macrophages ) were more susceptible to pentostam compared to non transfected strain . transfection of this protein in an indian clinical isolate ( l. donovani ) induced sensitization of this strain to a lower dose of pentostam compared to untransfected one . ( 2 ) nonenzymatic reductionthis pathway involves parasite thiols which are represented by cysteine , spermidine , gsh , and trypanothione ( tsh ) , the latter corresponds to the major thiol of the parasite . in vitro experiments indicated that the sbv reducing activity of tsh was more relevant at ph7 than at ph5 and in all cases faster than sbv reduction with gsh . this metabolic route involves two key enzymes , ornithine decarboxylase ( odc ) and gammaglutamylcystein synthase ( gcs ) . production of thiols is crucial for the parasite in order to maintain a reduced environment inside the cell that counters the effects of oxidative compounds such as antimonials . in fact , the exposure of the parasite to heavy metals can lead to an impairment of its redox potential causing an efflux of intracellular thiols , the accumulation of intraparasitic disulfite , and the inhibition of two enzymes : trypanothione reductase and glutathione synthetase [ 81 , 82 ] . through conjugation to sbiii it can so be inferred that enhancement of this machinery may result in antimony resistance . in fact , in l. tarentolae mutants selected for asiii resistance and shown to be cross resistant to sbiii and sbv , thiols levels were higher than in wild type strain mainly for tsh .molecular approaches pointed out an amplification of the gsh gene within a linear amplicon described in l. tarentolae , selected for asiii or sbiii resistance [ 17 , 83 ] . amplification and overexpression of gsh are not always correlated as shown for in vitro l. tarentolae selected for sbiii resistance ; gsh1 and gsh2 were overexpressed but no amplification was found for gsh2 and it was discrete for gsh1 . tarentolae revertant strain , a drop of gsh1 amplification was associated to a significant decrease in thiol levels without reaching the basal one . transfection experiments carried out in this revertant strain with gsh gene resume resistance profile unlike wild type l. tarentolae which remains susceptible despite displaying an increase in thiols level that can be greater than that in resistant strains to asiii . therefore , the intracellular tsh level is crucial to confer resistance pattern but is not sufficient alone to provide it . the level of thiols measured in l. donovani promastigotes was significantly higher in resistant strains with increased metabolic turnover and faster regeneration of thiols in order to counteract the production of ros by antimonials . this effect is abolished by h2o2 which causes a depletion of thiols thus restoring the efficiency of antimony .for odc , an overexpression without amplification was found in both l. tarentolae asiii resistant and revertant strains and for in vitro selected l. tropica and l. mexicana . since there was not gene rearrangement nor increased translation , this overexpression in the absence of amplification may be the consequence of an enhanced rna stability . in clinical strains , odc gene was found amplified ; this amplicon was not extrachromosomal and protein amount was increased . in clinical isolates , odc overexpression was found in resistant strains of l. braziliensis and was associated with gsh overexpression in l. donovani [ 57 , 86].specific inhibition of gsh and odc with buthionine sulfoximine ( bso ) and difluoromethylornithine ( dfmo ) , respectively , further supported their role in resistance . this suppression resulted in a partial reversal of resistance in resistant strains and in a decrease in tsh eventhough remaining at residual level higher than in wild type strains [ 16 , 32 , 37 , 85 ] . reversal of resistance was also obtained by bso in unresponsive l. donovani and l. tropica clinical isolates [ 5 , 39].inhibition of gsh synthesis by bso was also tested in vivo in mice infected with resistant and susceptible strains of l. donovani . however , bso had no effect on l. panamensis clinical strains resistant to sbv .as reported above , intracellular glutathione is crucial for cell survival by maintaining a redox balance and protection against toxic agents that cause oxidative or chemical stress . impairment of production of gsh is toxic to cells and its inhibition has been successfully used to increase the sensitivity of number of intracellular pathogens and cancer cells to different drugs . infection of mice with sbv sensitive l. donovani strains is associated with increased macrophage gcs mrna unlike resistant strains which induce a reduction in intramacrophagic expression of gcs which leads to a loss of gsh and generates an oxidative environment into the host cell . consequently , the reduction of sbv to its highly active sbiii is diminished in macrophage . in contrast , these same resistant strains display an overexpression of gcs compared to susceptible ones leading to a higher rate of thiols in the parasite in order to compensate an oxidizing environment generated in macrophage .in l. infantum axenic amastigotes selected for sbiii , sahh gene , which is responsible for the conversion of s adenosyl homocystein to homocystein , the cystein precursor was overexpressed . interestingly , cystein which is a glutathione precursor was the only thiol with increased concentration in this study . this pathway involves parasite thiols which are represented by cysteine , spermidine , gsh , and trypanothione ( tsh ) , the latter corresponds to the major thiol of the parasite . in vitro experiments indicated that the sbv reducing activity of tsh was more relevant at ph7 than at ph5 and in all cases faster than sbv reduction with gsh . this metabolic route involves two key enzymes , ornithine decarboxylase ( odc ) and gammaglutamylcystein synthase ( gcs ) . production of thiols is crucial for the parasite in order to maintain a reduced environment inside the cell that counters the effects of oxidative compounds such as antimonials . in fact , the exposure of the parasite to heavy metals can lead to an impairment of its redox potential causing an efflux of intracellular thiols , the accumulation of intraparasitic disulfite , and the inhibition of two enzymes : trypanothione reductase and glutathione synthetase [ 81 , 82 ] . through conjugation to sbiii it can so be inferred that enhancement of this machinery may result in antimony resistance . in fact , in l. tarentolae mutants selected for asiii resistance and shown to be cross resistant to sbiii and sbv , thiols levels were higher than in wild type strain mainly for tsh . molecular approaches pointed out an amplification of the gsh gene within a linear amplicon described in l. tarentolae , selected for asiii or sbiii resistance [ 17 , 83 ] . amplification and overexpression of gsh are not always correlated as shown for in vitro l. tarentolae selected for sbiii resistance ; gsh1 and gsh2 were overexpressed but no amplification was found for gsh2 and it was discrete for gsh1 . tarentolae revertant strain , a drop of gsh1 amplification was associated to a significant decrease in thiol levels without reaching the basal one . transfection experiments carried out in this revertant strain with gsh gene resume resistance profile unlike wild type l. tarentolae which remains susceptible despite displaying an increase in thiols level that can be greater than that in resistant strains to asiii . therefore , the intracellular tsh level is crucial to confer resistance pattern but is not sufficient alone to provide it . the level of thiols measured in l. donovani promastigotes was significantly higher in resistant strains with increased metabolic turnover and faster regeneration of thiols in order to counteract the production of ros by antimonials . this effect is abolished by h2o2 which causes a depletion of thiols thus restoring the efficiency of antimony . for odc , an overexpression without amplification was found in both l. tarentolae asiii resistant and revertant strains and for in vitro selected l. tropica and l. mexicana . since there was not gene rearrangement nor increased translation , this overexpression in the absence of amplification may be the consequence of an enhanced rna stability . in clinical strains , odc gene was found amplified ; this amplicon was not extrachromosomal and protein amount was increased . in clinical isolates , odc overexpression was found in resistant strains of l. braziliensis and was associated with gsh overexpression in l. donovani [ 57 , 86 ] . specific inhibition of gsh and odc with buthionine sulfoximine ( bso ) and difluoromethylornithine ( dfmo ) , respectively , further supported their role in resistance . this suppression resulted in a partial reversal of resistance in resistant strains and in a decrease in tsh eventhough remaining at residual level higher than in wild type strains [ 16 , 32 , 37 , 85 ] . reversal of resistance was also obtained by bso in unresponsive l. donovani and l. tropica clinical isolates [ 5 , 39 ] . inhibition of gsh synthesis by bso was also tested in vivo in mice infected with resistant and susceptible strains of l. donovani . however , bso had no effect on l. panamensis clinical strains resistant to sbv . as reported above , intracellular glutathione is crucial for cell survival by maintaining a redox balance and protection against toxic agents that cause oxidative or chemical stress . impairment of production of gsh is toxic to cells and its inhibition has been successfully used to increase the sensitivity of number of intracellular pathogens and cancer cells to different drugs . infection of mice with sbv sensitive l. donovani strains is associated with increased macrophage gcs mrna unlike resistant strains which induce a reduction in intramacrophagic expression of gcs which leads to a loss of gsh and generates an oxidative environment into the host cell . consequently , the reduction of sbv to its highly active sbiii is diminished in macrophage . in contrast , these same resistant strains display an overexpression of gcs compared to susceptible ones leading to a higher rate of thiols in the parasite in order to compensate an oxidizing environment generated in macrophage . in l. infantum axenic amastigotes selected for sbiii , sahh gene , which is responsible for the conversion of s adenosyl homocystein to homocystein , the cystein precursor was overexpressed . interestingly , cystein which is a glutathione precursor was the only thiol with increased concentration in this study . atp binding cassette ( abc ) family is a set of proteins involved in resistance either by drug efflux or by its sequestration . this efflux pumps system was first described in tumor cells , and the derived phenotype was named multidrug resistance ( mdr ) resulting in refractoriness to chemotherapy . in leishmania , 8 subfamilies of abc transporters genes , present among 19 different chromosomes , were identified when l. major whole genome was compared with established abc proteins sequences . among these , two subclasses of abc transporters seem to be involved in leishmania resistance : carriers with high similarity to p - glycoproteins in mammals that confer an mdr phenotype similar to that seen in cancer cells , homologous to the multidrug resistance - related protein ( mrp ) in humans . mdr profile was found in resistant strains of leishmania and the mdr1 gene was amplified in mutants selected for resistance to vinblastine , daunomycin , or to puromycin [ 8992 ] . mdr - class transporters are involved in extrusion of hydrophobic molecules but not hydrophilic ones as sbiii . mrpa formerly called pgpa for p - glycoprotein a is the most studied abc transporter in leishmania and its role in resistance is well documented in vitro . mrpa gene is often located on an amplified h - circle extrachromosomal dna [ 21 , 83 , 88 , 9395 ] which initially belongs to a locus in chromosome 23 . this locus is flanked at each side by repeated sequences of 1389 bp that present a sequence homology of 100% in a l. infantum strain selected for sbiii resistance . these repeats were also found on the same chromosome of l. major and l. braziliensis . however , in l. mexicana selected for asiii resistance , it was located in a linear amplicon . mrpa gene is frequently amplified / overexpressed in strains resistant to heavy metals [ 60 , 83 ] . the level of resistance is associated with this amplification as demonstrated by a comparative study between in vitro selected l. major and l. braziliensis strains to antimonials . in fact , resistance was associated to h - circle amplification in l. major but not in l. braziliensis and the latter remained significantly more sensitive to antimony . similarly , decreased amplification was observed in revertant strains . initially susceptible strains transfected with mrpa gene acquire a certain level of resistance to sbiii or asiii but this level is variable depending on species even if equal band intensity can be found in wb for mrpa recombinant protein [ 95 , 97 , 98 ] . evidence of combined action between mrpa and thiols in heavy metals resistance was confirmed by cotransfection experiments of mrpa and gsh genes which confer resistance in wild type l. tarentolae and restore a high resistance level in revertant strain unlike gsh transfection alone . unlike classical abc proteins in fact , this protein is located in an intracellular compartment close to the flagellar pocket of the parasite recognized as the site of endocytosis and exocytosis and acts by the sequestration of the sbiii - tsh complex since free sbiii or asiii is not transported by mrpa [ 62 , 78 ] . therefore , thiols act by two opposite mechanisms : on one hand , they are involved in drug activation and on the other , in its inactivation via its sequestration . in parallel with drug sequestration , mrpa amplification seems to cause a decrease in drug entry rather than increasing its efflux . this can be explained by a possible dominant negative role played by mrpa on membrane proteins . plasma membrane efflux mechanism was demonstrated in leishmania in membrane - enriched everted vesicles of l. tarentolae in which an atp calcium - dependent protein was involved in asiii - gsh transport not mediated by mrpa . in fact , double knock - out of mrpa gene did not alter this transport which was inhibited to some extent by pentostam indicating competition between the two related metals . in addition , the activity of this transporter is not increased in membranes from mutants that overexpress mrpa suggesting that this thiol x efflux pump is encoded by another gene . functional studies of the abc family pumps were performed on clinical isolates of l. donovani ( promastigotes and axenic amastigotes ) using rhodamine123 ( r123 ) as substrate for mdr , calcein for mrp , and by employing specific inhibitors . the r123 accumulation was found greater in resistant strains and its rate was not modified by verapamil , a specific inhibitor of mdr , suggesting the absence of a classical mdr activity . the accumulation of calcein was lower among these resistant strains for both promastigotes and axenic amastigotes . use of probenecid , an inhibitor of mrp , did not lead to a significant increase in calcein unlike atp depletion indicating the existence of an mrp - like pump more active in resistant isolates . the role of mrpa and the level of resistance depend on the rate of residual sbiii as demonstrated in a study that initially showed no correlation between the in vivo efficacy of sbv and the residual sbiii in parasite but highlighted the importance of this residual rate for the action of mrpa . indeed , although strains that overexpress mrpa were all resistant to sbiii , cross - resistance to sbv was observed only in strains that exhibited a high sbiii residual rate , indicating that this refractoriness was directed against the antimony trivalent form . found that mrpa was overexpressed in sbiii resistant l. infantum , compared to wild type by microarray approach . interestingly , two other proteins of the abc family were overexpressed , namely , abca3 and abch1 . identical results were obtained in another independently selected strain . however , in wild - type strains , only mrpa transfection confers sbiii resistance . implication of abca3 in vesicular trafficking and exocytosis pathway supports an indirect implication in leishmania resistance . all these proteins were located in intracellular compartments . their role in resistance to antimonials was investigated in a revertant strain of l. tarentolae . only transfections of abcc4-gfp and abcc5-gfp were accompanied with a two - fold increase of resistance level . other proteins of the mrp subfamily of abc have been identified as pgp b , c , d , and e. however , single transfection of their genes does not confer resistance . the role and action of these proteins remain unknown , but their sequence similarity with mrpa indicates that they may correspond to membrane transporters involved in antimonials efflux . recently , a second protein belonging to the abc family transporters , and named prp1 , was identified by cloning after selection by pentamidine . some genes identified in vitro as involved in resistance to antimonials were investigated in clinical isolates from sudan and france . mdr1 was amplified in 65% of isolates and was the only statistically associated with l. infantum . amplification of mrpa in concomitance with gsc was observed in some strains confirming the joint action of thiols metabolism and sequestration of conjugated antimony . nevertheless , it was not possible to establish a correlation between therapeutic response and gene amplification observed in this survey since the french patients were treated with liposomal amphotericin b while the clinical data were lacking for sudanese isolates . however , it supported the presence of an amplification of some genes identified in vitro as taking part in resistance . the singularity of this study is the presence of an amplification of the mdr1 gene which is found amplified in in vitro strains selected for resistance to molecules other than heavy metals . the significance of this issue remains difficult to elucidate and we can only speculate on the role of mdr1 in antimony resistance in clinical isolates . this phenomenon can be explained by adaptative functions of the parasites when exposed in vivo to drugs used for treatment of another disease . this drug can select in leishmania a new function , such as mdr1 amplification in this case , without any linkage to antimony therapy . this gene is related to folates pathway and antifolates are used in several affections such as malaria where dhfr is amplified in plasmodium resistant strains . although antifolates are not used in leishmania treatment , amplification of dhfr gene occurs in vitro after methotrexate selection and in clinical strains [ 56 , 83 ] . indeed , miltefosine , a drug recently introduced in antileishmanial therapy , has plasma membrane as targets and it was suggested that antimony resistant leishmania also shows membrane modifications . combined with antimonials wide use in some endemic areas , this mode of action led to cross - resistance between these two molecules . table 1 summarizes the most important molecular targets identified either in in vitro selected mutants or in field isolates of leishmania . a comparative proteomic analysis between two l. donovani indian clinical isolates resistant and susceptible to sbv identified proteins having already known roles in programmed cell death ( pcd ) as the 14 - 3 - 3 protein present in higher quantities in the resistant strain , and which belongs to a family of conserved proteins able to bind other phosphorylated proteins involved in apoptosis . heat shock proteins ( hsps ) are also involved in pcd by modulating some steps in the apoptosis pathway . in fact , antimonials induce the production of a variety of hsps ( hsp70 , hsp81 , and hsp65 ) and their role is supposed to be protective against the toxic effect of these drugs . transfection of a cosmid library in leishmania parasites and their selection with sbiii identified in resistant strains a cosmid containing hsp70 and hsc70 . . however , transfection experiments of hsp70 or hsc genes did not give rise to any resistance thus assuming a minor role in resistance . similarly , brochu et al . showed that level of hsps of different molecular weights is increased in the presence of arsenicals . however , transfection of hsp gene does not confer resistance directly but rather increases parasite tolerance to heavy metals . small kinetoplastid calpain - related protein 1.14 ( skcrp1.14 ) , which was underexpressed in l. donovani resistant strain , is often associated to other proteins identified as effectors of pcd in kinetoplastidae . thus , it is conceivable that the process of apoptosis in resistant strains is impaired . this is corroborated by evidence of reduced dna fragmentation and formation of bridges in resistant strains compared to susceptible ones . tryparedoxin peroxidase ( trper ) , an enzyme responsible for peroxides ions detoxification in leishmania , also plays a role in antimonial resistance as shown in l. donovani promastigotes and amastigotes transfected with this protein . in fact , either in vitro selected strains or field isolates became less susceptible to sbiii [ 105 , 106 ] . likewise , the resistance profile was also demonstrated in resistant mutants of l. tarentolae strains by overexpression of the recombinant protein in initially susceptible strains . h1 showed differential expression pattern between susceptible and resistant strains of l. donovani clinical isolates . nevertheless , h2a gene transfection in l. donovani susceptible strains displayed no significant increase in the ed50 for sbiii . differential expression also concerned mapk , a mediator which plays an important role in intracellular multiplication of the parasite , the morphogenesis of the flagellum and consequently in the virulence of the parasite during host infection [ 117119 ] . linj05_v3.0830 was 4 times overexpressed in l. infantum resistant strains selected for sbiii resistance . however , transfection experiments in sensitive and revertant strains indicated no significant change in sensitivity . leishmania synthetizes phosphoproteoglycans ( ppgs ) that are secreted or present at the parasite membrane in the two stages . various other proteins were overexpressed in resistant clinical isolates of l. donovani at membrane and cytosolic levels such as gpi transaminase protein , a cysteine - rich leucine protein , a 60s ribosomal protein l23a , proliferative cell nuclear antigen ( pcna ) , the alpha5 subunit of the proteasome , carboxypeptidase , enolase , fructose-1,6-bisphosphate aldolase , and a beta tubulin chain . in l. infantum axenic amastigotes , proteomic approach highlighted overexpression of arginosuccinate synthetase and an underexpression of kinetoplastidae membrane protein ( kmp-11 ) with an expression level correlated with the resistance phenotype . other l. infantum strains overexpressing a protein which belongs to the superfamily of leucine - rich repeat ( lrr ) proteins , became resistant to sbiii for axenic amastigotes and to sbv for intramacrophagic ones . in an l. infantum mutant strain selected for sbiii resistance , the expression of whole chromosomes was modulated , compared to the sensitive one . gene overexpression involved rather chromosomes 1 , 11 , and 25 , in opposition with chromosome 9 , 12 , and 32 where genes were found underexpressed . interestingly , aneuploidy and the presence of the circular amplicon carrying mrpa gene disappeared in the revertant strain . however , haploidy for chromosomes 12 and 32 has persisted ; this possibly explains the remaining of resistance at residual level . in a study on resistant field isolates of l. donovani , increase of thiols level was much lower than that observed in mutant strains selected in vitro explaining in part the higher resistance observed in mutants compared with clinical unresponsive strains . trypanothione reductase gene was 2.5 times amplified in resistant isolates thus maintaining a high level in thiols and counteracting the effect of sbiii . however , gcs gene was not amplified like in l. tropica clinical strains . unlike in vitro selected mutants in which tsh level is usually increased , some clinical resistant strains display cystein and gsh overproduction but not tsh . as that of aqp1 , gcs , and odc genes were surprisingly underexpressed in resistant strains in intracellular amastigotes . this modulation was also observed for gsc in the same proportions in promastigotes ; it was lower for aqp1 and absent in the case odc . these findings are in opposition with most studies on in vitro resistant mutants and other surveys on clinical isolates . this difference can be attributed at least for the in vitro mutant strains to different mechanisms depending on the used antimonial form . indeed , in this study it was observed that sbv exposure of amastigotes induced an underexpression of aqp1 involved in the entry of antimony in the parasite cell , but also inhibition of enzymatic and nonenzymatic pathways involved in pentavalent compound reduction . the number of copies of aqp1 gene may not correlate with the level of expression as determined in l. donovani resistant clinical isolates , which displayed lowered sbiii accumulation , an underexpression of aqp1 , but increased copy number of its gene . inconsistency between in vitro resistance tests and clinical outcome was also found in cutaneous leishmaniasis isolates from peruvian subjects . some molecular targets were tested in promastigotes ofamerican cutaneous leishmaniasis clinical isolates ( l. braziliensis , l. guyanensis ) . for l. braziliensis there was no difference in expression for mrpa , gsh , trypanothione reductase , and tdr1 in relation to treatment issue , while for l. guyanensis , gsc gene was overexpressed in strains that caused treatment failure . recently , an amplified sequence of 1254 kb was found in l. donovani resistant field isolates . hybridization experiments have shown that this sequence did not match any gene of abc family or with other genes involved in resistance . however , antibodies against the recombinant protein of this sequence allowed assessing its membrane localization . the functional study of this sequence confirmed the absence of an mdr - like pump that can be inhibited by verapamil or an mrp - like pump . another experiment using intramacrophagic amastigotes labeled with luciferase from susceptible and resistant l. donovani clinical isolates showed that refractory clinical strains to sbv displayed also in vitro resistance to this compound . this strain was also significantly more resistant to sbiii than the susceptible one that allowed performing experiences on promastigotes , easier to carry on . genes usually involved in in vitro resistance as aqp1 , mrpa , or gsh were not modulated in the resistant strain . antimonials remain the first line treatment of leishmaniasis in medical and veterinary fields and the emergence and spreading of resistance is still an important public health problem . field studies gave partial information and definition of the resistant phenotypes appeared to be difficult because of the influence of host factors and lack of exhaustivity . in vitro sensitivity testing on infected cells remains the better global estimation of leishmania resistance and substitutive , informative , molecular tests are needed . progress is undeniable ; however , the discrepancies between in vitro and clinical resistance remain numerous , even for surveys held for clinical isolates of the same species in adjacent geographic areas . this review illustrated the difficulties to identify markers of resistance that can be standardized with a test easy to implement , replacing the time consuming determination of ic50 in the amastigote - macrophage model . additional work , focusing on search for reliable molecular markers , would be valuable for the community .

leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 million cases . there are few available treatments and antimonials are still of major importance in the therapeutic strategies used in most endemic regions . however , resistance toward these compounds has recently emerged in areas where the replacement of these drugs is mainly limited by the cost of alternative molecules . in this paper , we reviewed the studies carried out on antimonial resistance in leishmania . several common limitations of these works are presented before prevalent approaches to evidence antimonial resistance are related . afterwards , phenotypic determination of resistance is described , then confronted to clinical outcome . finally , we detail molecular mechanisms and targets involved in resistance and already identified in vitro within selected mutant strains or in clinical isolates .

1. Introduction 2. Methodologic Aspects 3. 4. Field Isolates Can Exhibit Resistance Pattern Different from 5. Conclusion

leishmaniases are a set of vector - borne diseases caused by a flagellate protozoan transmitted by the bite of an insect vector , the phlebotomine sandfly . this group of diseases affects 98 countries with three distinct entities : cutaneous , mucocutaneous , and visceral leishmaniasis . this accounts for the still predominant place of antimony derivatives , sodium stibiogluconate ( pentostam ) , and meglumine antimoniate ( glucantime ) which have been used in the treatment of the majority of cases of leishmaniases for more than 60 years worldwide . low dosage or insufficient duration of treatment led to the selection of resistant mutants that were transmitted more easily in this anthroponotic form than in areas where anthropozoonotic cycle occurs . currently , in the most heavily affected areas of india , resistance to antimonials may reach more than 60% of the cases , thus representing a public health problem . in other mediterranean areas such as maghreb , albania , and middle east except israel , most infected dogs are left untreated ; antimonials are still the first line treatment and display high efficiency [ 11 , 12 ] . drug resistance of leishmania may be natural , acquired when the parasite is exposed to suboptimal doses of the drug , or induced in vitro after selection of mutants by exposure to gradually increasing concentrations of the drug . first , we relate methodology , general limitations , and tools used for resistance surveys . in a second step , finally , we focus on molecular resistance pathways and already identified targets within in vitro selected mutants and field isolates . before discussing the different ways of investigation , we present some general considerations related to leishmania isolates , the evolution of their characteristics during maintenance in vitro , selection of mutants , and postulates about cross - resistance mechanisms . by using this species , different resistance mechanisms and molecular targets were identified and their presence confirmed in other species selected in vitro or from clinical isolates . however , intrinsic susceptibility to heavy metals and thiols levels found in l. tarentolae can be very different from those encountered in leishmania species of clinical interest . by using this species , different resistance mechanisms and molecular targets were identified and their presence confirmed in other species selected in vitro or from clinical isolates . however , intrinsic susceptibility to heavy metals and thiols levels found in l. tarentolae can be very different from those encountered in leishmania species of clinical interest . carriers belonging to the abc system and involved in the efflux or sequestration of antimonials have indeed been identified in promastigotes resistant mutants and few studies on the role of these proteins have been conducted on amastigotes . however , they do not represent the parasitic stage on which antimonials act in vivo . carriers belonging to the abc system and involved in the efflux or sequestration of antimonials have indeed been identified in promastigotes resistant mutants and few studies on the role of these proteins have been conducted on amastigotes . however , the sensitivity was reduced in strains isolated from either patients or dogs treated with antimonials [ 9 , 10 , 27 ] . ( 2 ) impact of in vitro culture on primary phenotype leishmania parasites isolated from an infected host may present a polyclonal population with a varied expression of different phenotypes such as growth capacity in vitro , infectivity , and drug resistance [ 29 , 30 ] . indeed , the virulence of a strain and its capacity for growth and adaptation in culture can alter the clonal composition of strain over successive passages in the medium and so modify the expression of resistance phenotype . however , experiments have shown that during induction of in vitro resistance by discontinuous exposition of parasites to the drug , the resistance phenotype does not vary either during successive passages in culture in the absence of the drug or after in vivo infection [ 29 , 30 ] . leishmania parasites isolated from an infected host may present a polyclonal population with a varied expression of different phenotypes such as growth capacity in vitro , infectivity , and drug resistance [ 29 , 30 ] . indeed , the virulence of a strain and its capacity for growth and adaptation in culture can alter the clonal composition of strain over successive passages in the medium and so modify the expression of resistance phenotype . however , experiments have shown that during induction of in vitro resistance by discontinuous exposition of parasites to the drug , the resistance phenotype does not vary either during successive passages in culture in the absence of the drug or after in vivo infection [ 29 , 30 ] . ( 3 ) use of arsenicals for in vitro selectionseveral experiments on leishmania resistance were performed with a progressive selection of mutant strains by exposing promastigotes to increasing concentrations of arsenicals [ 17 , 20 , 32 , 33 ] . these studies suffer the selection bias of arsenic derivatives not used in antileishmanial therapy but whose use in vitro showed that they confer cross - resistance to antimonials . it is therefore conceivable that the mutant strains selected in vitro against arsenic derivatives and then tested against antimonials may have developed resistance mechanisms specific to arsenite along with other common mechanisms , thus explaining the phenomenon of cross - resistance . several experiments on leishmania resistance were performed with a progressive selection of mutant strains by exposing promastigotes to increasing concentrations of arsenicals [ 17 , 20 , 32 , 33 ] . these studies suffer the selection bias of arsenic derivatives not used in antileishmanial therapy but whose use in vitro showed that they confer cross - resistance to antimonials . however , arsenicals induce an increase in intracellular calcium that does not occur with antimonials . conversely , the level of intracellular glutathione which is not affected during treatment with arsenicals is lowered when antimonials are used . it is therefore conceivable that the mutant strains selected in vitro against arsenic derivatives and then tested against antimonials may have developed resistance mechanisms specific to arsenite along with other common mechanisms , thus explaining the phenomenon of cross - resistance . ( 4 ) sbiii , and sbv - selected mutantsthe use of sbiii that is not the prescribed form in clinical practice but represents the biologically active form of the drug would lead to the selection of resistant strains with different mechanisms and targets that are not necessarily involved in clinical isolates resistance since these are first exposed to sbv . the use of sbiii that is not the prescribed form in clinical practice but represents the biologically active form of the drug would lead to the selection of resistant strains with different mechanisms and targets that are not necessarily involved in clinical isolates resistance since these are first exposed to sbv . the infection of mice or hamsters , which was among the first approaches for studying the sensitivity to antimonials in vivo , still faces the problem of variability related to the host and genetic background of these vertebrate hosts may have a major influence upon evolution after infection . however , it remains a reference technique for studying the resitance phenotype of clinical isolates . evidenced that in clinical isolates , among both hiv and immunocompetent patients , the ic50 of amastigotes within macrophages was significantly higher in patients who have not improved , compared to those who have responded to treatment . thus , in vitro susceptibility of clinical isolates correlated well with clinical outcome in immediate treatment . in contrast , the long - term treatment did not appear correlated with in vitro tests since relapses occurred even in patients harboring susceptible strains . correlation between the clinical outcome and in vitro susceptibility tests was also observed in indian field isolates . results showed a statistically significant improvement of cure rate reaching 86% in the first group compared to 35% in patients treated with sbv without in vitro tests . nevertheless , clinical outcome and in vitro susceptibility tests can be contradictory as shown for l. donovani clinical isolates tested for their sensitivity to sbv and sbiii . as mentioned above , these tests are carried out on selected in vitro mutants , on clinical human isolates , or on strains affecting other vertebrates such as l. tarentolae , a species widely used in vitro . gene amplification was the first molecular resistance mechanism identified in l. major resistant to methotrexate ( mtx ) and displayed by the presence of h - circle . quantitative reverse transcription pcr ( qrt - pcr ) is an approach used in this context to highlight gene overexpression of rna which can be independent of amplification . flow cytometry was used in two aims : as a tools for leishmania quantification in the last stage of sensitivity testing of infected cells and more interestingly for functional analysis of drugs transporter by using fluorescent drug analogs and specific inhibitors . specific inhibitors are commonly used for two purposes : the first is in vitro study of inhibiting an enzyme supposed to participate in resistance phenotype ; the second is withdrawal of resistance profile in animal model using such inhibitor in combination with antimonials . injection - coupled plasma mass spectrometry ( icpms ) is used in leishmania resistance surveys to measure differences in uptake and accumulation of sbv or sbiii depending on strain sensitivity or its stage . sbiii entry is in part dependent on a parasitic plasma membrane protein , aquaglyceroporine ( aqp1 ) , also involved in the entry of other metbolites . in fact , the replacement of alanine at position 163 by serine or threonine induced the decrease of the entry of the active form of antimony . ( 1 ) enzymatic reductiontwo parasitic enzymes have been identified as being involved in sbv reduction , a thiol dependent reductase 1 ( tdr1 ) , and another one , called lmacr2.the sequence of tdr1 was identified in l. major by in silico analysis , cloned , and the recombinant protein showed high conversion rate of sbv to sbiii using gsh as reducing agent . in fact , in l. tarentolae mutants selected for asiii resistance and shown to be cross resistant to sbiii and sbv , thiols levels were higher than in wild type strain mainly for tsh .molecular approaches pointed out an amplification of the gsh gene within a linear amplicon described in l. tarentolae , selected for asiii or sbiii resistance [ 17 , 83 ] . transfection experiments carried out in this revertant strain with gsh gene resume resistance profile unlike wild type l. tarentolae which remains susceptible despite displaying an increase in thiols level that can be greater than that in resistant strains to asiii . this effect is abolished by h2o2 which causes a depletion of thiols thus restoring the efficiency of antimony .for odc , an overexpression without amplification was found in both l. tarentolae asiii resistant and revertant strains and for in vitro selected l. tropica and l. mexicana . since there was not gene rearrangement nor increased translation , this overexpression in the absence of amplification may be the consequence of an enhanced rna stability . in clinical isolates , odc overexpression was found in resistant strains of l. braziliensis and was associated with gsh overexpression in l. donovani [ 57 , 86].specific inhibition of gsh and odc with buthionine sulfoximine ( bso ) and difluoromethylornithine ( dfmo ) , respectively , further supported their role in resistance . this suppression resulted in a partial reversal of resistance in resistant strains and in a decrease in tsh eventhough remaining at residual level higher than in wild type strains [ 16 , 32 , 37 , 85 ] . reversal of resistance was also obtained by bso in unresponsive l. donovani and l. tropica clinical isolates [ 5 , 39].inhibition of gsh synthesis by bso was also tested in vivo in mice infected with resistant and susceptible strains of l. donovani . in vitro experiments indicated that the sbv reducing activity of tsh was more relevant at ph7 than at ph5 and in all cases faster than sbv reduction with gsh . amplification and overexpression of gsh are not always correlated as shown for in vitro l. tarentolae selected for sbiii resistance ; gsh1 and gsh2 were overexpressed but no amplification was found for gsh2 and it was discrete for gsh1 . transfection experiments carried out in this revertant strain with gsh gene resume resistance profile unlike wild type l. tarentolae which remains susceptible despite displaying an increase in thiols level that can be greater than that in resistant strains to asiii . in clinical isolates , odc overexpression was found in resistant strains of l. braziliensis and was associated with gsh overexpression in l. donovani [ 57 , 86 ] . this suppression resulted in a partial reversal of resistance in resistant strains and in a decrease in tsh eventhough remaining at residual level higher than in wild type strains [ 16 , 32 , 37 , 85 ] . reversal of resistance was also obtained by bso in unresponsive l. donovani and l. tropica clinical isolates [ 5 , 39 ] . atp binding cassette ( abc ) family is a set of proteins involved in resistance either by drug efflux or by its sequestration . among these , two subclasses of abc transporters seem to be involved in leishmania resistance : carriers with high similarity to p - glycoproteins in mammals that confer an mdr phenotype similar to that seen in cancer cells , homologous to the multidrug resistance - related protein ( mrp ) in humans . mrpa formerly called pgpa for p - glycoprotein a is the most studied abc transporter in leishmania and its role in resistance is well documented in vitro . the level of resistance is associated with this amplification as demonstrated by a comparative study between in vitro selected l. major and l. braziliensis strains to antimonials . plasma membrane efflux mechanism was demonstrated in leishmania in membrane - enriched everted vesicles of l. tarentolae in which an atp calcium - dependent protein was involved in asiii - gsh transport not mediated by mrpa . functional studies of the abc family pumps were performed on clinical isolates of l. donovani ( promastigotes and axenic amastigotes ) using rhodamine123 ( r123 ) as substrate for mdr , calcein for mrp , and by employing specific inhibitors . their role in resistance to antimonials was investigated in a revertant strain of l. tarentolae . only transfections of abcc4-gfp and abcc5-gfp were accompanied with a two - fold increase of resistance level . the role and action of these proteins remain unknown , but their sequence similarity with mrpa indicates that they may correspond to membrane transporters involved in antimonials efflux . some genes identified in vitro as involved in resistance to antimonials were investigated in clinical isolates from sudan and france . however , it supported the presence of an amplification of some genes identified in vitro as taking part in resistance . the significance of this issue remains difficult to elucidate and we can only speculate on the role of mdr1 in antimony resistance in clinical isolates . this drug can select in leishmania a new function , such as mdr1 amplification in this case , without any linkage to antimony therapy . although antifolates are not used in leishmania treatment , amplification of dhfr gene occurs in vitro after methotrexate selection and in clinical strains [ 56 , 83 ] . table 1 summarizes the most important molecular targets identified either in in vitro selected mutants or in field isolates of leishmania . a comparative proteomic analysis between two l. donovani indian clinical isolates resistant and susceptible to sbv identified proteins having already known roles in programmed cell death ( pcd ) as the 14 - 3 - 3 protein present in higher quantities in the resistant strain , and which belongs to a family of conserved proteins able to bind other phosphorylated proteins involved in apoptosis . heat shock proteins ( hsps ) are also involved in pcd by modulating some steps in the apoptosis pathway . in fact , antimonials induce the production of a variety of hsps ( hsp70 , hsp81 , and hsp65 ) and their role is supposed to be protective against the toxic effect of these drugs . transfection of a cosmid library in leishmania parasites and their selection with sbiii identified in resistant strains a cosmid containing hsp70 and hsc70 . however , transfection experiments of hsp70 or hsc genes did not give rise to any resistance thus assuming a minor role in resistance . tryparedoxin peroxidase ( trper ) , an enzyme responsible for peroxides ions detoxification in leishmania , also plays a role in antimonial resistance as shown in l. donovani promastigotes and amastigotes transfected with this protein . in fact , either in vitro selected strains or field isolates became less susceptible to sbiii [ 105 , 106 ] . h1 showed differential expression pattern between susceptible and resistant strains of l. donovani clinical isolates . however , transfection experiments in sensitive and revertant strains indicated no significant change in sensitivity . however , haploidy for chromosomes 12 and 32 has persisted ; this possibly explains the remaining of resistance at residual level . in a study on resistant field isolates of l. donovani , increase of thiols level was much lower than that observed in mutant strains selected in vitro explaining in part the higher resistance observed in mutants compared with clinical unresponsive strains . these findings are in opposition with most studies on in vitro resistant mutants and other surveys on clinical isolates . this difference can be attributed at least for the in vitro mutant strains to different mechanisms depending on the used antimonial form . indeed , in this study it was observed that sbv exposure of amastigotes induced an underexpression of aqp1 involved in the entry of antimony in the parasite cell , but also inhibition of enzymatic and nonenzymatic pathways involved in pentavalent compound reduction . inconsistency between in vitro resistance tests and clinical outcome was also found in cutaneous leishmaniasis isolates from peruvian subjects . hybridization experiments have shown that this sequence did not match any gene of abc family or with other genes involved in resistance . another experiment using intramacrophagic amastigotes labeled with luciferase from susceptible and resistant l. donovani clinical isolates showed that refractory clinical strains to sbv displayed also in vitro resistance to this compound . genes usually involved in in vitro resistance as aqp1 , mrpa , or gsh were not modulated in the resistant strain . antimonials remain the first line treatment of leishmaniasis in medical and veterinary fields and the emergence and spreading of resistance is still an important public health problem . in vitro sensitivity testing on infected cells remains the better global estimation of leishmania resistance and substitutive , informative , molecular tests are needed . progress is undeniable ; however , the discrepancies between in vitro and clinical resistance remain numerous , even for surveys held for clinical isolates of the same species in adjacent geographic areas . this review illustrated the difficulties to identify markers of resistance that can be standardized with a test easy to implement , replacing the time consuming determination of ic50 in the amastigote - macrophage model .

this cross - sectional study investigates the possible relationship between identity development , parenting , and adolescent problems , which may manifest through internalized modes ( phobias , obsessions , depression , eating disorders , entropy ) and externalized ones ( alcohol use and school discomfort ) , in a group of 198 italian students . recent literature emphasizes the importance of examining the risk and protection factors for the internalizing and externalizing symptoms in adolescence , which appear in different contexts : social and interpersonal , such as family , school , and peer group ; and individual factors , such as identity development , and cognitive and behavioral patterns . compared to the interpersonal factors,14 studies show that a high level of parental control57 is related to a high level of adolescent s implication in problematic behaviors ( such as alcohol abuse),8,9 above all in the age group between 18 and 19 years.10,11 conversely , when the parental presence is significant and authoritative , the adolescents are better protected12,13 and the probability of risk - taking behavior decreases.14,15 in reference to the individual dimension , adolescence represents a crucial stage in the development of identity . according to marcia s model , the psychosocial identity construct is defined as a dynamic structure and not a static one , whose formation depends on different factors such as the decisions taken during life . the styles through which identity problems are faced are classified into four identity statuses : achievement ( characterized by positive self - image , flexibility , and cognitive independence ) , moratorium ( fears for the future , limited flexibility , and reduced cooperation ) , foreclosure ( conventionality , rigidity , low self - esteem , and conflictual relationships ) , and diffusion status ( flexibility and cognitive complexity).14 literature shows that less sophisticated identity status seems to be correlated with a higher level of alcohol consumption;15 so , adolescents with low level of development identity seem to be classified as binge drinkers and heavy drinkers . however , the social and family dimensions can influence the early risk behavior experimentation in adolescence , but the outcome of these conditioning issues could have different consequences in relation to the individual features , such as identity development and the possible presence of internalizing symptoms.1618 during adolescence , development tasks can be faced without serious difficulties , and the overcoming of these development tasks ( such as autonomy from parents , the comparison with peer group and the acquisition of useful tools to experience new roles ) could be a significant factor of protection from possible evolutionary breakdown;19,20 but transitional or problematic states can lead the person to manifest internalizing symptoms , especially during adolescence.21,22 the main risk factors that seem to match with the development of internalizing symptoms are as follows : reduced identity development,23 lack of emotional support,24 frequent adoption of punitive parenting behavior,25,26 parental psychological control,27,28 and also disadvantaged sociocultural characteristics.29,30 compared to parenting , adolescents exposed to parental psychological control seem to adopt the same rigorous and strict standards proposed by parents , living inadequately with the inability to reach the standards , both socially prescribed and those self - imposed.31,32 in particular , literature underlines the harmful effects of maternal control , which are less evident toward their daughters and more evident toward their sons , over the level of perfectionism in adolescent children . in the mother daughter dyad , rather than perfectionism , it seems that the responsible mechanism for the link between controlling parenting and internalizing symptoms is relational dependency.33,34 moreover , adolescents with low - profile identity ( foreclosure and diffusion status ) who find themselves living in an adverse family situation could experience a low perception of control over their environment , which may predispose them to develop the perception of reduced control over mental and physical states.35,36 in fact , recent studies show how high levels of warmth or support are related to lower levels of internalization in adolescents with high - profile identity;3739 on the other hand , excessive control and lack of parental empathy appear to be related to the onset of interior symptoms such as depression , anxiety,4042 lack of self - control , or irritability in low - profile adolescents.4345 in particular , the result of internalizing symptoms refers to the superimposition of anxious and depressive syndromes;46,47 the presence of anxiety symptoms and feelings of worry and depression in low - profile adolescents are associated with the feeling of being perceived negatively by parents.48,49 even hypercontrol is interpreted by adolescents as the confirmation of a general devaluation by their parents.5052 recent research has made refusal and parental control effects more detailed and complex , highlighting how they may be related to both internalizing and externalizing symptoms.48,53 in particular , research has demonstrated the adaptive function of paternal empathy and support toward sons;54,55 these variables would act on both internalizing symptoms such as anxiety , depression , impulse control and externalizing symptoms such as aggression , taking drugs , alcohol use , and school discomfort . in addition , some theories about deviance in adolescence argue that certain deviant behaviors , such as delinquency and alcohol use , are no more than external manifestations of a difficult management of internalizing symptoms.56 the objective of this study is to examine the relationship between identity , parenting , and adolescent problems , which may manifest through internalized modes and externalized ones in a group of adolescents . in light of the recent literature , we assumed the following research hypotheses : 1 ) identity development and , in particular , the importance conferred to values , religion , family,50,57 and friendship16 influence alcohol use in adolescents;57 2 ) the presence of low - profile identity and excessive maternal control affect the relational addiction26 and the tendency to perfectionism;32 3 ) a low - profile identity and paternal dysfunctional parenting in adolescent males influence the levels of phobia , obsession , depression , and entropy;35,58 4 ) among the predictors of the use of alcohol are socioeconomic status ( ses),29,59 a low - profile identity,21,57 high parental control,9,10,15,60 and the presence of internalizing symptoms in adolescents.36 during adolescence , development tasks can be faced without serious difficulties , and the overcoming of these development tasks ( such as autonomy from parents , the comparison with peer group and the acquisition of useful tools to experience new roles ) could be a significant factor of protection from possible evolutionary breakdown;19,20 but transitional or problematic states can lead the person to manifest internalizing symptoms , especially during adolescence.21,22 the main risk factors that seem to match with the development of internalizing symptoms are as follows : reduced identity development,23 lack of emotional support,24 frequent adoption of punitive parenting behavior,25,26 parental psychological control,27,28 and also disadvantaged sociocultural characteristics.29,30 compared to parenting , adolescents exposed to parental psychological control seem to adopt the same rigorous and strict standards proposed by parents , living inadequately with the inability to reach the standards , both socially prescribed and those self - imposed.31,32 in particular , literature underlines the harmful effects of maternal control , which are less evident toward their daughters and more evident toward their sons , over the level of perfectionism in adolescent children . in the mother daughter dyad , rather than perfectionism , it seems that the responsible mechanism for the link between controlling parenting and internalizing symptoms is relational dependency.33,34 moreover , adolescents with low - profile identity ( foreclosure and diffusion status ) who find themselves living in an adverse family situation could experience a low perception of control over their environment , which may predispose them to develop the perception of reduced control over mental and physical states.35,36 in fact , recent studies show how high levels of warmth or support are related to lower levels of internalization in adolescents with high - profile identity;3739 on the other hand , excessive control and lack of parental empathy appear to be related to the onset of interior symptoms such as depression , anxiety,4042 lack of self - control , or irritability in low - profile adolescents.4345 in particular , the result of internalizing symptoms refers to the superimposition of anxious and depressive syndromes;46,47 the presence of anxiety symptoms and feelings of worry and depression in low - profile adolescents are associated with the feeling of being perceived negatively by parents.48,49 even hypercontrol is interpreted by adolescents as the confirmation of a general devaluation by their parents.5052 recent research has made refusal and parental control effects more detailed and complex , highlighting how they may be related to both internalizing and externalizing symptoms.48,53 in particular , research has demonstrated the adaptive function of paternal empathy and support toward sons;54,55 these variables would act on both internalizing symptoms such as anxiety , depression , impulse control and externalizing symptoms such as aggression , taking drugs , alcohol use , and school discomfort . in addition , some theories about deviance in adolescence argue that certain deviant behaviors , such as delinquency and alcohol use , are no more than external manifestations of a difficult management of internalizing symptoms.56 the objective of this study is to examine the relationship between identity , parenting , and adolescent problems , which may manifest through internalized modes and externalized ones in a group of adolescents . in light of the recent literature , we assumed the following research hypotheses : 1 ) identity development and , in particular , the importance conferred to values , religion , family,50,57 and friendship16 influence alcohol use in adolescents;57 2 ) the presence of low - profile identity and excessive maternal control affect the relational addiction26 and the tendency to perfectionism;32 3 ) a low - profile identity and paternal dysfunctional parenting in adolescent males influence the levels of phobia , obsession , depression , and entropy;35,58 4 ) among the predictors of the use of alcohol are socioeconomic status ( ses),29,59 a low - profile identity,21,57 high parental control,9,10,15,60 and the presence of internalizing symptoms in adolescents.36 the research involved 198 italian students ( 104 males and 94 females ) in the 4th year ( mean [ m ] = 16.94 years , standard deviation [ sd ] = 0.35 ) and 5th year ( m = 17.94 years , sd = 0.43 ) of senior secondary schools , who live in caltanissetta , a town located in sicily , italy . the research lasted for 1 year ; the group of participants involved all students attending the last 2 years of high school , through authorization of headmasters and teachers . although all subjects agreed to be part of the research , there was a mortality rate of 12% . this happened because the instruments were administered on 2 different days and the possible absence of students made it difficult to complete the compilation of all protocols . the consent of the school authorities and the students involved in the study was sought before the distribution and collection of the instruments . the questionnaires were anonymous , and the participants were informed of the aim and structure of the study . the research was approved by the internal review board of the faculty of human and social sciences at the kore university of enna . participants completed an anamnestic questionnaire , the ego identity process questionnaire , the parental bonding instrument , and the constraints of mind . anamnestic data were collected through the administration of a questionnaire constructed ad hoc and divided into three parts : the first to acquire basic information , age , sex , year attended , academic qualifications and professions of parents ; the second for establishing school performances , such as absences , permission to enter late or leave early , and marks obtained in the last 30 days ; and the third for ascertaining the frequency of alcohol use , average number of drinks consumed per occasion in the past 30 days and the reasons for it , and the beliefs about alcohol use . family income was defined on an eight - point scale , ranging from 700 euros to 4,000 euros per month . family education was measured with the highest level of parents education on a seven - point scale , ranging from primary school to a master s degree.61 the ego identity process questionnaire is a tool investigating the identity status development according to marcia s model31 through the dimensions of exploration and commitment . the exploration level is measured through the analysis of four ideological domains ( occupation , religion , politics , and values ) , and the commitment level is investigated through four interpersonal domains ( family , friendship , sex roles , and sentimental relationships ) . literature reports the estimates of internal validity of the tool as 0.80 for the results that indicate commitment and 0.86 for the scores that indicate exploration ; the scores that indicate reliability are 0.90 for commitment and 0.76 for exploration ; the internal consistency is 0.72 and 0.71 for commitment and exploration , respectively.62 the parental bonding instrument is a questionnaire consisting of 25 items , divided into two parts ( one for the mother and the other one for the father ) , which retrospectively measure the perception of behavior of the parents during childhood.63 the instrument investigates the processes of parenting across two domains , parental care and control or overprotection , from the combination of which four types of attachment were classified : affectionate constraint : high scores in both scales ; optimal parenting : high care and low protection ; affectionless control : high protection and low care ; and neglectful parenting : low care and low protection . assignment to high or low categories of domains is based on the following cutoff scores : for mothers , a care score of 27.0 and a protection score of 13.5 are the minimum scores for inclusion in the high category ; for fathers , a care score of 24.0 and a protection score of 12.5 are the cutoff points . the italian adaptation reports the estimates of internal consistency of the tool as 0.75 for the results that indicate mother s care , 0.84 for the scores that indicate mother s overprotection , 0.83 for father s care , and 0.88 for father s overprotection.64 the constraints of mind is a questionnaire consisting of 150 items , which measures 30 mind constraints grouped into five topicals:65 phobias : catastrophism , frailty , risk avoidance , health apprehension , distrust of others , relational addiction;obsessions : perfectionism , emotional inhibition , fear of making mistakes , control , superstition , cleaning and contamination;depression : failure , loneliness , inadequacy , forced esteem , compulsion to provide care for others , pessimism;entropy : distrust , odd thoughts , perceptual social control , social isolation , deterministic causality , ambivalence;diet : somatic discomfort , obsession of one s body , eating behavior , compulsive physical activity , dependence of other s judgment , relational dynamic . phobias : catastrophism , frailty , risk avoidance , health apprehension , distrust of others , relational addiction ; obsessions : perfectionism , emotional inhibition , fear of making mistakes , control , superstition , cleaning and contamination ; depression : failure , loneliness , inadequacy , forced esteem , compulsion to provide care for others , pessimism ; entropy : distrust , odd thoughts , perceptual social control , social isolation , deterministic causality , ambivalence ; diet : somatic discomfort , obsession of one s body , eating behavior , compulsive physical activity , dependence of other s judgment , relational dynamic . assignment to high or low categories of domains is based on the following cutoff scores : any score of 4 or more is considered meaningful ( both the score of each mind constraint , that the score of each topical ) . all analyses were conducted with statistical package for the social sciences 23.0 ( ibm corporation , armonk , ny , usa ) . in reference to preliminary data , t - test for independent samples was used to compare the mean between groups ( males versus females ) . in reference to the mind constraints and alcohol consumption , multivariate analysis of variance ( manova ) was conducted to verify the influence of independent variables ( sex and age ) on the identity and the mind constraints . in order to verify the influence of ideological and interpersonal domains on the use of alcohol , univariate analysis of variance if a low level of identity combined with excessive maternal control influences the relational dependence and the tendency to perfectionism , manova was carried out . the same analysis was carried out to verify if a low level of identity combined with dysfunctional paternal parenting influences the constraints of mind in the group of boys . in order to explore the predictive variables of alcohol use , hierarchical regression for separate blocks was used : 1 ) sex , age , and ses in the first block ; 2 ) the level of school performance in the second block ; 3 ) parenting in the third block ; and 4 ) mind constraints in the fourth block . each block of independent variables was evaluated in terms of what they added to the explanation of the variability of the dependent variable at the time of their entry , evaluating the weight of all predictors . the research involved 198 italian students ( 104 males and 94 females ) in the 4th year ( mean [ m ] = 16.94 years , standard deviation [ sd ] = 0.35 ) and 5th year ( m = 17.94 years , sd = 0.43 ) of senior secondary schools , who live in caltanissetta , a town located in sicily , italy . the research lasted for 1 year ; the group of participants involved all students attending the last 2 years of high school , through authorization of headmasters and teachers . although all subjects agreed to be part of the research , there was a mortality rate of 12% . this happened because the instruments were administered on 2 different days and the possible absence of students made it difficult to complete the compilation of all protocols . the consent of the school authorities and the students involved in the study was sought before the distribution and collection of the instruments . the questionnaires were anonymous , and the participants were informed of the aim and structure of the study . the research was approved by the internal review board of the faculty of human and social sciences at the kore university of enna . participants completed an anamnestic questionnaire , the ego identity process questionnaire , the parental bonding instrument , and the constraints of mind . anamnestic data were collected through the administration of a questionnaire constructed ad hoc and divided into three parts : the first to acquire basic information , age , sex , year attended , academic qualifications and professions of parents ; the second for establishing school performances , such as absences , permission to enter late or leave early , and marks obtained in the last 30 days ; and the third for ascertaining the frequency of alcohol use , average number of drinks consumed per occasion in the past 30 days and the reasons for it , and the beliefs about alcohol use . family income was defined on an eight - point scale , ranging from 700 euros to 4,000 euros per month . family education was measured with the highest level of parents education on a seven - point scale , ranging from primary school to a master s degree.61 the ego identity process questionnaire is a tool investigating the identity status development according to marcia s model31 through the dimensions of exploration and commitment . the exploration level is measured through the analysis of four ideological domains ( occupation , religion , politics , and values ) , and the commitment level is investigated through four interpersonal domains ( family , friendship , sex roles , and sentimental relationships ) . literature reports the estimates of internal validity of the tool as 0.80 for the results that indicate commitment and 0.86 for the scores that indicate exploration ; the scores that indicate reliability are 0.90 for commitment and 0.76 for exploration ; the internal consistency is 0.72 and 0.71 for commitment and exploration , respectively.62 the parental bonding instrument is a questionnaire consisting of 25 items , divided into two parts ( one for the mother and the other one for the father ) , which retrospectively measure the perception of behavior of the parents during childhood.63 the instrument investigates the processes of parenting across two domains , parental care and control or overprotection , from the combination of which four types of attachment were classified : affectionate constraint : high scores in both scales ; optimal parenting : high care and low protection ; affectionless control : high protection and low care ; and neglectful parenting : low care and low protection . assignment to high or low categories of domains is based on the following cutoff scores : for mothers , a care score of 27.0 and a protection score of 13.5 are the minimum scores for inclusion in the high category ; for fathers , a care score of 24.0 and a protection score of 12.5 are the cutoff points . the italian adaptation reports the estimates of internal consistency of the tool as 0.75 for the results that indicate mother s care , 0.84 for the scores that indicate mother s overprotection , 0.83 for father s care , and 0.88 for father s overprotection.64 the constraints of mind is a questionnaire consisting of 150 items , which measures 30 mind constraints grouped into five topicals:65 phobias : catastrophism , frailty , risk avoidance , health apprehension , distrust of others , relational addiction;obsessions : perfectionism , emotional inhibition , fear of making mistakes , control , superstition , cleaning and contamination;depression : failure , loneliness , inadequacy , forced esteem , compulsion to provide care for others , pessimism;entropy : distrust , odd thoughts , perceptual social control , social isolation , deterministic causality , ambivalence;diet : somatic discomfort , obsession of one s body , eating behavior , compulsive physical activity , dependence of other s judgment , relational dynamic . phobias : catastrophism , frailty , risk avoidance , health apprehension , distrust of others , relational addiction ; obsessions : perfectionism , emotional inhibition , fear of making mistakes , control , superstition , cleaning and contamination ; depression : failure , loneliness , inadequacy , forced esteem , compulsion to provide care for others , pessimism ; entropy : distrust , odd thoughts , perceptual social control , social isolation , deterministic causality , ambivalence ; diet : somatic discomfort , obsession of one s body , eating behavior , compulsive physical activity , dependence of other s judgment , relational dynamic . assignment to high or low categories of domains is based on the following cutoff scores : any score of 4 or more is considered meaningful ( both the score of each mind constraint , that the score of each topical ) . all analyses were conducted with statistical package for the social sciences 23.0 ( ibm corporation , armonk , ny , usa ) . in reference to preliminary data , t - test for independent samples was used to compare the mean between groups ( males versus females ) . in reference to the mind constraints and alcohol consumption , multivariate analysis of variance ( manova ) was conducted to verify the influence of independent variables ( sex and age ) on the identity and the mind constraints . in order to verify the influence of ideological and interpersonal domains on the use of alcohol , univariate analysis of variance if a low level of identity combined with excessive maternal control influences the relational dependence and the tendency to perfectionism , manova was carried out . the same analysis was carried out to verify if a low level of identity combined with dysfunctional paternal parenting influences the constraints of mind in the group of boys . in order to explore the predictive variables of alcohol use , hierarchical regression for separate blocks was used : 1 ) sex , age , and ses in the first block ; 2 ) the level of school performance in the second block ; 3 ) parenting in the third block ; and 4 ) mind constraints in the fourth block . each block of independent variables was evaluated in terms of what they added to the explanation of the variability of the dependent variable at the time of their entry , evaluating the weight of all predictors . a descriptive analysis was conducted in order to investigate the mind constraints , comparing the mean scores of boys and girls and the cutoff scores for the italian population . in the participant group , the t - test shows that in the entropy domain , boys had higher scores than girls in perceptual social control ( f=4.60 ; p<0.05 ) ; in the diet domain , girls reported average scores significantly higher than boys in somatic discomfort ( f=12.13 ; p<0.01 ) . compared to alcohol consumption , t - test shows a significant difference for the variable sex ( t = 2.69 , p<0.001 ) : boys reported monthly use of alcohol more frequently than girls ( males : m = 3.95 , sd = 6.73 ; females : m = 1.95 , sd = 0.28 ) . manova , done to verify the influence of independent variables on the ideological and interpersonal domains , emphasizes the main effect of age ( wilks s lambda = 0.92 , f=2.30 , p<0.05 ) , but no effect due to sex ( wilks s lambda = 0.86 , f=1.22 , p = not significant [ ns ] ) . breakdown of the univariate effects shows that older students got higher scores in the family dimension than younger ones ( f=3.40 , p<0.05 ) . a further manova done to verify the influence of independent variables on the mind constraints emphasizes a main effect linked to the sex variable ( wilks s lambda = 0.30 , f=7.73 , p<0.001 ) , age ( wilks s lambda = 0.31 , f=1.55 , p<0.01 ) , and ses ( wilks s lambda = 0.003 , f=1.30 , p<0.001 ) . in reference to the sex variable , breakdown of the univariate effects shows differences in emotional inhibition ( f=4.93 , p<0.05 ) and pessimism ( f=110.26 , p<0.001 ) : tukey s post hoc shows that boys obtained higher values in emotional inhibition than girls , who obtained higher scores in the pessimism dimension . in reference to age , there is a difference in the pessimism dimension ( f=14.23 , p<0.001 ) : tukey s post hoc shows that older adolescents manifested higher values in pessimism than those who were younger . in reference to ses , there are differences in pessimism ( f=11.34 , p<0.001 ) and fear of making mistakes ( f=2.55 , p<0.05 ) ; in particular , adolescents with a greater ses showed lower average scores in pessimism than the adolescents with low ses , who reported greater emotional inhibition . in reference to the first hypothesis , the analysis of variance shows that between the ideological dimensions , only politics ( f=4.56 , p<0.01 ) and religion ( f=3.03 , p<0.05 ) appear to influence alcohol use in adolescents ; between the interpersonal dimensions , only the family ( f=2.93 , p<0.05 ) seems to affect this risk behavior . the analysis of mean scores shows how adolescents who give more emphasis to politics , religion , and family , respectively , declare a lower level of alcohol consumption . the data confirm , in part , the first research hypothesis because the importance conferred to friendship does not seem to affect alcohol use in adolescents . in confirmation of the second research hypothesis , manova shows the main effect of identity ( wilks s lambda = 0.41 , f=1.46 , p<0.05 ) and the type of parenting mother ( wilks s lambda = 0.001 , f=0.32 , p<0.05 ) and no interaction effect ( wilks s lambda = 0.65 ; f=0.92 , p = ns ) . the univariate effect shows how the identity influences relational addiction ( f=5.11 , p<0.01 ) , perfectionism ( f=3.20 , p<0.05 ) , fear of error ( f=2.78 , p<0.05 ) , sense of inadequacy ( f=2.95 , p<0.05 ) , and compulsion to provide care for others ( f=3.83 , p<0.05 ) . post hoc shows that students in foreclosure status have the highest average scores in relational addiction , the fear of making mistakes , and the compulsion to care ; students in achievement manifest a greater perfectionism ; and those in moratorium perceive greater sense of inadequacy . the type of maternal parenting seems to affect significantly the following constraints : distrust of others ( f=4.90 , p<0.01 ) , relational addiction ( f=3.26 , p<0.05 ) , sense of failure ( f=5.25 , p<0.01 ) , pessimism ( f=3.13 , p<0.05 ) , distrust ( f=4.29 , p<0.01 ) , social control ( f=4.24 , p<0.01 ) , social isolation ( f=3.28 , p<0.05 ) , and ambivalence ( f=6.50 , p<0.001 ) . post hoc shows that adolescents with affectionless control parenting show higher scores in relational dependence , sense of failure , mistrust for the next , perceptual social control , isolation , and ambivalence ; otherwise , adolescents with neglectful parenting manifest higher levels of mistrust of others . in reference to the third hypothesis , manova shows the effect of parenting ( wilks s lambda = 0.65 , f=1.85 , p<0.05 ) on the following symptoms : catastrophism ( f=3.45 , p<0.05 ) , health apprehension ( f=3.56 , p<0.05 ) , distrust of others ( f=04.03 , p<0.05 ) , relational addiction ( f=3.09 , p<0.05 ) , emotional inhibition ( f=4.99 , p<0.01 ) , superstition ( f=3.17 , p<0.05 ) , failure ( f=3.78 , p<0.05 ) , inadequacy ( f=4.83 , p<0.01 ) , and social isolation ( f=2.99 , p<0.05 ) , depending on other s judgment ( f=5.05 , p<0.01 ) and relational dynamic ( f=3.18 , p<0.05 ) . tukey s post hoc shows that adolescent boys with neglectful parenting have high scores in the aforementioned symptoms except two , sense of failure and inadequacy , which are manifested by boys with affectionate constraint parenting . it , therefore , seems to confirm the third research hypothesis . in reference to the last hypothesis , hierarchical regression with separate blocks shows that the predictors of alcohol consumption are ( table 2 ) : sex , age , ses , the level of depression , and entropy ( 21.2% of the overall variance explained ) . a descriptive analysis was conducted in order to investigate the mind constraints , comparing the mean scores of boys and girls and the cutoff scores for the italian population . in the participant group , the t - test shows that in the entropy domain , boys had higher scores than girls in perceptual social control ( f=4.60 ; p<0.05 ) ; in the diet domain , girls reported average scores significantly higher than boys in somatic discomfort ( f=12.13 ; p<0.01 ) . compared to alcohol consumption , t - test shows a significant difference for the variable sex ( t = 2.69 , p<0.001 ) : boys reported monthly use of alcohol more frequently than girls ( males : m = 3.95 , sd = 6.73 ; females : m = 1.95 , sd = 0.28 ) . manova , done to verify the influence of independent variables on the ideological and interpersonal domains , emphasizes the main effect of age ( wilks s lambda = 0.92 , f=2.30 , p<0.05 ) , but no effect due to sex ( wilks s lambda = 0.86 , f=1.22 , p = not significant [ ns ] ) . breakdown of the univariate effects shows that older students got higher scores in the family dimension than younger ones ( f=3.40 , p<0.05 ) . a further manova done to verify the influence of independent variables on the mind constraints emphasizes a main effect linked to the sex variable ( wilks s lambda = 0.30 , f=7.73 , p<0.001 ) , age ( wilks s lambda = 0.31 , f=1.55 , p<0.01 ) , and ses ( wilks s lambda = 0.003 , f=1.30 , p<0.001 ) . in reference to the sex variable , breakdown of the univariate effects shows differences in emotional inhibition ( f=4.93 , p<0.05 ) and pessimism ( f=110.26 , p<0.001 ) : tukey s post hoc shows that boys obtained higher values in emotional inhibition than girls , who obtained higher scores in the pessimism dimension . in reference to age , there is a difference in the pessimism dimension ( f=14.23 , p<0.001 ) : tukey s post hoc shows that older adolescents manifested higher values in pessimism than those who were younger . in reference to ses , there are differences in pessimism ( f=11.34 , p<0.001 ) and fear of making mistakes ( f=2.55 , p<0.05 ) ; in particular , adolescents with a greater ses showed lower average scores in pessimism than the adolescents with low ses , who reported greater emotional inhibition . in reference to the first hypothesis , the analysis of variance shows that between the ideological dimensions , only politics ( f=4.56 , p<0.01 ) and religion ( f=3.03 , p<0.05 ) appear to influence alcohol use in adolescents ; between the interpersonal dimensions , only the family ( f=2.93 , p<0.05 ) seems to affect this risk behavior . the analysis of mean scores shows how adolescents who give more emphasis to politics , religion , and family , respectively , declare a lower level of alcohol consumption . the data confirm , in part , the first research hypothesis because the importance conferred to friendship does not seem to affect alcohol use in adolescents . in confirmation of the second research hypothesis , manova shows the main effect of identity ( wilks s lambda = 0.41 , f=1.46 , p<0.05 ) and the type of parenting mother ( wilks s lambda = 0.001 , f=0.32 , p<0.05 ) and no interaction effect ( wilks s lambda = 0.65 ; f=0.92 , p = ns ) . the univariate effect shows how the identity influences relational addiction ( f=5.11 , p<0.01 ) , perfectionism ( f=3.20 , p<0.05 ) , fear of error ( f=2.78 , p<0.05 ) , sense of inadequacy ( f=2.95 , p<0.05 ) , and compulsion to provide care for others ( f=3.83 , p<0.05 ) . post hoc shows that students in foreclosure status have the highest average scores in relational addiction , the fear of making mistakes , and the compulsion to care ; students in achievement manifest a greater perfectionism ; and those in moratorium perceive greater sense of inadequacy . the type of maternal parenting seems to affect significantly the following constraints : distrust of others ( f=4.90 , p<0.01 ) , relational addiction ( f=3.26 , p<0.05 ) , sense of failure ( f=5.25 , p<0.01 ) , pessimism ( f=3.13 , p<0.05 ) , distrust ( f=4.29 , p<0.01 ) , social control ( f=4.24 , p<0.01 ) , social isolation ( f=3.28 , p<0.05 ) , and ambivalence ( f=6.50 , p<0.001 ) . post hoc shows that adolescents with affectionless control parenting show higher scores in relational dependence , sense of failure , mistrust for the next , perceptual social control , isolation , and ambivalence ; otherwise , adolescents with neglectful parenting manifest higher levels of mistrust of others . in reference to the third hypothesis , manova shows the effect of parenting ( wilks s lambda = 0.65 , f=1.85 , p<0.05 ) on the following symptoms : catastrophism ( f=3.45 , p<0.05 ) , health apprehension ( f=3.56 , p<0.05 ) , distrust of others ( f=04.03 , p<0.05 ) , relational addiction ( f=3.09 , p<0.05 ) , emotional inhibition ( f=4.99 , p<0.01 ) , superstition ( f=3.17 , p<0.05 ) , failure ( f=3.78 , p<0.05 ) , inadequacy ( f=4.83 , p<0.01 ) , and social isolation ( f=2.99 , p<0.05 ) , depending on other s judgment ( f=5.05 , p<0.01 ) and relational dynamic ( f=3.18 , p<0.05 ) . post hoc shows that adolescent boys with neglectful parenting have high scores in the aforementioned symptoms except two , sense of failure and inadequacy , which are manifested by boys with affectionate constraint parenting . it , therefore , seems to confirm the third research hypothesis . in reference to the last hypothesis , hierarchical regression with separate blocks shows that the predictors of alcohol consumption are ( table 2 ) : sex , age , ses , the level of depression , and entropy ( 21.2% of the overall variance explained ) . the study investigates the possible relationship between identity development , parenting , and internalizing externalizing symptoms during adolescence . it involves 198 italian students , aged between 16 and 19 , who live in caltanissetta , a town located in sicily , italy . as a partial confirmation of the first research hypothesis , identity seems to influence alcohol use in adolescents . in coherence with the literature,50,57 adolescents who confer greater importance to politics , religion , and family reduce their use of alcohol , although the dimension of friendship does not seem to influence the risk behavior . probably , the partial confirmation of the hypothesis is due to the age of the participants , characterized by instability typical of adolescence.19,21 as the second research hypothesis states , both identity and the type of parenting affect internalizing symptoms , in particular . adolescents in the foreclosure status manifest greater compulsion to caregiving with a resulting relational addiction and fear of error , probably due to the lack of critical evaluation of the various identity alternatives.26,32 students in moratorium status perceive a greater sense of inadequacy , typical of those who have started an exploration path of the possible identity alternatives , without assuming any commitment yet.14,19 at least , the adolescents in achievement show high levels of perfectionism ; this finding could be explained by the fact that in sicilian culture , roles within the family are highly emphasized and adolescents have a strong need to meet parental and social expectations.6668 this process , which is very common among south italians , to make a good impression ( fare bella figura ) when interacting with other people , is very much a matter of showing integrity and bringing people to have respect for them ; when they are not able to adopt to social pressure that is , when they make a bad impression so , exposure to perfectionism and to an authoritarian parenting style may bring the adolescent to the perception of rigorous expectations , self - esteem linked to success , and fear of disappointing others.69 in confirmation of the third hypothesis , adolescent boys with paternal parenting marked by excessive control would perceive a greater sense of failure and inadequacy in dealing with everyday problems , which is typical in adolescence.70 the latest research hypothesis seems to be confirmed as well . among the predictor variables to the use of alcohol , in particular , it seems that the older kids with greater emotional inhibition and perception of external control consume more alcohol ; such a risky behavior is shown mainly by people belonging to higher ses , or in families whose parents have a good level of education and culture , and high professional status , which is in disagreement with the international literature.71,72 in fact , there was little empirical evidence to support the commonly held view that social deprivation or low ses is associated with belated and reduced alcohol use . in reference to the relationship between low ses and alcohol consumption , there may be two opposing mechanisms.73 first , social deprivation is associated with almost all forms of morbidity and mortality.74 the second mechanism underlines that individuals with more money can afford more alcohol because there is a consistent inverse relationship across time between the cost of alcohol and the amount consumed . our data support the second mechanism and appear to confirm the recent research conducted by the organization for economic cooperation and development , according to which in economically developed countries , such as italy , the consumption of alcohol is higher among young people . based on the results described herein , it is appropriate to emphasize the limits of this work , namely , the absence of a sampling method , which prevents the presence of a representative sample , generalization of the results , and external validity . an additional limitation is the absence of a longitudinal - type study design , which is more suitable for research involving adolescents and their identity development . finally , the absence of a cross - sectional survey method makes it difficult to determine if the adverse relational family conditions ( eg , low support or high control ) are antecedents , links , or consequences of the antisocial behavior of children . therefore , identification of risk factors in individuals or in their environment is not enough to predict the future development , but it is necessary to consider the way in which certain features interact with the environment , modifying it and being , in turn , influenced by it . it is necessary , in other words , to take into consideration the features of the subject , the features of the environment , and the way in which these two sets of influences interact over time.75 researchers have underlined the importance of parent training interventions for adolescents with alcohol use and comorbidity of externalizing and internalizing disorders.76,77 participation in parent training , in fact , increased parental monitoring78 and decreased family conflict adolescent substance use and adolescent behavior problems.79

backgroundliterature has demonstrated the adaptive function of identity development and parenting toward manifestation of problem behaviors in adolescence . these dimensions act on both internalizing and externalizing symptoms.methodsthe objective is to investigate the relationship between identity status , parenting , and adolescent problems , which may manifest through internalized ( phobias , obsessions , depression , eating disorders , entropy ) and externalized modes ( alcohol use and school discomfort ) . the research involved 198 italian students ( 104 males and 94 females ) in the 4th year ( mean = 16.94 years , standard deviation = 0.35 ) and 5th year ( mean = 17.94 years , standard deviation = 0.43 ) of senior secondary schools , who live in caltanissetta , a town located in sicily , italy . the research lasted for 1 school year . the general group consisted of 225 students with a mortality rate of 12% . they completed an anamnestic questionnaire to provide 1 ) basic information , 2 ) alcohol consumption attitude in the past 30 days , and 3 ) their beliefs about alcohol ; the ego identity process questionnaire to investigate identity development ; the parental bonding instrument to measure the perception of parenting during childhood ; and the constraints of mind to value the presence of internalizing symptoms.resultsdata show that identity status influences alcohol consumption . low - profile identity and excessive maternal control affect the relational dependence and the tendency to perfectionism in adolescents . among the predictors of alcohol use , there are socioeconomic status , parental control , and the presence of internalizing symptoms.conclusionfamily is the favored context of learning beliefs , patterns , and values that affect the broader regulatory social environment , and for this reason , it is considered the privileged context on which to intervene to reduce the adolescents behavior problems . this deviance could be an external manifestation of the difficulty in management of internalizing symptoms in adolescence .

Introduction Identity, parenting, and internalizing symptoms in adolescence Methods Participants and procedure Measures Data analysis Results Descriptive statistics Preliminary data Discussion Conclusion

this cross - sectional study investigates the possible relationship between identity development , parenting , and adolescent problems , which may manifest through internalized modes ( phobias , obsessions , depression , eating disorders , entropy ) and externalized ones ( alcohol use and school discomfort ) , in a group of 198 italian students . recent literature emphasizes the importance of examining the risk and protection factors for the internalizing and externalizing symptoms in adolescence , which appear in different contexts : social and interpersonal , such as family , school , and peer group ; and individual factors , such as identity development , and cognitive and behavioral patterns . however , the social and family dimensions can influence the early risk behavior experimentation in adolescence , but the outcome of these conditioning issues could have different consequences in relation to the individual features , such as identity development and the possible presence of internalizing symptoms.1618 during adolescence , development tasks can be faced without serious difficulties , and the overcoming of these development tasks ( such as autonomy from parents , the comparison with peer group and the acquisition of useful tools to experience new roles ) could be a significant factor of protection from possible evolutionary breakdown;19,20 but transitional or problematic states can lead the person to manifest internalizing symptoms , especially during adolescence.21,22 the main risk factors that seem to match with the development of internalizing symptoms are as follows : reduced identity development,23 lack of emotional support,24 frequent adoption of punitive parenting behavior,25,26 parental psychological control,27,28 and also disadvantaged sociocultural characteristics.29,30 compared to parenting , adolescents exposed to parental psychological control seem to adopt the same rigorous and strict standards proposed by parents , living inadequately with the inability to reach the standards , both socially prescribed and those self - imposed.31,32 in particular , literature underlines the harmful effects of maternal control , which are less evident toward their daughters and more evident toward their sons , over the level of perfectionism in adolescent children . in the mother daughter dyad , rather than perfectionism , it seems that the responsible mechanism for the link between controlling parenting and internalizing symptoms is relational dependency.33,34 moreover , adolescents with low - profile identity ( foreclosure and diffusion status ) who find themselves living in an adverse family situation could experience a low perception of control over their environment , which may predispose them to develop the perception of reduced control over mental and physical states.35,36 in fact , recent studies show how high levels of warmth or support are related to lower levels of internalization in adolescents with high - profile identity;3739 on the other hand , excessive control and lack of parental empathy appear to be related to the onset of interior symptoms such as depression , anxiety,4042 lack of self - control , or irritability in low - profile adolescents.4345 in particular , the result of internalizing symptoms refers to the superimposition of anxious and depressive syndromes;46,47 the presence of anxiety symptoms and feelings of worry and depression in low - profile adolescents are associated with the feeling of being perceived negatively by parents.48,49 even hypercontrol is interpreted by adolescents as the confirmation of a general devaluation by their parents.5052 recent research has made refusal and parental control effects more detailed and complex , highlighting how they may be related to both internalizing and externalizing symptoms.48,53 in particular , research has demonstrated the adaptive function of paternal empathy and support toward sons;54,55 these variables would act on both internalizing symptoms such as anxiety , depression , impulse control and externalizing symptoms such as aggression , taking drugs , alcohol use , and school discomfort . in addition , some theories about deviance in adolescence argue that certain deviant behaviors , such as delinquency and alcohol use , are no more than external manifestations of a difficult management of internalizing symptoms.56 the objective of this study is to examine the relationship between identity , parenting , and adolescent problems , which may manifest through internalized modes and externalized ones in a group of adolescents . in light of the recent literature , we assumed the following research hypotheses : 1 ) identity development and , in particular , the importance conferred to values , religion , family,50,57 and friendship16 influence alcohol use in adolescents;57 2 ) the presence of low - profile identity and excessive maternal control affect the relational addiction26 and the tendency to perfectionism;32 3 ) a low - profile identity and paternal dysfunctional parenting in adolescent males influence the levels of phobia , obsession , depression , and entropy;35,58 4 ) among the predictors of the use of alcohol are socioeconomic status ( ses),29,59 a low - profile identity,21,57 high parental control,9,10,15,60 and the presence of internalizing symptoms in adolescents.36 during adolescence , development tasks can be faced without serious difficulties , and the overcoming of these development tasks ( such as autonomy from parents , the comparison with peer group and the acquisition of useful tools to experience new roles ) could be a significant factor of protection from possible evolutionary breakdown;19,20 but transitional or problematic states can lead the person to manifest internalizing symptoms , especially during adolescence.21,22 the main risk factors that seem to match with the development of internalizing symptoms are as follows : reduced identity development,23 lack of emotional support,24 frequent adoption of punitive parenting behavior,25,26 parental psychological control,27,28 and also disadvantaged sociocultural characteristics.29,30 compared to parenting , adolescents exposed to parental psychological control seem to adopt the same rigorous and strict standards proposed by parents , living inadequately with the inability to reach the standards , both socially prescribed and those self - imposed.31,32 in particular , literature underlines the harmful effects of maternal control , which are less evident toward their daughters and more evident toward their sons , over the level of perfectionism in adolescent children . in the mother daughter dyad , rather than perfectionism , it seems that the responsible mechanism for the link between controlling parenting and internalizing symptoms is relational dependency.33,34 moreover , adolescents with low - profile identity ( foreclosure and diffusion status ) who find themselves living in an adverse family situation could experience a low perception of control over their environment , which may predispose them to develop the perception of reduced control over mental and physical states.35,36 in fact , recent studies show how high levels of warmth or support are related to lower levels of internalization in adolescents with high - profile identity;3739 on the other hand , excessive control and lack of parental empathy appear to be related to the onset of interior symptoms such as depression , anxiety,4042 lack of self - control , or irritability in low - profile adolescents.4345 in particular , the result of internalizing symptoms refers to the superimposition of anxious and depressive syndromes;46,47 the presence of anxiety symptoms and feelings of worry and depression in low - profile adolescents are associated with the feeling of being perceived negatively by parents.48,49 even hypercontrol is interpreted by adolescents as the confirmation of a general devaluation by their parents.5052 recent research has made refusal and parental control effects more detailed and complex , highlighting how they may be related to both internalizing and externalizing symptoms.48,53 in particular , research has demonstrated the adaptive function of paternal empathy and support toward sons;54,55 these variables would act on both internalizing symptoms such as anxiety , depression , impulse control and externalizing symptoms such as aggression , taking drugs , alcohol use , and school discomfort . in addition , some theories about deviance in adolescence argue that certain deviant behaviors , such as delinquency and alcohol use , are no more than external manifestations of a difficult management of internalizing symptoms.56 the objective of this study is to examine the relationship between identity , parenting , and adolescent problems , which may manifest through internalized modes and externalized ones in a group of adolescents . in light of the recent literature , we assumed the following research hypotheses : 1 ) identity development and , in particular , the importance conferred to values , religion , family,50,57 and friendship16 influence alcohol use in adolescents;57 2 ) the presence of low - profile identity and excessive maternal control affect the relational addiction26 and the tendency to perfectionism;32 3 ) a low - profile identity and paternal dysfunctional parenting in adolescent males influence the levels of phobia , obsession , depression , and entropy;35,58 4 ) among the predictors of the use of alcohol are socioeconomic status ( ses),29,59 a low - profile identity,21,57 high parental control,9,10,15,60 and the presence of internalizing symptoms in adolescents.36 the research involved 198 italian students ( 104 males and 94 females ) in the 4th year ( mean [ m ] = 16.94 years , standard deviation [ sd ] = 0.35 ) and 5th year ( m = 17.94 years , sd = 0.43 ) of senior secondary schools , who live in caltanissetta , a town located in sicily , italy . although all subjects agreed to be part of the research , there was a mortality rate of 12% . participants completed an anamnestic questionnaire , the ego identity process questionnaire , the parental bonding instrument , and the constraints of mind . anamnestic data were collected through the administration of a questionnaire constructed ad hoc and divided into three parts : the first to acquire basic information , age , sex , year attended , academic qualifications and professions of parents ; the second for establishing school performances , such as absences , permission to enter late or leave early , and marks obtained in the last 30 days ; and the third for ascertaining the frequency of alcohol use , average number of drinks consumed per occasion in the past 30 days and the reasons for it , and the beliefs about alcohol use . literature reports the estimates of internal validity of the tool as 0.80 for the results that indicate commitment and 0.86 for the scores that indicate exploration ; the scores that indicate reliability are 0.90 for commitment and 0.76 for exploration ; the internal consistency is 0.72 and 0.71 for commitment and exploration , respectively.62 the parental bonding instrument is a questionnaire consisting of 25 items , divided into two parts ( one for the mother and the other one for the father ) , which retrospectively measure the perception of behavior of the parents during childhood.63 the instrument investigates the processes of parenting across two domains , parental care and control or overprotection , from the combination of which four types of attachment were classified : affectionate constraint : high scores in both scales ; optimal parenting : high care and low protection ; affectionless control : high protection and low care ; and neglectful parenting : low care and low protection . the italian adaptation reports the estimates of internal consistency of the tool as 0.75 for the results that indicate mother s care , 0.84 for the scores that indicate mother s overprotection , 0.83 for father s care , and 0.88 for father s overprotection.64 the constraints of mind is a questionnaire consisting of 150 items , which measures 30 mind constraints grouped into five topicals:65 phobias : catastrophism , frailty , risk avoidance , health apprehension , distrust of others , relational addiction;obsessions : perfectionism , emotional inhibition , fear of making mistakes , control , superstition , cleaning and contamination;depression : failure , loneliness , inadequacy , forced esteem , compulsion to provide care for others , pessimism;entropy : distrust , odd thoughts , perceptual social control , social isolation , deterministic causality , ambivalence;diet : somatic discomfort , obsession of one s body , eating behavior , compulsive physical activity , dependence of other s judgment , relational dynamic . in order to verify the influence of ideological and interpersonal domains on the use of alcohol , univariate analysis of variance if a low level of identity combined with excessive maternal control influences the relational dependence and the tendency to perfectionism , manova was carried out . in order to explore the predictive variables of alcohol use , hierarchical regression for separate blocks was used : 1 ) sex , age , and ses in the first block ; 2 ) the level of school performance in the second block ; 3 ) parenting in the third block ; and 4 ) mind constraints in the fourth block . the research involved 198 italian students ( 104 males and 94 females ) in the 4th year ( mean [ m ] = 16.94 years , standard deviation [ sd ] = 0.35 ) and 5th year ( m = 17.94 years , sd = 0.43 ) of senior secondary schools , who live in caltanissetta , a town located in sicily , italy . although all subjects agreed to be part of the research , there was a mortality rate of 12% . participants completed an anamnestic questionnaire , the ego identity process questionnaire , the parental bonding instrument , and the constraints of mind . anamnestic data were collected through the administration of a questionnaire constructed ad hoc and divided into three parts : the first to acquire basic information , age , sex , year attended , academic qualifications and professions of parents ; the second for establishing school performances , such as absences , permission to enter late or leave early , and marks obtained in the last 30 days ; and the third for ascertaining the frequency of alcohol use , average number of drinks consumed per occasion in the past 30 days and the reasons for it , and the beliefs about alcohol use . literature reports the estimates of internal validity of the tool as 0.80 for the results that indicate commitment and 0.86 for the scores that indicate exploration ; the scores that indicate reliability are 0.90 for commitment and 0.76 for exploration ; the internal consistency is 0.72 and 0.71 for commitment and exploration , respectively.62 the parental bonding instrument is a questionnaire consisting of 25 items , divided into two parts ( one for the mother and the other one for the father ) , which retrospectively measure the perception of behavior of the parents during childhood.63 the instrument investigates the processes of parenting across two domains , parental care and control or overprotection , from the combination of which four types of attachment were classified : affectionate constraint : high scores in both scales ; optimal parenting : high care and low protection ; affectionless control : high protection and low care ; and neglectful parenting : low care and low protection . the italian adaptation reports the estimates of internal consistency of the tool as 0.75 for the results that indicate mother s care , 0.84 for the scores that indicate mother s overprotection , 0.83 for father s care , and 0.88 for father s overprotection.64 the constraints of mind is a questionnaire consisting of 150 items , which measures 30 mind constraints grouped into five topicals:65 phobias : catastrophism , frailty , risk avoidance , health apprehension , distrust of others , relational addiction;obsessions : perfectionism , emotional inhibition , fear of making mistakes , control , superstition , cleaning and contamination;depression : failure , loneliness , inadequacy , forced esteem , compulsion to provide care for others , pessimism;entropy : distrust , odd thoughts , perceptual social control , social isolation , deterministic causality , ambivalence;diet : somatic discomfort , obsession of one s body , eating behavior , compulsive physical activity , dependence of other s judgment , relational dynamic . in order to verify the influence of ideological and interpersonal domains on the use of alcohol , univariate analysis of variance if a low level of identity combined with excessive maternal control influences the relational dependence and the tendency to perfectionism , manova was carried out . in order to explore the predictive variables of alcohol use , hierarchical regression for separate blocks was used : 1 ) sex , age , and ses in the first block ; 2 ) the level of school performance in the second block ; 3 ) parenting in the third block ; and 4 ) mind constraints in the fourth block . in reference to the last hypothesis , hierarchical regression with separate blocks shows that the predictors of alcohol consumption are ( table 2 ) : sex , age , ses , the level of depression , and entropy ( 21.2% of the overall variance explained ) . in confirmation of the second research hypothesis , manova shows the main effect of identity ( wilks s lambda = 0.41 , f=1.46 , p<0.05 ) and the type of parenting mother ( wilks s lambda = 0.001 , f=0.32 , p<0.05 ) and no interaction effect ( wilks s lambda = 0.65 ; f=0.92 , p = ns ) . in reference to the last hypothesis , hierarchical regression with separate blocks shows that the predictors of alcohol consumption are ( table 2 ) : sex , age , ses , the level of depression , and entropy ( 21.2% of the overall variance explained ) . the study investigates the possible relationship between identity development , parenting , and internalizing externalizing symptoms during adolescence . it involves 198 italian students , aged between 16 and 19 , who live in caltanissetta , a town located in sicily , italy . adolescents in the foreclosure status manifest greater compulsion to caregiving with a resulting relational addiction and fear of error , probably due to the lack of critical evaluation of the various identity alternatives.26,32 students in moratorium status perceive a greater sense of inadequacy , typical of those who have started an exploration path of the possible identity alternatives , without assuming any commitment yet.14,19 at least , the adolescents in achievement show high levels of perfectionism ; this finding could be explained by the fact that in sicilian culture , roles within the family are highly emphasized and adolescents have a strong need to meet parental and social expectations.6668 this process , which is very common among south italians , to make a good impression ( fare bella figura ) when interacting with other people , is very much a matter of showing integrity and bringing people to have respect for them ; when they are not able to adopt to social pressure that is , when they make a bad impression so , exposure to perfectionism and to an authoritarian parenting style may bring the adolescent to the perception of rigorous expectations , self - esteem linked to success , and fear of disappointing others.69 in confirmation of the third hypothesis , adolescent boys with paternal parenting marked by excessive control would perceive a greater sense of failure and inadequacy in dealing with everyday problems , which is typical in adolescence.70 the latest research hypothesis seems to be confirmed as well . among the predictor variables to the use of alcohol , in particular , it seems that the older kids with greater emotional inhibition and perception of external control consume more alcohol ; such a risky behavior is shown mainly by people belonging to higher ses , or in families whose parents have a good level of education and culture , and high professional status , which is in disagreement with the international literature.71,72 in fact , there was little empirical evidence to support the commonly held view that social deprivation or low ses is associated with belated and reduced alcohol use . in reference to the relationship between low ses and alcohol consumption , there may be two opposing mechanisms.73 first , social deprivation is associated with almost all forms of morbidity and mortality.74 the second mechanism underlines that individuals with more money can afford more alcohol because there is a consistent inverse relationship across time between the cost of alcohol and the amount consumed . based on the results described herein , it is appropriate to emphasize the limits of this work , namely , the absence of a sampling method , which prevents the presence of a representative sample , generalization of the results , and external validity . it is necessary , in other words , to take into consideration the features of the subject , the features of the environment , and the way in which these two sets of influences interact over time.75 researchers have underlined the importance of parent training interventions for adolescents with alcohol use and comorbidity of externalizing and internalizing disorders.76,77 participation in parent training , in fact , increased parental monitoring78 and decreased family conflict adolescent substance use and adolescent behavior problems.79

working memory ( wm ) is generally described as a memory system that temporarily maintains and manipulates different types of information during a task at hand and baddeley 's most recent definition of wm further highlights the role of verbal and visuospatial components and cross - modal integration during tasks that require different levels of executive processing . moreover , wm functions have been shown to play an important role in many complex cognitive abilities , such as reading , problem solving , and spatial orientation . wm capacity , for example , reflects the ability to maintain goal - relevant information processing in the presence of competing or interfering information . interestingly , although studies on wm and emotion are few , recently , researchers have begun stressing the role of wm in emotional processing as well , showing how wm may be crucial for emotion regulation strategies . in particular , a series of studies have shown how individuals with higher wm capacity were better at suppressing emotional facial expressions and more successful at adopting an unemotional attitude while viewing emotionally charged stimuli . in addition , even though age - related difficulties are well established in classical wm tasks , many studies have repeatedly shown an age - related enhancement effect in wm tasks when emotional stimuli are involved [ 5 , 6 ] . in fact , a new corpus of data seems to indicate that emotions may have a special status in wm to the extent that age - related differences may even disappear . in addition , many neuroimaging studies have highlighted less pronounced age - related changes to brain regions , for example , amygdala , typically involved in emotion processing . based on this new evidence , various studies [ 3 , 6 ] support the possibility that there may be an affective wm , that is , a specific wm system to maintain and manipulate affective information . affective wm tasks are thus classical wm tasks that require the processing of affective stimuli ( e.g. , positive and negative pictures or words ) . most interestingly , this system seems to show a different trajectory in the aging mind compared to classical wm functions and although wm deficits , in general , are typically cited as one of the principal cognitive indexes of pathological aging , studies about emotional effects in wm in dementia of alzheimer 's type ( dat ) patients are only at the beginning . consequently , here , we aimed to review a series of studies with dat patients that show emotion modulation in wm performance during the active manipulation of affective to - be - remembered stimuli . in particular , we extrapolated three major classical wm functions that may be crucial for emotional processing and examined the relevant studies conducted with dat patients . anticipating our conclusions , we generally found that studies using wm tasks did not provide very strong evidence for emotional enhancement effects in dat and neuroimaging studies repeatedly showed that brain structures involved in emotion processes are disrupted in dat . however , it may be that dat patients do not show a general emotional enhancement effect but that they have more specific emotional effects linked to the wm function involved . after discussing the interpretation of these data , we offer suggestions about how issues of emotional processing in wm and dat may be explored in the future , especially in terms of trying to disentagle the role of different factors that may affect emotional enhancement effects in the wm of dat patients . recent research has evidenced an age - related wm resource shift towards emotional processing in alzheimer 's disease as well . in fact , there are studies that have shown preserved emotion processing in dat patients and this efficiency of wm functions for emotional stimuli seems to be further supported by neuroimaging studies that have highlighted how the amygdala may modulate wm resources ( prefrontal cortex ) despite degenerative processes . these findings raise interesting questions about the integrity of wm capacity in pathological aging and this review aims at clarifying whether there may be an emotional benefit effect in dat patients ' wm . to identify relevant studies , a comprehensive literature search in a variety of electronic databases was performed until april 12 , 2013 ( pubmed , psychinfo , and web of science ) . entered search terms were emotion , alzheimer , emotional processing , emotion regulation , working memory , and affective working memory . in addition , reference lists from the retrieved articles were screened to identify additional papers . articles were included for review if they met the following criteria : the study sample(s ) comprise(s ) people with a diagnosis of dementia . participants in the intervention studies have cognitive impairments resulting from neurodegenerative diseases , that is , a diagnosis of alzheimer 's disease . participants fulfill either the criteria for dementia as outlined in the diagnostic and statistical manual of mental disorder ( dsm - iv - tr ) or disease - specific criteria such as those for alzheimer 's dementia as formulated by the national institute of neurological and communicative diseases and the stroke / alzheimer 's disease . in addition , studies were included if diagnoses were based on historic information , neurologic examination , and neuropsychological assessment and supported by findings on structural and functional imaging.the study used a working memory task that included emotional stimuli.the actual definition of working memory shows a large variation in the literature and may include a combination of different component processes . provided that emotional processing is involved , all studies with alzheimer were included.as for type of dementia and severity , based on mini - mental state examination scores ( mmse ) , 23 subdivided into four categories : minimal ( mmse > 23 ) , mild ( mmse 1823 ) , moderate ( mmse 1017 ) , and severe ( mmse < 10).experimental design.altogether , we reviewed 19 studies . the study sample(s ) participants in the intervention studies have cognitive impairments resulting from neurodegenerative diseases , that is , a diagnosis of alzheimer 's disease . participants fulfill either the criteria for dementia as outlined in the diagnostic and statistical manual of mental disorder ( dsm - iv - tr ) or disease - specific criteria such as those for alzheimer 's dementia as formulated by the national institute of neurological and communicative diseases and the stroke / alzheimer 's disease . in addition , studies were included if diagnoses were based on historic information , neurologic examination , and neuropsychological assessment and supported by findings on structural and functional imaging . the study used a working memory task that included emotional stimuli . the actual definition of working memory shows a large variation in the literature and may include a combination of different component processes . provided that emotional processing is involved , all studies with alzheimer were included . as for type of dementia and severity , based on mini - mental state examination scores ( mmse ) , 23 subdivided into four categories : minimal ( mmse > 23 ) , mild ( mmse 1823 ) , moderate ( mmse 1017 ) , and severe ( mmse < 10 ) . emotionally charged events are generally remembered better than neutral events even to the extent that age - related differences in wm may disappear . this pattern of performance is typically found in healthy older adults and recent data investigating the encoding of emotional information in dat patients also shows similar results . therefore , to better clarify the interaction between emotions and wm in dat , we present a series of studies according to the main wm function involved , maintenance , binding , and inhibition ( although no single task taps just one unique wm function ) . in order for information to be processed maintenance , therefore , is a crucial function that allows other processes to intervene and manipulate information in different ways ( e.g. , repetition , inhibition , binding , etc . ) . typically , researchers infer short - term maintenance of information by asking participants to complete a recognition task in which old and new items are mixed and participants are asked to recognize which stimuli they had already seen or to freely recall a series of items immediately after their presentation . schultz et al . tested immediate recall for valenced pictures and found a general emotional enhancement effect in dat patients . on the contrary , a study by satler and tomaz showed better performance with negative trials compared to positive or neutral trials . this finding is in line with the negativity bias previously found in a study by dhnel and colleagues with mild cognitive impairment ( mci ) patients . this negativity bias was also found in another study by fleming and colleagues which showed better immediate free recall for negative words in dat patients and in the study by boller et al . negative emotions , therefore , seem to be maintained in wm and remembered better by dat patients compared to positive and neutral ones . when we perceive the world around us , we must first simultaneously process separate features and subsequently bind them together in order to form unique and memorable objects , scenes , and/or episodes . above all , this function is crucial in all cognitive tasks that require integration of information coming from different types of material ( e.g. , verbal and visuospatial ) , modalities ( e.g. , visual and acoustic ) , and domains ( cognitive versus emotional ) . typically this function is studied by asking participants to remember a series of objects or pictures in their studied locations . for example , huijbers and colleagues used a picture recollection task in which their participants studied a grid where 9 pictures appeared sequentially . at the end of each trial , participants were required to replace each picture in its exact location . in this case , dat patients performed better on negative trials compared to neutral ones , as did the control group . in a similar binding task , borg et al . results showed how dat patients remembered the locations of the positive pictures better than the locations of the negative and neutral pictures . on the contrary , nashiro and mather did not find any valence effects on dat performance when they controlled for arousal and not only valence . data on emotions and binding functions therefore are ambiguous and binding functions seem to be influenced in different ways according to the type of stimuli and task used . inhibition in wm involves paying attention to only some of the to - be - processed information . selection and inhibition allow only relevant information to be further manipulated in wm . generally speaking , inhibition refers to an individual 's control over the access of relevant and irrelevant information and has been studied with different complex tasks ( e.g. , stroop test and daneman & carpenter 's reading span , to cite only a few ) . in a typical wm span task [ 17 , 18 ] , participants are required to process ( read and judge ) a series of sentences , words , or operations and remember final words / digits in their correct serial order . these tasks have been shown to entail concurrent processing and short - term storage demands coupled with an attentional control component necessary for limiting interference between processing and storage . mammarella et al . assessed age - related differences in healthy older adults and dat patients using a modified version of the operation wm span test that included positive and negative words as well as neutral words ( as in the classical working memory task ) . participants judged simple math operations during the processing phase and then had to actively maintain a set of unrelated words that were either neutral , as in the original task , positive or negative in memory . finally participants were asked to remember all of the final target words ( affective and neutral ones ) . results showed that while healthy older adults performed better than dat patients with valenced words , dat patients did not show any emotional benefit . differently , doniger and bylsma found that moderate dat patients showed larger interference effects with positive and negative trials in an emotional version of the stroop task . these results seem to indicate that suppressing the emotional valence of items is more difficult and suggest that dat performance was influenced by emotion . on one hand , the complex evidence presented above is consistent with the idea that emotional information has a preferential access to wm even in dat patients . this assumption is based on the idea that the emotional effect in wm , although linked to more automatic and early attentional responses ( e.g. , fast detection of emotional stimuli ) may rely on wm representations associated with emotional personal events . on the other hand , it also highlights that the processing of emotional information in wm may fail due to the fact that additional control is required in order to orient attention towards this aspect and prevent distraction . moreover , it may also indicate that emotional valence effects do not occur early in the processing stage but rather arise later during the maintenance and use of emotional information when a greater amount of cognitive resources is required . in line with this , only valenced information that does not recruit a greater amount of cognitive resources will be favoured ( e.g. , negative information ) . in fact , studies on healthy aging have shown that the tendency to remember positive information better than negative information is based on recruitment of control processes . the ability to manipulate valenced information in wm is a critical component of emotion regulation processes . for example , negative and positive stimuli elaboration may be attenuated or amplified in a way that it may increase / decrease people 's vulnerability to mood disorders . thus , the interaction between valence and wm functions may have direct effects on emotion regulation abilities . . conducted one of the first studies that tested the ability of dat patients to apply different emotion regulation strategies online . in particular , they asked a group of healthy older adults and a group of dat patients to suppress ( e.g. , hide their feelings as much as they can ) or amplify ( e.g. , show their feelings as much as they can ) their feelings while watching a film clip . results showed that behavioral amplification of expressed emotion was disrupted in dat , while the ability to inhibit the expression of ongoing emotions was relatively preserved . similarly and more recently , goodkind and colleagues showed that when dat patients were told when the aversive event would occur but not given instructions about downregulation , they were able to spontaneously suppress emotional facial behavior just as well as normal controls . this small set of data points to the idea that the difficulty found in amplifying emotions might reflect decreasing controlled processes in dat such as wm resources , while relatively intact suppression of emotions could reflect more reliance on automatic inhibitory mechanisms . reduced wm functions may thus be related to the observed difficulties in emotion upregulating processes in dat . one explanation may be the fact that prefrontal regions show greater regulation - related activation and the functional efficacy of those structures depends on underlying cognitive ability . although several aspects of emotion seem to be intact in dat patients , emerging evidence shows that patients have an impaired ability to adaptively regulate their emotions . in addition , evidence regarding emotion regulation processes and their relationship with wm in dat is scarce . undoubtedly part of the reason for this is that it is difficult to study emotion regulation strategies such as cognitive reappraisal in patients with cognitive difficulties . in fact , depending on task demands , measures of emotion regolation invariably involve additional skills such as face perception , expressive speech , and/or semantic knowledge pertaining to an emotion label , all of which are impaired in dat patients . what is clearly needed , similar to what has been developed for wm tasks targeting cognition , is an effort to develop new wm tasks based on the use of different automatic emotion regulation strategies . neuroimaging studies have explained the emotional enhancement effect in long - term memory in terms of an interaction between the amygdala and brain regions that are involved during encoding and retrieval of emotional events . specifically , memory for emotional events relies on a fronto - amygdala - hippocampal circuit with the amydala modulating prefrontal cortex activity ( orbital and dorsolateral ) and the hippocampus . fmri studies have evidenced amygdala activity together with activation in frontoparietal regions during wm tasks with emotional stimuli as well . . found greater activation in prefrontal and parietal regions during an n - back wm task with emotional faces associated with a greater amygdala activation . these data have been explained in terms of an interaction between the amygdala and the orbitofrontal cortex which plays a crucial role in attributing emotional qualities to stimuli . this information is subsequently maintained and manipulated in the dorsolateral prefrontal cortex ( pfc ) . given the well established role of dorsolateral pfc in wm functions , it is likely that emotional events are remembered better because they capture a greater amount of perceptual and attentional resources ( as the contemporary activation of visual brain regions may indicate ) . in particular , with regards to aging studies , neuroimaging evidence shows that the brain regions and circuits involved in emotion processing are less sensitive to aging when compared to other brain regions . in addition , when mather and colleagues asked a group of younger ( mean age 23 ) and a group of older ( mean age 78 ) adults to rate a series of positive , negative , and neutral pictures on a 7-point scale in terms of arousal ( from calm to excitement ) , fmri data show similar levels of amygdala activation across the two groups and a greater activation for positive pictures in the older adults group alone . findings with older adults do not seem to reflect only early activation following automatic limbic responses . they also reflect later involvement of neural circuits that allow emotional stimuli to be maintained in wm and bound to autobiographical events . for example , kensinger and schacter detected greater activation in the medial pfc and the cingulate gyrus in a group of older adults compared to younger adults . these regions are known to be typically involved in autoreferential processing , highlighting the tendency for older adults to link online information to personal events or to the generation of regulation strategies . studies of emotional long - term memory in dat have repeatedly explained the absence / presence of emotional enhancement effects in alzheimer 's disease as due to the specific stage of atrophy in amygdala and hippocampal structures . for example , horinek et al . found the same degree of hippocampal and amygdalar volume loss . this data is in line with previous studies which found the same rate of degeneration in both structures . poulin et al . also found that amygdala atrophy is comparable to that of the hippocampus from the earlier stages of dementia on . in detail , with regards to wm functions , perrin et al . found that ad patients ' picture recall did not benefit from positive emotional context ( a positive sound associated to the picture ) and again explained this data in terms of wm limitations due to amygdala atrophy and early frontotemporal dysfunctions . more interesting is the pet study by rosenbaum et al . which found preserved influence from the left amygdala and left hippocampus on left and then right inferior pfc , in the absence of a direct amygdala - hippocampus influence using a match - to - sample face recognition task . this data seems to indicate that dat patients may still use some emotional brain circuits and show an emotional enhancement effect in wm despite their well - known amygdala and hippocampus deficits . in summary , since memory for emotional stimuli involves a variety of encoding and retrieval processes , it seems reasonable to assume that different brain regions may be involved at different moments . generally speaking , neuroimaging studies have shown that the prefrontal cortex supports short - term maintenance and manipulation of emotional stimuli in wm and that the amygdala modulates the activity in these brain regions during the different stages of emotion processing . this circuit seems to be less sensitive to pathological aging processes compared to other brain regions . the complex picture of emotional effects in wm of dat leads us to several considerations . first , it is important to further our knowledge about the relationship between emotion and wm measures . in fact , wm tasks are generally grouped in recall - based more active tasks such as the classical daneman and carpenter 's reading / listening span , the operation span , the active visuospatial task , and recognition - based more passive tasks such as n - back , recency - probe paradigms , matching - to - sample , and binding tasks to cite only a few . all of these tasks , however , differ in the type and the amount of processing required as well as in the nature of the information to be temporarily maintained . an interesting attempt to reconcile different pattern of results with emotion , wm , and dat would be to use different tasks and highlight the role of specific wm functions involved and specialized for affective processing . in fact , the majority of studies did not investigate the ability of actively maintaining and storing information to achieve the task goals as classical wm tasks require . second , the majority of studies with dat did not carefully control for different affective dimensions of study material . for example , some studies used high- versus medium - arousal valenced pictures , while others used arousing versus nonarousing items . still others did not mention whether arousal was controlled . in sum , it is not clear whether emotional enhancement effects in dat patients are mainly due to arousal effects , as suggested by nashiro and mather , valence effects , or both . from the wm studies , it is likely that wm for high arousing and negative valenced stimuli is preserved in dat , but we need further studies to assess the role valence in wm modulation . in fact , differences among the samples of dat patients may also explain the mixed findings of whether dat patients demonstrated an emotional benefit in their wm performance . these differences may be linked to different stages of deterioration in the brain regions involved in emotion and cognition processing . finally , the majority of studies reviewed here used affective visuospatial material . a long - term memory study by kensinger and colleagues that used verbal material , in fact , 's study that used a verbal wm task and did not detect an emotional effect with dat patients . pictures , in fact , may create richer memory traces that may generate larger emotional enhancement effect not detectable with affective words . a concluding thought must go to a motivational explanation of this complex picture of data as motivation towards emotional goals has been shown to be crucial to understanding the trajectory of the aging mind towards alzheimer 's disease . according to this approach , lack of motivation given that motivation towards meaningful emotional goals needs recruitment of cognitive resources towards emotion processing , results typically obtained in wm emotional tasks with dat may depend on the lack of attentional and wm processes motivated towards emotion processing . importantly , this hypothesis may highlight how different levels of motivation towards emotional goals may lead to different degree of emotional effects in wm also in dat . however , general positive effects may be obtained in the early stages of dementia across a variety of wm functions . undeniably , people with dementia can still use emotional stimuli and this may have relevant daily life implications . studies conducted in our lab show that emotional wm tasks can best be used with dat provided that the experimenter models each task step and provides verbal cues to guide the patient . correct verbal instructions and asking patients not to guess are very important steps for the outcomes of an emotional wm task . emotional wm integrity may help build up general activity levels , motivation necessary for undertaking new activities , and the sense of competence , which together may result in better quality of life for people with dementia . finally , the concept of emotional wm may provide health professionals with an opportunity to interact with their patients in a more effective manner , focusing on residual emotional abilities and learning capacities rather than deficits and decline . we hope that the evidence reviewed in this study fosters both emotional wm researches and gives new insights to improve emotion - based clinical practice with dat .

a number of recent studies have reported that working memory does not seem to show typical age - related deficits in healthy older adults when emotional information is involved . differently , studies about the short - term ability to encode and actively manipulate emotional information in dementia of alzheimer 's type are few and have yielded mixed results . here , we review behavioural and neuroimaging evidence that points to a complex interaction between emotion modulation and working memory in alzheimer 's . in fact , depending on the function involved , patients may or may not show an emotional benefit in their working memory performance . in addition , this benefit is not always clearly biased ( e.g. , towards negative or positive information ) . we interpret this complex pattern of results as a consequence of the interaction between multiple factors including the severity of alzheimer 's disease , the nature of affective stimuli , and type of working memory task .

1. Introduction 2. Overview 3. Working Memory Functions and Emotional Processing in DAT 4. Working Memory, Emotion Regulation, and DAT 5. Neural Correlates of WM Emotional Processing in DAT 6. Recommendations for Future Research 7. Conclusions

working memory ( wm ) is generally described as a memory system that temporarily maintains and manipulates different types of information during a task at hand and baddeley 's most recent definition of wm further highlights the role of verbal and visuospatial components and cross - modal integration during tasks that require different levels of executive processing . moreover , wm functions have been shown to play an important role in many complex cognitive abilities , such as reading , problem solving , and spatial orientation . wm capacity , for example , reflects the ability to maintain goal - relevant information processing in the presence of competing or interfering information . interestingly , although studies on wm and emotion are few , recently , researchers have begun stressing the role of wm in emotional processing as well , showing how wm may be crucial for emotion regulation strategies . in particular , a series of studies have shown how individuals with higher wm capacity were better at suppressing emotional facial expressions and more successful at adopting an unemotional attitude while viewing emotionally charged stimuli . in addition , even though age - related difficulties are well established in classical wm tasks , many studies have repeatedly shown an age - related enhancement effect in wm tasks when emotional stimuli are involved [ 5 , 6 ] . in fact , a new corpus of data seems to indicate that emotions may have a special status in wm to the extent that age - related differences may even disappear . in addition , many neuroimaging studies have highlighted less pronounced age - related changes to brain regions , for example , amygdala , typically involved in emotion processing . based on this new evidence , various studies [ 3 , 6 ] support the possibility that there may be an affective wm , that is , a specific wm system to maintain and manipulate affective information . affective wm tasks are thus classical wm tasks that require the processing of affective stimuli ( e.g. most interestingly , this system seems to show a different trajectory in the aging mind compared to classical wm functions and although wm deficits , in general , are typically cited as one of the principal cognitive indexes of pathological aging , studies about emotional effects in wm in dementia of alzheimer 's type ( dat ) patients are only at the beginning . consequently , here , we aimed to review a series of studies with dat patients that show emotion modulation in wm performance during the active manipulation of affective to - be - remembered stimuli . in particular , we extrapolated three major classical wm functions that may be crucial for emotional processing and examined the relevant studies conducted with dat patients . anticipating our conclusions , we generally found that studies using wm tasks did not provide very strong evidence for emotional enhancement effects in dat and neuroimaging studies repeatedly showed that brain structures involved in emotion processes are disrupted in dat . however , it may be that dat patients do not show a general emotional enhancement effect but that they have more specific emotional effects linked to the wm function involved . after discussing the interpretation of these data , we offer suggestions about how issues of emotional processing in wm and dat may be explored in the future , especially in terms of trying to disentagle the role of different factors that may affect emotional enhancement effects in the wm of dat patients . recent research has evidenced an age - related wm resource shift towards emotional processing in alzheimer 's disease as well . in fact , there are studies that have shown preserved emotion processing in dat patients and this efficiency of wm functions for emotional stimuli seems to be further supported by neuroimaging studies that have highlighted how the amygdala may modulate wm resources ( prefrontal cortex ) despite degenerative processes . these findings raise interesting questions about the integrity of wm capacity in pathological aging and this review aims at clarifying whether there may be an emotional benefit effect in dat patients ' wm . to identify relevant studies , a comprehensive literature search in a variety of electronic databases was performed until april 12 , 2013 ( pubmed , psychinfo , and web of science ) . entered search terms were emotion , alzheimer , emotional processing , emotion regulation , working memory , and affective working memory . in addition , reference lists from the retrieved articles were screened to identify additional papers . participants in the intervention studies have cognitive impairments resulting from neurodegenerative diseases , that is , a diagnosis of alzheimer 's disease . participants fulfill either the criteria for dementia as outlined in the diagnostic and statistical manual of mental disorder ( dsm - iv - tr ) or disease - specific criteria such as those for alzheimer 's dementia as formulated by the national institute of neurological and communicative diseases and the stroke / alzheimer 's disease . in addition , studies were included if diagnoses were based on historic information , neurologic examination , and neuropsychological assessment and supported by findings on structural and functional imaging.the study used a working memory task that included emotional stimuli.the actual definition of working memory shows a large variation in the literature and may include a combination of different component processes . provided that emotional processing is involved , all studies with alzheimer were included.as for type of dementia and severity , based on mini - mental state examination scores ( mmse ) , 23 subdivided into four categories : minimal ( mmse > 23 ) , mild ( mmse 1823 ) , moderate ( mmse 1017 ) , and severe ( mmse < 10).experimental design.altogether , we reviewed 19 studies . the study sample(s ) participants in the intervention studies have cognitive impairments resulting from neurodegenerative diseases , that is , a diagnosis of alzheimer 's disease . participants fulfill either the criteria for dementia as outlined in the diagnostic and statistical manual of mental disorder ( dsm - iv - tr ) or disease - specific criteria such as those for alzheimer 's dementia as formulated by the national institute of neurological and communicative diseases and the stroke / alzheimer 's disease . in addition , studies were included if diagnoses were based on historic information , neurologic examination , and neuropsychological assessment and supported by findings on structural and functional imaging . the study used a working memory task that included emotional stimuli . the actual definition of working memory shows a large variation in the literature and may include a combination of different component processes . provided that emotional processing is involved , all studies with alzheimer were included . as for type of dementia and severity , based on mini - mental state examination scores ( mmse ) , 23 subdivided into four categories : minimal ( mmse > 23 ) , mild ( mmse 1823 ) , moderate ( mmse 1017 ) , and severe ( mmse < 10 ) . emotionally charged events are generally remembered better than neutral events even to the extent that age - related differences in wm may disappear . this pattern of performance is typically found in healthy older adults and recent data investigating the encoding of emotional information in dat patients also shows similar results . therefore , to better clarify the interaction between emotions and wm in dat , we present a series of studies according to the main wm function involved , maintenance , binding , and inhibition ( although no single task taps just one unique wm function ) . in order for information to be processed maintenance , therefore , is a crucial function that allows other processes to intervene and manipulate information in different ways ( e.g. . typically , researchers infer short - term maintenance of information by asking participants to complete a recognition task in which old and new items are mixed and participants are asked to recognize which stimuli they had already seen or to freely recall a series of items immediately after their presentation . on the contrary , a study by satler and tomaz showed better performance with negative trials compared to positive or neutral trials . negative emotions , therefore , seem to be maintained in wm and remembered better by dat patients compared to positive and neutral ones . when we perceive the world around us , we must first simultaneously process separate features and subsequently bind them together in order to form unique and memorable objects , scenes , and/or episodes . above all , this function is crucial in all cognitive tasks that require integration of information coming from different types of material ( e.g. , verbal and visuospatial ) , modalities ( e.g. , visual and acoustic ) , and domains ( cognitive versus emotional ) . typically this function is studied by asking participants to remember a series of objects or pictures in their studied locations . for example , huijbers and colleagues used a picture recollection task in which their participants studied a grid where 9 pictures appeared sequentially . at the end of each trial , participants were required to replace each picture in its exact location . results showed how dat patients remembered the locations of the positive pictures better than the locations of the negative and neutral pictures . on the contrary , nashiro and mather did not find any valence effects on dat performance when they controlled for arousal and not only valence . data on emotions and binding functions therefore are ambiguous and binding functions seem to be influenced in different ways according to the type of stimuli and task used . inhibition in wm involves paying attention to only some of the to - be - processed information . generally speaking , inhibition refers to an individual 's control over the access of relevant and irrelevant information and has been studied with different complex tasks ( e.g. , stroop test and daneman & carpenter 's reading span , to cite only a few ) . in a typical wm span task [ 17 , 18 ] , participants are required to process ( read and judge ) a series of sentences , words , or operations and remember final words / digits in their correct serial order . these tasks have been shown to entail concurrent processing and short - term storage demands coupled with an attentional control component necessary for limiting interference between processing and storage . assessed age - related differences in healthy older adults and dat patients using a modified version of the operation wm span test that included positive and negative words as well as neutral words ( as in the classical working memory task ) . participants judged simple math operations during the processing phase and then had to actively maintain a set of unrelated words that were either neutral , as in the original task , positive or negative in memory . finally participants were asked to remember all of the final target words ( affective and neutral ones ) . results showed that while healthy older adults performed better than dat patients with valenced words , dat patients did not show any emotional benefit . differently , doniger and bylsma found that moderate dat patients showed larger interference effects with positive and negative trials in an emotional version of the stroop task . these results seem to indicate that suppressing the emotional valence of items is more difficult and suggest that dat performance was influenced by emotion . on one hand , the complex evidence presented above is consistent with the idea that emotional information has a preferential access to wm even in dat patients . this assumption is based on the idea that the emotional effect in wm , although linked to more automatic and early attentional responses ( e.g. on the other hand , it also highlights that the processing of emotional information in wm may fail due to the fact that additional control is required in order to orient attention towards this aspect and prevent distraction . moreover , it may also indicate that emotional valence effects do not occur early in the processing stage but rather arise later during the maintenance and use of emotional information when a greater amount of cognitive resources is required . in line with this , only valenced information that does not recruit a greater amount of cognitive resources will be favoured ( e.g. , negative information ) . in fact , studies on healthy aging have shown that the tendency to remember positive information better than negative information is based on recruitment of control processes . the ability to manipulate valenced information in wm is a critical component of emotion regulation processes . for example , negative and positive stimuli elaboration may be attenuated or amplified in a way that it may increase / decrease people 's vulnerability to mood disorders . thus , the interaction between valence and wm functions may have direct effects on emotion regulation abilities . conducted one of the first studies that tested the ability of dat patients to apply different emotion regulation strategies online . in particular , they asked a group of healthy older adults and a group of dat patients to suppress ( e.g. , hide their feelings as much as they can ) or amplify ( e.g. results showed that behavioral amplification of expressed emotion was disrupted in dat , while the ability to inhibit the expression of ongoing emotions was relatively preserved . this small set of data points to the idea that the difficulty found in amplifying emotions might reflect decreasing controlled processes in dat such as wm resources , while relatively intact suppression of emotions could reflect more reliance on automatic inhibitory mechanisms . one explanation may be the fact that prefrontal regions show greater regulation - related activation and the functional efficacy of those structures depends on underlying cognitive ability . although several aspects of emotion seem to be intact in dat patients , emerging evidence shows that patients have an impaired ability to adaptively regulate their emotions . in addition , evidence regarding emotion regulation processes and their relationship with wm in dat is scarce . undoubtedly part of the reason for this is that it is difficult to study emotion regulation strategies such as cognitive reappraisal in patients with cognitive difficulties . in fact , depending on task demands , measures of emotion regolation invariably involve additional skills such as face perception , expressive speech , and/or semantic knowledge pertaining to an emotion label , all of which are impaired in dat patients . what is clearly needed , similar to what has been developed for wm tasks targeting cognition , is an effort to develop new wm tasks based on the use of different automatic emotion regulation strategies . neuroimaging studies have explained the emotional enhancement effect in long - term memory in terms of an interaction between the amygdala and brain regions that are involved during encoding and retrieval of emotional events . specifically , memory for emotional events relies on a fronto - amygdala - hippocampal circuit with the amydala modulating prefrontal cortex activity ( orbital and dorsolateral ) and the hippocampus . fmri studies have evidenced amygdala activity together with activation in frontoparietal regions during wm tasks with emotional stimuli as well . these data have been explained in terms of an interaction between the amygdala and the orbitofrontal cortex which plays a crucial role in attributing emotional qualities to stimuli . this information is subsequently maintained and manipulated in the dorsolateral prefrontal cortex ( pfc ) . in particular , with regards to aging studies , neuroimaging evidence shows that the brain regions and circuits involved in emotion processing are less sensitive to aging when compared to other brain regions . in addition , when mather and colleagues asked a group of younger ( mean age 23 ) and a group of older ( mean age 78 ) adults to rate a series of positive , negative , and neutral pictures on a 7-point scale in terms of arousal ( from calm to excitement ) , fmri data show similar levels of amygdala activation across the two groups and a greater activation for positive pictures in the older adults group alone . findings with older adults do not seem to reflect only early activation following automatic limbic responses . for example , kensinger and schacter detected greater activation in the medial pfc and the cingulate gyrus in a group of older adults compared to younger adults . these regions are known to be typically involved in autoreferential processing , highlighting the tendency for older adults to link online information to personal events or to the generation of regulation strategies . studies of emotional long - term memory in dat have repeatedly explained the absence / presence of emotional enhancement effects in alzheimer 's disease as due to the specific stage of atrophy in amygdala and hippocampal structures . for example , horinek et al . found the same degree of hippocampal and amygdalar volume loss . poulin et al . also found that amygdala atrophy is comparable to that of the hippocampus from the earlier stages of dementia on . in detail , with regards to wm functions , perrin et al . this data seems to indicate that dat patients may still use some emotional brain circuits and show an emotional enhancement effect in wm despite their well - known amygdala and hippocampus deficits . in summary , since memory for emotional stimuli involves a variety of encoding and retrieval processes , it seems reasonable to assume that different brain regions may be involved at different moments . generally speaking , neuroimaging studies have shown that the prefrontal cortex supports short - term maintenance and manipulation of emotional stimuli in wm and that the amygdala modulates the activity in these brain regions during the different stages of emotion processing . first , it is important to further our knowledge about the relationship between emotion and wm measures . in fact , wm tasks are generally grouped in recall - based more active tasks such as the classical daneman and carpenter 's reading / listening span , the operation span , the active visuospatial task , and recognition - based more passive tasks such as n - back , recency - probe paradigms , matching - to - sample , and binding tasks to cite only a few . all of these tasks , however , differ in the type and the amount of processing required as well as in the nature of the information to be temporarily maintained . an interesting attempt to reconcile different pattern of results with emotion , wm , and dat would be to use different tasks and highlight the role of specific wm functions involved and specialized for affective processing . in fact , the majority of studies did not investigate the ability of actively maintaining and storing information to achieve the task goals as classical wm tasks require . second , the majority of studies with dat did not carefully control for different affective dimensions of study material . for example , some studies used high- versus medium - arousal valenced pictures , while others used arousing versus nonarousing items . still others did not mention whether arousal was controlled . in sum , it is not clear whether emotional enhancement effects in dat patients are mainly due to arousal effects , as suggested by nashiro and mather , valence effects , or both . in fact , differences among the samples of dat patients may also explain the mixed findings of whether dat patients demonstrated an emotional benefit in their wm performance . these differences may be linked to different stages of deterioration in the brain regions involved in emotion and cognition processing . finally , the majority of studies reviewed here used affective visuospatial material . a long - term memory study by kensinger and colleagues that used verbal material , in fact , 's study that used a verbal wm task and did not detect an emotional effect with dat patients . pictures , in fact , may create richer memory traces that may generate larger emotional enhancement effect not detectable with affective words . a concluding thought must go to a motivational explanation of this complex picture of data as motivation towards emotional goals has been shown to be crucial to understanding the trajectory of the aging mind towards alzheimer 's disease . according to this approach , lack of motivation given that motivation towards meaningful emotional goals needs recruitment of cognitive resources towards emotion processing , results typically obtained in wm emotional tasks with dat may depend on the lack of attentional and wm processes motivated towards emotion processing . importantly , this hypothesis may highlight how different levels of motivation towards emotional goals may lead to different degree of emotional effects in wm also in dat . undeniably , people with dementia can still use emotional stimuli and this may have relevant daily life implications . correct verbal instructions and asking patients not to guess are very important steps for the outcomes of an emotional wm task . emotional wm integrity may help build up general activity levels , motivation necessary for undertaking new activities , and the sense of competence , which together may result in better quality of life for people with dementia . finally , the concept of emotional wm may provide health professionals with an opportunity to interact with their patients in a more effective manner , focusing on residual emotional abilities and learning capacities rather than deficits and decline .

there are three principal reasons to develop methods for the asymmetric synthesis of chiral palladacycles , an area in which many examples are metallocene - based planar chiral complexes . first is the use of these metallacycles as catalysts for the synthesis of non - racemic organic compounds . conspicuous success has been achieved in catalysis of the asymmetric allylic imidate rearrangement and closely related reactions using planar chiral palladacycles based upon bulky cobalt sandwich complexes ( e.g. , 1 and 2 ) or related pentaphenylferrocene frameworks ( scheme 1).[46 ] in these reactions the palladium - carbon palladacycle bond is maintained throughout the catalytic cycle . second is the use of palladacycles as precatalysts for the in situ generation of pd species.[8 , 9 ] finally , the development of enantioselective methods for palladium catalysed asymmetric c h activation is informed by the synthesis of complexes related closely to the intermediate chiral palladacycles generated in these reactions . diastereoselective synthesis of cobalt oxazoline palladacycle 1 [ ( s , rp)-cop - oac ] and transformation into chloride - bridged dimer 2 [ ( s , rp)-cop - cl ] . central to the synthesis of 1 , as with many related planar chiral metallocene - based palladacycles , is an auxiliary - mediated diastereoselective c palladium bond.[2b , 11 ] there are far fewer examples of planar chiral palladacycles derived from enantioselective c non - racemic planar chiral palladacycles have been generated from a limited number of enantioselective transcyclopalladation reactions , and in 1979 sokolov reported the n - acetyl amino acid mediated enantioselective palladation of n , n - dimethylaminomethylferrocene ( 3 ) . recent re - investigation of this latter chemistry within our group identified conditions for the synthesis of palladacycle ( sp)-4 in 96 % ee using ( r)-n - acetylphenylalanine ( 5 ) , and an extension of the methodology to the kinetic resolution of [ 2.2]paracyclophane ( 6 ) gave palladacycle ( sp)-7 in more than 99 % ee ( scheme 2 ) . in this study we report on the application of enantioselective palladation to the facile synthesis of non - racemic amine palladacycles containing a cobalt sandwich complex , and on the application of these to asymmetric transcyclopalladation and asymmetric allylic imidate rearrangement . amine 8 , a cobalt sandwich complex analogue of 3 , was first synthesised as previously reported from the mannich - type reaction of ( -cyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt with bis(dimethylamino)methane . the low yield of this reaction ( typically 40 % ) led us to an alternative and more productive procedure in which acid 9[11 , 17 ] was converted to dimethylamide 10 followed by reduction ( scheme 3 ) . the palladation of amine 8 has been reported previously with a mixture of lithium tetrachloropalladate and sodium acetate in methanol . this result suggested that the prochiral cobalt complex 8 would be a suitable substrate for a chiral carboxylate mediated asymmetric palladation . application of the room temperature conditions optimised previously for the enantioselective palladation of 3 , with a reaction time of 16 h , resulted in a new chloride bridged palladacycle 11 in 64 % yield ( scheme 4 ) . the product was determined to have an enantiomeric excess of 92 % following treatment with ( s)-proline and analysis of the resulting diastereomeric adducts 12 and 13 . their ratio was determined readily by h nmr spectroscopy , in particular by comparison of one of the base - line resolved methyl singlets [ 12 : 2.31 ppm ( 3 h , s ) , 13 2.36 ppm ( 3 h , s ) ] , or by comparison of two of the signals arising from the cyclopentadienyl rings [ ( 12 : 4.26 ppm ( 1 h , br s ) , 13 4.30 ppm ( 2 h , br s ) ] . performing the palladation reaction at 0 c resulted in no change in enantioselectivity and a longer reaction time was required to ensure complete palladation . a racemic acetate - bridged palladacycle 14 was synthesised by heating together 8 and palladium acetate in toluene at reflux for 2 h ( scheme 5 ) . subsequent treatment with ( s)-proline as before gave a 1:1 mixture of 12 and 13 , confirming that these proline adducts are planar chiral stereoisomers and not cis / trans coordination stereoisomers . enantioselective palladation of 8 and derivatisation with ( s)-proline . non - enantioselective palladation of 8 with pd(oac)2 . following recrystallisation from ch2cl2/hexane of the proline adducts derived from asymmetric palladation , a small quantity of the major diastereoisomer was obtained pure and the configuration of the element of planar chirality was determined as sp by x - ray crystallography ( figure 1 ) . the pyrrolidine ring is disordered , with alternative sites for one methylene group , at c(24a ) and c(24b ) . the other four members of this ring are approximately co - planar , so that the five - membered ring adopts an envelope shape with the flap on one side , for example , c(24a ) , or the other , c(24b ) . principal bond lengths [ ] include : pd c(11 ) 1.973(4 ) , pd principal angles [ ] include : c(11)-pd - n(17 ) 82.68(18 ) , n(21)-pd - o(27 ) 82.50(16 ) . the high enantioselectivity observed in the palladation reaction points to the involvement of a palladium intermediate containing a coordinated carboxylate ligand obtained by deprotonation of ( r)-n - acetylphenylalanine . palladation reactions with palladium acetate and other palladium(ii)-carboxylate species have been shown to proceed by a concerted metallation - deprotonation ( cmd ) pathway , a mechanism consistent with a kinetic isotope effect of more than 1 . to determine if this mechanism may be operating in the n - acetyl amino acid mediated formation of 11 , a racemic 2-deutertated sample of the starting amine d-(rac)-8 ( 90 % deuterium incorporation ) was synthesised by treatment of the racemic palladacycle 14 with liald4 ( scheme 6 ) . repetition of the na2pdcl4/n - acetyl amino acid palladation conditions , with n - acetylglycine in place of ( r)-n - acetylphenylalanine , followed by ligand substitution with sodium hexafluoroacetylacetonate , gave monomeric palladacycle d-(rac)-15/h-(rac)-15 ( 64 % deuterium incorporation ) . the intramolecular isotope effect of 2.5 is very similar to the value of 2.3 determined for the palladation of the ferrocene analogue 3 under the same conditions . furthermore , use of the same sample of d-(rac)-8 in a reaction with palladium acetate in toluene at reflux followed by ligand substitution revealed an intramolecular isotope effect of 2.0 ( d-(rac)-15/h-(rac)-15=60:40 ) . all of these values are consistent with a carboxylate ligand accelerated cmd pathway , with the reactions containing ( r)-n - acetylphenylalanine resulting in the preferential formation of the ( sp)-palladacycle . determination of the intramolecular isotope effect for the n - acetyl amino acid promoted palladation of 8 . a pathway for the chiral carboxylate mediated palladation of 8 is outlined in scheme 7 . this is based on the dft calculated mechanism of dimethylbenzylamine cyclometalation by palladium acetate , and a suggested extension of this process to the n - acetylphenylalanine mediated enantioselective palladation of phosphines containing a 2-phenylferrocene substituent . in this pathway an initially formed amine and -carboxylate ligated complex 16 leads to transition state 17 with the carbonyl oxygen of the now -carboxylate ligand participating in deprotonation simultaneously with the formation of the new carbon replacement of ligand x in 17 by the nitrogen of the amino acid derived ligand to give a chelate would appear to be geometrically incompatible with the participation of the carbonyl group of this ligand as a base . instead the conformational properties of the ligand are controlled by its dipeptide - like properties . variation of the ee of the ( r)-n - acetylphenylalanine employed in cyclometallation resulted in a small positive non - linear effect with respect to the ee of metallacycle ( sp)-11 , an outcome compatible with coordination of a second -carboxylate 18 and chirality matched rate accelerated cyclometallation ( figure 2 ) . a possible pathway for the enantioselective palladation of 8 . an investigation into the relationship between the ee of n - acetylphenylalanine and the ee of product palladacycle ( sp)-11 . this was most conveniently performed by first treating ( sp)-11 with ( r)-proline as the major diastereoisomer ( r , sp)-13 has a higher rf ( 0.24 ) than the minor diastereoisomer ( r , rp)-12 ( 0.16 ) in 2.5 % meoh / ch2cl2 , such that the majority of the former can be eluted with little or no contamination from the latter . if required , a subsequent recrystallisation can ensure diastereomeric purity ( > 99:1 as determined by h nmr spectroscopy ) . the x - ray crystal structure of ( r , sp)-13 ( see the supporting information ) confirmed further the absolute configuration and the trans nitrogen geometry . conversion back to the cobalt amine palladacycle ( sp)-11 [ ( sp)-me2-cap - cl ] was carried out by stirring , overnight , a biphasic mixture of ( r , sp)-13 in ch2cl2 and aqueous 0.5 m hcl ( scheme 8) . the enantiopure chloride - bridged dimer is formed in good yield as an approximately 1:1 mixture of isomers with respect to the cis / trans arrangements of the two bridged c addition of silver acetate to ( sp)-11 resulted in the clean formation of acetate bridged dimer ( sp)-14 [ ( sp)-me2-cap - oac ] , and in common with other examples of planar chiral acetate - bridged palladacycles this is a single , presumably trans , stereoisomer . treatment of ( sp)-11 with sodium hexafluoroacetylacetonate [ na(hfacac ) ] gave ( sp)-15 [ ( sp)-me2-cap - hfacac ] , although attempts to synthesise this directly from proline adduct ( r , sp)-13 were unsuccessful . a representation of the x - ray crystal structure of ( rac)-15 ( obtained from ( rac)-14 and na(hfacac ) ) is shown in figure 3 . the hfacac ligand allows comparison of this structure with the hfacac derivative of palladacycles 1 and 2 [ ( s , rp)-cop - hfacac].[5c ] in common with that structure is the longer length of the o(2)pd bond compared to o(1)pd , indicative of the larger trans influence of the carbanion ligand compared to nitrogen . that both these bond lengths in 15 are longer than the corresponding bonds in the cop derivative point to more electron density on the palladium atom of 15 due to the greater basicity of the amine nitrogen compared to the oxazoline nitrogen , and the presence of the electron - withdrawing oxazoline substituent in the cop derivative this is supported further by the larger chemical shift of the methine proton in the hfacac ligand of the cop derivative ( 5.95 ppm ) compared to that in 15 ( 5.87 ppm ) . principal bond lengths [ ] and angles [ ] [ corresponding data for ( s , rp - cop - hfacac ) in parenthesis][5c ] include : c(5)pd 1.955(3 ) [ 1.962(6 ) ] , n(1)pd 2.085(3 ) [ 2.026(5 ) ] , o(1)pd 2.046(2 ) [ 2.020(4 ) ] , o(2)pd 2.119(2 ) [ 2.102(4 ) ] , c(5)-pd - n(1 ) 81.87(12 ) [ 80.8(2 ) ] , o(1)-pd - o(2 ) 91.33(9 ) [ 92.77(15 ) ] . a number of amines related to the n , n - dimethylamino containing substrate 8 were synthesised to examine further the asymmetric palladation methodology . following reduction of methyl ester 19 , the alcohol 20 was converted in situ with n - bromosuccinimide / triphenylphosphine into the corresponding -bromomethyl sandwich complex followed by treatment with a variety of secondary amines to give products 21 a e ( scheme 9 ) . oxidation of alcohol 20 to aldehyde 22 followed by reductive amination with benzylamine gave secondary amine 23 . hydrogenolysis of this resulted only in debenzylation to give the primary amine 24 with no formation of ( -methylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt , an alternative reduction product which would have resulted from hydrogenolysis of the nitrogen - c(-sandwich complex ) bond . finally , introduction of a cbz group followed by reduction of 25 with lithium aluminium hydride gave the n - methyl amine 26 . synthesis of further amine substrates 21 a e , 23 , 24 and 26 . application to these new amines of the standard asymmetric palladation conditions resulted in only two new palladacycles , ( sp)-27 and ( sp)-28 derived from the n , n - diethylamine 21 a and pyrrolidinyl complex 21 b , respectively ( scheme 10 ) . ( s)-proline derivatisation revealed the ee of 27 as 82 % and 28 as more than 98 % . in the latter case the minor diastereoisomer ( s , rp)-32 could not be detected by h nmr spectroscopy . the determination of the ratio of isomers as more than 100:1 was made following the synthesis of ( r , sp)-32 from ( r)-proline , and the use of this to spike the h nmr sample . the absolute configuration of these new palladacycles was assigned initially by the sign of the specific rotation [ ( sp)=ve , ( rp)=+ve ] , and confirmed by the correspondence between the cd spectra of ( sp)-27 and ( sp)-28 with that of ( sp)-11 ( see the supporting information ) . that the diethylamine and pyrrolidine derivatives are limiting substrates under these reaction conditions is revealed by the reduced yield obtained . the n , n - dimethyl , n , n - diethyl and pyrrolidinyl substrates appear to have the correct balance of nitrogen basicity and steric accessibility to permit palladation . it is significant that treatment of 21 c e with pd(oac)2 in toluene at either 7080 c , or heating at reflux , also did not result in palladation . preliminary investigations into the use of these new cap complexes in asymmetric synthesis began with the n , n - dimethylamino derivatives obtained following proline - mediated purification . transcyclometallation is a term coined to describe the exchange of cyclometalated ligands without the formation of dissociated metal salts . following a demonstration of asymmetric transcyclopalladation using palladacycles derived from ( r)-3-amino-3-phenyl-2,2-dimethylpropane,[12a ] one of us reported that the reaction between ( s , rp)-cop - oac ( 1 ) and prochiral phosphines 33 or 34 resulted in the clean formation of phosphapalladacycles 35 and 36 in up to 95 % ee ( r = cy).[12b ] the applicability of cap complexes to this reaction was investigated by combining ( sp)-me2-cap - oac and phosphine 33 ( r = ph ) followed by heating at 70 c in toluene for 24 h ( scheme 11 ) . the initially formed acetate - bridged phosphapalladacycle was converted into the monomeric acac ligated complex 35 for which a 86:14 ratio of rp and sp enantiomers was determined by chiral hplc analysis . in the same way phosphine 34 ( r = cy ) gave a 89:11 ratio of rp and sp isomers of 36 . the initial reaction between a phosphine substrate and the amine - coordinated palladacycle results in the formation of a monomeric adduct , as revealed by the synthesis of ( sp)-37 from 33 and ( sp)-11 ( scheme 11 ) . a representation of the x - ray crystal structure of ( sp)-37 is shown in figure 4 . in common with most other nitrogen ligand based palladacycles , the added phosphine is incorporated trans to nitrogen , the thermodynamic ligand substitution product . the triarylphosphine ligand displays an induced p configuration , and the tetraphenylcyclobutadiene moiety is m. this latter configuration is also displayed in the solid state structure of ( rac)-15 ( relative to sp ) , but ( s , sp)-12 and ( r , sp)-13 are p , revealing no correlation between the planar and induced propeller chirality of the -tetraphenylcyclobutadiene group . a molecule of ( sp)-37 from x - ray analysis . principal bond lengths [ ] include : pd cl 2.383(4 ) , pd c(51 ) 2.004(16 ) , pd p(6 ) 2.271(4 ) , mean co c(c4 ring ) 1.97(4 ) , mean co c(cp ) 2.05(7 ) , mean fe c(substd - cp ) 2.00(4 ) , fe principal angles [ ] include : c(51)-pd - n(522 ) 80.9(6 ) , p(6)-pd - cl 87.8(2 ) . the x - ray crystal structure of ( sp)-37 also reveals the orientation of the ferrocenyl group above the palladium centred square - plane , as beneath lie phenyl groups attached to the -cyclobutadiene moiety . this and the trans to nitrogen coordination geometry are instrumental in controlling the enantioselectivity of palladium transfer . a pathway for this process is outlined in scheme 12 based , as before , on dimethylbenzylamine cyclopalladation and related studies.[21 , 22 ] following formation of 38 , dissociation of the amine ligand by formation of the -acetate complex 39 is followed by acetate assisted concerted metalation - deprotonation ( cmd ) via transition state 40 to give 41 . subsequent protonolysis of the cobalt complex carbon palladium bond by retro - cmd releases amine 8 and gives an acetate ligated phosphapalladacycle , replacement of which on addition of na(acac ) results in isolated complexes ( rp)-35 or ( rp)-36 . although rotation is possible about the carbon palladium bond in 39 , the conformer drawn is favoured by the orientation of the coordinated phosphine away from the dimethylaminomethyl moiety . thus the planar chirality of this monodentate species is also a factor in controlling the enantioselectivity . kinetic and molecular modelling studies on the cop - cl catalysed allylic imidate rearrangement revealed that the planar chirality is also the key factor in controlling the facial selectivity of nitrogen addition to the alkene moiety , this being bound trans to the oxazoline nitrogen in the rate and enantioselectivity determining anti - amino palladation step.[5e ] given the similarity of the coordination site trans to the amine nitrogen , it was anticipated that cap catalysis of the allylic imidate rearrangement by this pathway would result in usable levels of enantioselectivity , and a predictable correspondence between the configurations of planar and product chirality ( sp gives r ) . this was investigated first with the allylic imidate rearrangement of representative ( e)- and ( z)-n-(para - methoxyphenyl)trifluoroacetimidate substrates 42 ( scheme 13 , table 1 ) . the reactions were performed first with 5 mol % of ( sp)-11 at room temperature , which resulted in modest conversions for the formation of ( r)-43 ( 75 % ee ) and ( s)-43 ( 20 % ee ) from e and z substrates , respectively ( entries 1 and 2 ) . similar results were obtained with catalyst ( sp)-28 , the e substrate also resulting in higher conversion and enantioselectivity ( entries 3 and 4 ) . focusing on substrate ( e)-42 , and increasing the reaction temperature to 38 c with the addition of proton sponge , improved the ee to 86 % but the conversion was still modest ( entry 5 ) . under similar conditions 5 mol % of ( s , rp)-cop - cl ( 1 ) resulted in essentially complete conversion , and up to 92 % ee.[5a ] use of ( s)-cap - cl in catalysis of the rearrangement of ( e)- and ( z)-n-(para - methoxyphenyl)trifluoroacetimidates ( 42 ) . use of ( sp)-cap - cl in catalysis of the rearrangement of ( e)- and ( z)-n-(para - methoxyphenyl)trifluoroacetimidates ( 42 ) [ a ] 0.6 m 42 in ch2cl2 , reaction time 60 h at rt or 24 h at 38 c . [ d ] determined by chiral hplc of the secondary amine following trifluoroacetate removal . [ e ] with 4 x mol % 1,8-bis(dimethylamino)naphthylene . [ h ] isolated yield=80 % . assuming a correlation between conversion and the rate of catalysis , the reduced activity observed with the cap catalysts is attributed to the increased electron density on the palladium of the amine coordinated complex . related chloride - bridged ferrocene imidazoline palladacycles are essentially inactive as catalysts for the allylic imidate rearrangement due the electron - donating properties of the iron containing metallocene . activation is required by the addition of silver salts , a recent study having identified the resultant catalyst as a pd species obtained by initial chloride ligand abstraction followed by oxidation . addition of 3.8 equivalents with respect to ( sp)-28 of agno3 prior to the introduction of the substrate ( e)-42 resulted in complete conversion to give ( r)-43 in 81 % ee ( entry 6 ) . essentially the same outcome was obtained on addition of proton sponge ( entry 7 ) , though a tenfold reduction in catalyst loading to 0.5 mol % led to the erosion of enantioselectivity and yield ( entry 8) . encouraged by these results we applied the cap catalysts to the rearrangement of ( e)-trichloroacetimidates 44 ( scheme 14 ) . compared to n - aryltrifluoroacetimidates these are simpler to synthesise , and the products of rearrangement require only a single deprotection step to release an allylic amine building block . initial experiments with 1 , 2 and 5 mol % of ( sp)-11 gave a higher ee value with each increase in catalyst loading ( entries 13 ) , and use of 5 mol % of ( sp)-28 further increased the ee to 86 % ( table 2 ) . as these reactions all resulted in incomplete conversion to the product , the palladacycle ( sp)-28 was again activated by the addition of 3.8 equivalents of agno3 prior to the addition of the substrate . this gave complete conversion and an ee of 73 % ( entry 5 ) , which on repetition in the presence of proton sponge increased to 99 % ee ( entry 6 ) . decreasing the catalyst loading to 0.5 mol % again eroded significantly the ee and yield ( entry 7 ) , and so a small range of additional substrates were examined at 5 mol % loading ( entries 810 ) . the allyl containing trichloroacetimidate 44 b , which due to the additional alkene functionality capable of competitive palladium coordination is a challenging substrate for this reaction , resulted in ( r)-45 b in 71 % ee . in contrast , methyl and benzyl containing substrates 44 c / d reacted smoothly to give ( r)-45 b and ( r)-45 c , in 91 and 87 % ee , respectively . these results are comparable to ( s , rp)-cop - cl catalysed rearrangement of trichloroacetimidates,[5b , d ] but now using a catalyst available readily from highly enantioselective palladation of a simple prochiral substrate . use of ( sp)-cap - cl in catalysis of the rearrangement of ( e)-trichloroacetimidates 44 a d . use of ( sp)-cap - cl in catalysis of the rearrangement of ( e)-trichloroacetimidates ( 44 ) [ a ] 0.6 m 44 in ch2cl2 , reaction time 39 h at 38 c . [ d ] determined by chiral hplc . [ e ] with 3.8 x mol % agno3 . enantioselective palladation of n , n - dimethylaminomethyl - appended cobalt sandwich complex 8 with na2pdcl4 mediated by ( r)-n - acetylphenylalanine gave the chloride - bridged palladacycle ( sp)-11 in 92 % ee . the intramolecular isotope effect ( kh / kd=2.5 ) is consistent with a concerted metallation deprotonation pathway mediated by a chiral -carboxylate ligand . the enantiopurity of ( sp)-11 may be increased to more than 98 % ee by chromatographic separation of the minor diastereoisomer formed on addition of ( r)-proline followed by treatment of the major diastereoisomer with aqueous hcl . a number of related aminomethyl - substituted cobalt complexes were synthesised readily , but this enantioselective palladation protocol is limited to n , n - diethyl- ( 82 % ee ) and pyrrolidinyl ( > 98 % ee ) substituents . combining this enantioselective palladation with subsequent enantioselective transcyclopalladation enables the synthesis of ferrocene - based phosphapalladacycles in up to 78 % ee . the activity and enantioselectivity of a cobalt amine palladacycle catalyst for the allylic imidate rearrangement is increased significantly following the addition of 3.8 equivalents of silver nitrate . the catalyst generated from ( sp)-28 results in the rearrangement of ( e)-trichloroacetimidates with high enantioselectivity ( up to 99 % ee ) . combined with the simple highly enantioselective generation of ( sp)-28 , this methodology enables the straight - forward generation of highly scalemic allylic amine derivatives for application in organic synthesis . general : thin layer chromatography ( tlc ) was performed on merck silica gel 60 f254 and was visualised with uv light , iodine or potassium permanganate stain . nmr spectra were measured at 500 or 400 mhz for h and 126 or 100 mhz for c. the residual solvent protons ( h ) or the solvent carbons ( c ) were used as internal standards for chemical shift determinations . ir spectra were recorded on a fourier transform interferometer ; only diagnostic and/or intense peaks are reported . all reagents and solvents were purchased from commercial sources and were purified using standard methods where required . all imidate substrates used were synthesised according to the literature procedures from the corresponding allylic alcohols.[5a , b , d ] synthesis of ( -n , n - dimethylcarboxamidocyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 10 ) : a flask was charged with 9 ( 1.00 g , 1.91 mmol ) and it was then dissolved in ch2cl2 ( 20 ml ) . oxalyl chloride ( 0.33 ml , 3.8 mmol ) and dimethylformamide ( 3 drops ) were added sequentially . after 30 min the solution was concentrated in vacuo re - dissolved in ch2cl2 and re - concentrated in vacuo to give the crude acid chloride as a red / brown solid . a solution of the crude acid chloride in ch2cl2 ( 30 ml ) was added via cannula to a solution of dimethylamine hydrochloride ( 0.311 g , 3.81 mmol ) and triethylamine ( 2.30 ml , 16.5 mmol ) in ch2cl2 ( 20 ml ) . after 16 h the solution was washed with water ( 50 ml ) and brine ( 50 ml ) . the residue was dissolved in a minimum volume of ch2cl2 and purified by column chromatography ( sio2 , 7:3 hexanes / ethyl acetate ) to give the product 10 as an orange solid ( 1.01 g , 96 % ) . 249 c ; h nmr ( 500 mhz , cdcl3 ) : =7.557.44 ( m , 8 h , ar - h ) , 7.327.18 ( m , 12 h , ar - h ) , 5.16 ( br s , 2 h , cp - h ) , 4.75 ( br s , 2 h , cp - h ) , 2.79 ( br s , 3 h , ch3 ) , 2.64 ppm ( br s , 3 h , ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =135.64 , 129.04 , 128.12 , 126.64 , 85.46 , 84.89 , 77.36 , 76.26 ppm ( c = o and 2ch3 not observed ) ; ir ( neat ) : = 3052 , 2923 , 1967 , 1609 , 1596 , 1496 , 1388 , 1267 , 1162 , 1027 cm ; hrms ( esi ) : m / z calcd for c36h31cono : 552.1732 [ m+h ] ; found 552.1726 . alternative synthesis of ( -n , n - dimethylaminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 8) : a flask was charged with 10 ( 0.986 g , 1.78 mmol ) and it was then dissolved in thf ( 20 ml ) . the flask was cooled in an ice - water bath and lithium aluminium hydride ( 0.214 g , 5.63 mmol ) was added in two portions . water ( 20 ml ) was added and the aqueous layer was extracted with ch2cl2 ( 320 ml ) . the organic layer was dried over mgso4 , filtered and concentrated in vacuo to give the product 8 as an orange solid ( 0.959 g , 99 % ) . h nmr ( 500 mhz , cdcl3 ) : =7.517.40 ( m , 8 h , ar - h ) , 7.337.21 ( m , 12 h , ar - h ) , 4.73 ( t , j=2.0 hz , 2 h , cp - h ) , 4.68 ( d , j=2.1 hz , 2 h , cp - h ) , 2.90 ( s , 2 h , ch2 ) , 2.24 ppm ( s , 6 h , 2ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.42 , 128.95 , 128.10 , 126.30 , 93.84 , 84.21 , 83.66 , 74.89 , 56.53 , 44.95 ppm . synthesis of di--chlorobis[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 11 ) : a solution of ( r)-n - acetylphenylalanine ( 0.740 g , 3.57 mmol ) and naoh ( 0.066 g , 1.65 mmol ) in water ( 15 ml ) was added to a solution of na2pd2cl4 ( 0.439 g , 1.49 mmol ) in meoh ( 50 ml ) . the ph of the mixture was adjusted to 8.0 using either aqueous 50 % naoh(aq . ) or conc . a solution of 8 ( 0.800 g , 1.49 mmol ) in meoh / ch2cl2 ( 75/15 ml ) was then added in portions over 5 min . , the reaction mixture was diluted with ch2cl2 ( 150 ml ) and washed with brine ( 2100 ml ) . the organic phase was dried over mgso4 , filtered and the solvent was removed in vacuo . purification by column chromatography ( sio2 , 4:1 hexanes / etoac ) gave the product ( sp)-11 as an orange solid ( 0.650 g , 64 % ) , ee=92 % as determined by formation of the proline adducts . synthesis of proline adduct ( s , sp)-12 : a solution of ( sp)-11 ( 0.050 g , 0.04 mmol ) in acetone ( 1 ml ) was added to a solution of nahco3 ( 0.031 g , 0.37 mmol ) and ( s)-proline ( 0.043 g , 0.37 mmol ) in water ( 0.5 ml ) . during the addition a copious amount of precipitate was formed . the reaction was vigorously stirred for 16 h at rt and then diluted with ch2cl2 ( 50 ml ) . the phases were separated and the aqueous phase was washed with further portions of ch2cl2 ( 225 ml ) . the organic phases were combined , dried over mgso4 , filtered and solvent was removed in vacuo yielding the product as an orange solid ( 0.050 g , 90 % ) . crystals of ( s , sp)-12 suitable for x - ray crystallography were obtained by slow diffusion of hexane into ch2cl2 solution ( 50:1 hexane / ch2cl2 ) . 190192 c ; [ ]=99 ( c=1.29 mg ml in ch2cl2 ) ; h nmr ( 400 mhz , cdcl3 ) : =7.587.56 ( m , 8 h , ar - h ) , 7.267.22 ( m , 12 h , ar - h ) , 4.36 ( br s , 2 h , cp - h ) , 4.15 ( t , j=2 hz , 1 h , cp - h ) , 3.93 ( app . q , j=7.6 hz , 1 h , nhch ) , 3.20 ( d and br s , j=13.2 hz , 2 h , chhnme2 and nh ) , 2.87 ( d , j=13.2 hz , 1 h , chhnme2 ) , 2.65 ( s , 3 h , ch3 ) , 2.502.40 ( m , 1 h , nhchh ) , 2.38 ( s , 3 h , ch3 ) , 2.202.00 ( m , 3 h , 3ch ) , 1.601.50 ( m , 1 h , chh ) , 1.241.18 ppm ( m , 1 h , chh ) ; c nmr ( 100 mhz , cdcl3 ) : =136.72 , 129.05 , 128.23 , 126.25 , 103.75 , 101.58 , 84.33 , 82.67 , 77.85 , 74.01 , 64.28 , 52.60 , 51.70 , 51.17 , 29.89 , 26.10 ppm ( c = o not observed ) ; ir ( neat ) : = 2450 , 2919 , 1597 , 1496 , 1443 , 1379 , 1366 , 1259 , 1152 , 1066 , 1018 , 845 , 803 , 740 , 697 cm ; hrms ( ei ) : m / z calcd for c41h40con2o2pd : 757.1466 [ m+h ] ; found 757.1468 . synthesis of proline adduct ( r , sp)-13 : a solution of ( sp)-11 ( 0.050 g , 0.04 mmol ) in acetone ( 10 ml ) was added to a solution of nahco3 ( 0.088 g , 1.04 mmol ) and ( r)-proline ( 0.085 g , 0.74 mmol ) in water ( 5 ml ) . during the addition the reaction was vigorously stirred for 16 h at rt and then diluted with ch2cl2 ( 50 ml ) . the phases were separated and the aqueous phase was washed with further portions of ch2cl2 ( 225 ml ) . the organic phases were combined , dried over mgso4 , filtered and solvent was removed in vacuo yielding the crude product . purification by column chromatography eluting with ( sio2 , 97:3 ch2cl2/meoh ) gave exclusively ( r , sp)-13 as an orange / red solid ( 0.045 g , 81 % ) . crystals suitable for x - ray crystallography were obtained by slow diffusion of hexane into ch2cl2 solution ( 50:1 hexane / ch2cl2 ) . 236 c ; [ ]=+26 ( c=0.5 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) : =7.787.38 ( m , 8 h , ar - h ) , 7.356.99 ( m , 12 h , ar - h ) , 4.37 ( t , j=2.4 hz , 1 h , cp - h ) , 4.26 ( d , j=1.9 hz , 1 h , cp - h ) , 4.11 ( br s , 1 h , cp - h ) , 3.27 ( dd , j=13.7 , 8.6 hz , 1 h , nhch ) , 3.10 ( d , j=13.2 hz , 1 h , chhnme2 ) , 3.062.94 ( m , 1 h , nhchh ) , 2.902.78 ( m , 1 h , nhchh ) , 2.74 ( d , j=13.2 hz , 1 h , chhnme2 ) , 2.53 ( s , 3 h , ch3 ) , 2.36 ( s , 3 h , ch3 ) , 2.151.96 ( m , 2 h , chh and nh ) , 1.85 ( ddt , j=13.3 , 8.9 , 4.7 hz , 1 h , chh ) , 1.791.69 ( m , 1 h , chh ) , 1.431.28 ppm ( m , 1 h , chh ) ; c nmr ( 126 mhz , cdcl3 ) : =180.42 , 136.85 , 128.96 , 128.39 , 126.26 , 104.29 , 97.65 , 84.79 , 84.03 , 79.69 , 73.86 , 66.28 , 63.57 , 53.07 , 51.43 , 50.79 , 29.74 , 25.53 ppm ; ir ( neat ) : = 3056 , 2917 , 2849 , 2160 , 1972 , 1655 , 1596 , 1498 , 1446 , 1373 , 1263 , 1113 , 1067 , 1017 , 823 , 778 , 694 cm ; hrms ( esi ) : m / z calcd for c41h40o2n2pdco : 757.1466 [ m+h ] ; found : 757.1467 . conversion of ( r , sp)-13 into ( sp)-11 : dilute hydrochloric acid ( 0.64 ml of a 0.5 m solution ) was added to a solution of ( r , sp)-13 ( 0.100 g , 0.13 mmol ) in ch2cl2 ( 2 ml ) and the mixture was stirred vigorously for 16 h. the solution was diluted with ch2cl2 ( 5 ml ) and washed with brine ( 35 ml ) . the organic layer was collected , dried over mgso4 , filtered and concentrated in vacuo . purification by column chromatography ( sio2 , 4:1 hexanes / etoac ) gave the product ( sp)-11 as an orange solid ( 0.073 g , 82 % ) . 191 c ( decomp . ) ; [ ]=310 ( c=0.5 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) , 1:0.9 mixture of isomers : =7.777.53 ( m , 32 h , ar - h ) , 7.377.09 ( m , 48 h , ar - h ) , 4.53 ( d , j=1.2 hz , 2 h , cp - h ) , 4.38 ( d , j=1.5 hz , 2 h , cp - h ) , 4.33 ( br s , 4 h , cp - h ) , 4.19 ( t , j=2.4 hz , 2 h , cp - h ) , 4.09 ( t , j=2.4 hz , 2 h , cp - h ) , 3.18 ( d , j=13.4 hz , 2 h , chhnme2 ) , 3.13 ( d , j=13.3 hz , 2 h , chhnme2 ) , 2.81 ( d , j=13.4 hz , 2 h , chhnme2 ) , 2.77 ( d , j=13.2 hz , 2 h , chhnme2 ) , 2.67 ( s , 6 h , ch3 ) , 2.62 ( s , 6 h , ch3 ) , 2.19 ( s , 6 h , ch3 ) , 2.02 ppm ( s , 6 h , ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.83 , 136.78 , 129.42 , 129.36 , 128.09 , 128.06 , 125.95 , 125.87 , 103.09 , 102.71 , 102.31 , 101.91 , 85.03 , 83.46 , 81.07 , 80.36 , 77.73 , 74.84 , 74.81 , 64.64 , 64.55 , 53.57 , 52.21 , 51.87 , 51.52 ppm ; ir ( neat ) : = 3056 , 2886 , 1659 , 1597 , 1498 , 1446 , 1389 , 1352 , 1266 , 1155 , 1067 , 1024 , 984 , 957 , 910 , 842 , 809 , 780 , 739 , 697 , 563 cm ; elemental analysis calcd ( % ) for c72h62cl2co2n2pd2 : c 63.73 , h 4.61 , n 2.07 ; found c 63.75 , h 4.55 , n 2.16 . synthesis of di--acetatobis[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 14 ) : silver acetate ( 0.005 g , 0.03 mmol ) was added to a solution of ( sp)-11 ( 0.020 g , 0.02 mmol ) in acetone ( 1 ml ) . the solution was stirred vigorously , overnight , and then it was filtered through a short pad of celite , and eluted with ch2cl2 . the solvent was then removed in vacuo to give the product ( sp)-14 as an orange solid ( 0.020 g , 97 % ) . nmr ( 500 mhz , cdcl3 ) : =7.717.60 ( m , 16 h , ar - h ) , 7.257.14 ( m , 24 h , ar - h ) , 4.22 ( d , j=1.3 hz , 2 h , cp - h ) , 4.06 ( t , j=2.2 hz , 2 h , cp - h ) , 4.02 ( br s , 2 h , cp - h ) , 3.05 ( d , j=13.9 hz , 2 h , chhnme2 ) , 2.76 ( d , j=13.9 hz , 2 h , chhnme2 ) , 2.30 ( s , 6 h , nch3 ) , 2.15 ( s , 6 h , 2o2cch3 ) , 1.71 ppm ( s , 6 h , nch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =179.70 , 135.96 , 128.15 , 126.78 , 124.60 , 102.08 , 100.39 , 83.06 , 79.13 , 74.10 , 73.46 , 64.11 , 52.95 , 50.78 24.14 ppm ; ir ( neat ) : = 3055 , 2920 , 1577 , 1498 , 1412 , 1261 , 1176 , 1023 , 957 , 740 , 692 , 617 cm ; elemental analysis calcd ( % ) for c76h68co2n2o4pd2 : c 65.01 , h 4.88 , n 2.00 ; found c 65.18 , h 4.96 , n 2.04 . synthesis of ( rac)-14 : a solution of 8 ( 0.050 g , 0.09 mmol ) and pd(oac)2 ( 0.021 g , 0.09 mmol ) in toluene ( 1 ml ) was heated at reflux for 2 h. after being cooled to room temperature the solvent was removed in vacuo to give the product ( rac)-14 as an orange solid ( 0.062 g , 95 % ) . synthesis of hexafluoroacetylacetonate[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]palladium ( 15 ) : sodium hexafluoroacetylacetonate ( 0.007 g , 0.03 mmol ) was added to a solution of ( sp)-11 ( 0.020 g , 0.02 mmol ) in acetone / water ( 2:1 ml ) . the solution was stirred vigorously for 16 h. on completion , the solution was diluted with ch2cl2 ( 5 ml ) and washed with water ( 5 ml ) . the organic layer was collected , dried over mgso4 , filtered and concentrated in vacuo to give the product ( sp)-15 as an orange solid ( 0.012 g , 96 % ) . 219 c ; [ ]=160 ( c=1.0 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) : =7.717.47 ( m , 8 h , ar - h ) , 7.347.08 ( m , 12 h , ar - h ) , 5.87 ( s , 1 h , chco ) , 4.62 ( dd , j=2.3 , 1.0 hz , 1 h , cp - h ) , 4.50 ( d , j=1.5 hz , 1 h , cp - h ) , 4.37 ( t , j=2.4 hz , 1 h , cp - h ) , 3.41 ( d , j=13.9 hz , 1 h , chhnme2 ) , 2.92 ( d , j=13.9 hz , 1 h , chhnme2 ) , 2.76 ( s , 3 h , nch3 ) , 2.48 ppm ( s , 3 h , nch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =174.40 ( q , jcf=8.0 hz ) , 174.12 ( q , jcf=7.4 hz ) , 136.77 , 129.14 , 127.92 , 126.15 , 118.95 ( q , jcf=38.9 hz ) , 116.68 ( q , jcf=38.6 hz ) , 102.94 , 101.83 , 90.23 , 84.49 , 79.55 , 75.44 , 74.87 , 65.71 , 53.53 , 51.12 ppm ; ir ( neat ) : = 3056 , 2928 , 2160 , 1623 , 1597 , 1545 , 1498 , 1481 , 1458 , 1253 , 1195 , 1144 , 1024 , 950 , 779 , 695 cm ; elemental analysis calcd ( % ) for c41h32cof6no2pd2 h2o : c 55.58 , h 4.10 , n 1.58 ; found c 55.54 , h 3.90 , n 1.80 . crystals of ( rac)-15 for x - ray analysis , generated in the same way from ( rac)-14 , were obtained by slow diffusion of hexane into ch2cl2 solution ( 50:1 hexane / ch2cl2 ) . synthesis of ( -n , n - diethylaminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 21 a ) : a solution of 20 ( 0.100 g , 0.20 mmol ) and pph3 ( 0.051 g , 0.20 mmol ) in thf ( 3 ml ) was cooled to 20 c and nbs ( 0.035 g , 0.30 mmol ) was added in one portion . the mixture was stirred for 1015 min and et2nh ( 20 l , 0.2 mmol ) was added in one portion . the reaction was then allowed to warm to rt and was then heated at reflux for 1 h. the reaction mixture was cooled to rt and diluted with ch2cl2 ( 15 ml ) . the mixture was washed with 10 % hcl ( 10 ml ) , dried over mgso4 , filtered and concentrated in vacuo . purification by column chromatography ( sio2 , 19:1 ch2cl2/meoh ) gave the product 21 a as a yellow solid ( 0.045 g , 41 % ) . 140142 c ; h nmr ( 300 mhz , cdcl3 ) : =7.527.40 ( m , 8 h , ar - h ) , 7.317.18 ( m , 12 h , ar - h ) , 4.57 ( s , 4 h , cp - h ) , 2.92 ( s , 2 h , ch2net2 ) , 2.25 ( q , j=7.2 hz , 4 h , ch2ch3 ) , 0.88 ppm ( t , j=7.2 hz , 6 h , ch2ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.34 , 128.82 , 127.98 , 126.19 , 84.14 , 83.67 , 74.74 , 49.07 , 11.84 ppm ; ir ( neat ) : = 3057 , 2966 , 2921 , 1597 , 1499 , 1446 , 1068 , 1020 , 814 , 780 , 743 , 698 , 589 , 564 cm ; hrms ( esi ) : m / z calcd for c38h36con : 565.2180 [ m ] ; found 565.2170 . synthesis of ( -(1-pyrrolidinyl)methylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 21 b ) : the same method as described for 21 a was followed starting with 20 ( 0.127 g , 0.25 mmol ) . the product was isolated by column chromatography ( sio2 , 19:1 ch2cl2/meoh ) to give product 21 b as a yellow solid ( 0.100 g , 71 % ) . 180182 c ; h nmr ( 400 mhz , cdcl3 ) : =7.527.40 ( m , 8 h , ar - h ) , 7.317.18 ( m , 12 h , ar - h ) , 4.754.70 ( m , 2 h , cp - h ) , 4.624.58 ( m , 2 h , cp - h ) , 2.84 ( s , 2 h , ch2nme2 ) , 2.342.26 ( m , 4 h , n(ch2ch2)2 ) , 1.701.64 ppm ( m , 4 h , n(ch2ch2)2 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.35 , 128.92 , 128.08 , 126.31 , 84.06 , 83.75 , 74.93 , 53.68 , 52.60 , 23.41 ppm ; ir ( neat ) : = 2924 , 1596 , 1497 , 1446 , 1261 , 1023 , 816 , 781 , 746 , 701 , 589 , 564 cm ; hrms ( esi ) : m / z calcd for c38h35con : 564.2096 [ m+h ] ; found 564.2094 . synthesis of ( -n - benzylaminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 23 ) : 1,2-dichloroethane ( 35 ml ) was added to a mixture of 22 ( 2.61 g , 5.13 mmol ) and benzylamine ( 0.55 g , 5.13 mmol ) and then sodium triacetoxyborohydride ( 1.72 g , 8.12 mmol ) was added in one portion . the mixture was stirred at room temperature for 1.5 h. on completion , the reaction mixture was quenched by adding saturated aqueous nahco3 solution ( 50 ml ) and the product was extracted with etoac ( 240 ml ) . the etoac extract was dried over mgso4 , filtered and the solvent was removed in vacuo to give the crude product , which was purified by column chromatography ( sio2 , 19:1 ch2cl2/etoac ) to give product 23 as a yellow solid ( 3.00 g , 4.45 mmol , 97 % ) . 145147 c ; h nmr ( 400 mhz , cdcl3 ) : =7.43 ( dd , j=8.0 , 1.4 hz , 8 h , ar - h ) , 7.337.11 ( m , 17 h , ar - h ) , 4.66 ( t , j=1.8 hz , 2 h , cp - h ) , 4.57 ( t , j=1.9 hz , 2 h , cp - h ) , 3.52 ( s , 2 h , ch2ph ) , 3.13 ppm ( s , 2 h , ch2nh ) ; c nmr ( 126 mhz , cdcl3 ) : =136.33 , 135.43 , 129.01 , 128.88 , 128.14 , 128.10 , 126.62 , 83.68 , 82.56 , 74.89 , 53.56 , 45.38 ppm ; ir ( neat ) : = 3080 , 3059 , 3028 , 2924 , 2823 , 2246 , 1597 , 1499 , 1449 , 1379 , 909 , 733 , 697 cm ; hrms ( esi ) : m / z calcd for c41h35nco : 600.2102 [ m+h ] ; found : 600.2093 . synthesis of ( -aminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 24 ) : anhydrous ammonium formate ( 1.80 g , 28.5 mmol ) was added in a single portion to a stirred suspension of 23 ( 3.00 g , 5.00 mmol ) and an equal weight of 10 % pd / c in methanol ( 20 ml ) . the resulting reaction mixture was stirred at reflux for 2 h. on completion , the solution was filtered through a pad of celite and then washed with chloroform ( 20 ml ) . the combined organic filtrate was evaporated in vacuo and purified by column chromatography ( sio2 , 3:2 ch2cl2/etoac ) to give the product 24 as a yellow solid ( 1.09 g , 2.05 mmol , 43 % ) . 116 c ; h nmr ( 400 mhz , cdcl3 ) : =7.497.42 ( m , 8 h , ar - h ) , 7.257.18 ( m , 12 h , ar - h ) , 4.63 ( t , j=2.0 hz , 2 h , cp - h ) , 4.57 ( t , j=2.0 hz , 2 h , cp - h ) , 3.27 ppm ( br s , 2 h , ch2nh2 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.22 , 128.75 , 128.09 , 126.37 , 83.57 , 81.84 , 74.85 , 53.54 ppm ; ir ( neat ) : = 3052 , 2923 , 2162 , 1610 , 1596 , 1573 , 1453 , 1497 , 1453 , 1387 , 1267 , 1231 , 1106 , 1054 cm ; hrms ( esi ) : m / z calcd for c34h28ncona : 532.1451 [ m+na ] ; found : 532.1438 . synthesis of ( -n-(benzyloxycarbonyl)aminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 25 ) : iodine ( 0.006 g , 0.02 mmol ) was added to a stirred mixture of 24 ( 0.610 g , 1.20 mmol ) and benzylchloroformate ( 0.205 g , 1.20 mmol ) in 1:1 methanol / dichloromethane ( 5 ml ) . after being stirred for 2 h at rt , diethyl ether ( 10 ml ) was added . the reaction mixture was washed with 5 % na2s2o3 solution ( 5 ml ) and saturated nahco3 ( 5 ml ) , dried over mgso4 , filtered and the solvent was removed in vacuo . 4:1 ) gave the product as a yellow / brown solid ( 0.750 g , 97 % ) . 8890 c ; h nmr ( 400 mhz , cdcl3 ) : =7.45 ( d , j=6.4 hz , 8 h , ar - h ) , 7.417.17 ( m , 17 h , ar - h ) , 5.03 ( s , 2 h , och2ph ) , 4.63 ( br s , 2 h , cp - h ) , 4.58 ( d , j=1.8 hz , 2 h , cp - h ) , 4.31 ( br s , 1 h , nhcbz ) , 3.77 ppm ( d , j=5.6 hz , 2 h , ch2nh ) ; c nmr ( 126 mhz , cdcl3 ) : =156.23 , 136.78 , 136.18 , 128.77 , 128.54 , 128.27 , 128.09 , 127.09 , 126.56 , 96.05 , 83.61 , 81.87 , 75.04 , 69.75 , 66.58 , 65.46 , 54.97 ppm ; ir ( neat ) : = 3416 , 3080 , 3059 , 3030 , 2954 , 2247 , 1723 , 1597 , 1499 , 1444 , 1269 , 1026 , 735 , 697 cm ; hrms ( esi ) : m / z calcd for c42h35cono2 : 644.2000 [ m+h ] ; found : 644.1990 . synthesis of ( -n - methylaminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 26 ) : a stirred suspension of lialh4 , ( 0.090 g , 2.37 mmol ) in thf ( 30 ml ) was cooled to 0 c in an ice - water bath . to this was added a solution of 25 ( 0.700 g , 1.09 mmol ) in thf ( 40 ml ) and the mixture was heated to reflux for 1.5 h. after being cooled the reaction was quenched with saturated na2so4 solution ( 5 ml ) , filtered through a pad of celite and extracted with etoac ( 230 ml ) . the combined organic extracts were concentrated under reduced pressure to give a crude solid , which was purified by column chromatography ( sio2 , ch2cl2/etoac 7:3 ) to give the product 26 as yellow solid ( 0.560 g , 98 % ) . 177179 c ; h nmr ( 400 mhz , cdcl3 ) : =7.45 ( dd , j=7.2 , 1.9 hz , 8 h , ar - h ) , 7.227.26 ( m , 12 h , ar - h ) , 4.66 ( s , 2 h , cp - h ) , 4.57 ( s , 2 h , cp - h ) , 3.07 ( s , 2 h , ch2nhme ) , 2.14 ppm ( s , 3 h , nhch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.29 , 128.85 , 128.19 , 126.45 , 84.26 , 83.76 , 83.64 , 82.82 , 74.99 , 48.34 ppm ; ir ( neat ) : = 3080 , 3028 , 2938 , 2787 , 1597 , 1499 , 1444 , 1025 , 909 , 733 , 697 cm ; hrms ( esi ) : m / z calcd for c35h31con : 524.1778 [ m+h ] ; found : 524.1789 . synthesis of di--chlorobis[(-(sp)-n , n - diethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 27 ) : a solution of ( r)-n - acetylphenylalanine ( 0.250 g , 1.21 mmol ) and naoh ( 0.039 g , 0.98 mmol ) in water ( 15 ml ) was added to a solution of na2pd2cl4 ( 0.263 g , 0.89 mmol ) in meoh ( 50 ml ) . the ph of the mixture was adjusted to 8.0 using either aqueous naoh or hcl as required and the mixture was allowed to stir for 20 min . a solution of 21 a ( 0.500 g , 0.88 mmol ) in 5:1 meoh / ch2cl2 ( 90 ml ) was then added in portions over 5 min . , the reaction mixture was diluted with ch2cl2 ( 100 ml ) and washed with brine ( 2100 ml ) . the organic phase was dried over mgso4 , filtered and the solvent was removed in vacuo . purification by column chromatography ( sio2 , 4:1 hexanes / etoac ) gave the product ( sp)-27 as an orange solid ( 0.244 g , 39 % ) , ee=82 % as determined by formation of the proline adducts . m.p . > 200 c ( decomp ) ; [ ]=618 ( c=0.5 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) 1:0.6 mixture of isomers : =7.707.60 ( m , 32 h , ar - h ) , 7.297.15 ( m , 48 h , ar - h ) , 4.47 ( dd , j=2.3 , 1.1 hz , 2 h , cp - h ) , 4.32 ( t , j=2.2 hz , 4 h , cp - h ) , 4.304.27 ( m , 2 h , cp - h ) , 4.24 ( t , j=2.4 hz , 2 h , cp - h ) , 4.17 ( t , j=2.4 hz , 2 h , cp - h ) , 3.29 ( d , j=13.9 hz , 2 h , chhnet2 ) , 3.22 ( d , j=13.9 hz , 2 h , chhnet2 ) , 2.75 ( d , j=13.9 hz , 4 h , chhnet2 ) , 2.682.43 ( m , 16 h , ch2ch3 ) , 1.52 ( t , j=7.1 , hz , 6 h , ch2ch3 ) , 1.52 ( t , j=7.1 , hz , 6 h , ch2ch3 ) , 0.920.84 ppm ( m , 12 h , ch2ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.74 , 136.68 , 129.32 , 129.26 , 127.95 , 127.92 , 129.79 , 125.76 , 84.27 , 82.65 , 79.50 , 79.44 , 77.24 , 76.45 , 75.55 , 60.41 , 57.44 , 57.28 , 55.39 , 55.33 , 54.32 , 53.22 , 14.53 , 14.22 , 10.01 , 9.80 ppm ; ir ( neat ) : = 3056 , 2971 , 2929 , 1596 , 1498 , 1444 , 1387 , 909 , 734 , 695 cm ; elemental analysis calcd ( % ) for c76h70cl2co2n2pd2 : c 64.60 , h 4.99 , n 1.98 ; found c 64.70 , h 4.89 , n 2.04 . synthesis of proline adducts ( s , sp)-29 and ( s , rp)-30 : a solution of ( sp)-27 ( 0.020 g , 0.014 mmol ) in acetone ( 1 ml ) was added to a solution of nahco3 ( 0.003 g , 0.04 mmol ) and ( s)-proline ( 0.003 g , 0.03 mmol ) in water ( 0.5 ml ) . during the addition the reaction was then vigorously stirred for 16 h at rt and then diluted with ch2cl2 ( 5 ml ) . the phases were separated and the aqueous phase was washed with further portions of ch2cl2 ( 22 ml ) . the organic phases were combined , dried over mgso4 , filtered and the solvent was removed in vacuo yielding the product as an orange solid ( 0.021 g , 95 % ) . 206208 c ; [ ]=104 ( c=0.7 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) : =7.55 ( dd , j=6.6 , 3.0 hz , 8 h , ar - h ) , 7.257.21 ( m , 12 h , ar - h ) , 4.40 ( t , j=2.4 hz , 1 h , cp - h ) , 4.34 ( d , j=1.6 hz , 1 h , cp - h ) , 4.13 ( d , j=1.5 hz , 1 h , cp - h ) , 3.92 ( dd , j=15.2 , 7.6 hz , 1 h , nch ) , 3.29 ( d , j=13.9 hz , 2 h , 2chh ) , 2.88 ( d , j=13.7 hz , 2 h , 2chh ) , 2.782.55 ( m , 3 h , 3chh ) , 2.492.27 ( m , 2 h , 2chh ) , 2.172.06 ( m , 3 h , chh ) , 1.401.31 ( m , 3 h , ch2ch3 ) , 0.94 ppm ( t , j=7.4 hz , 3 h , ch2ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =180.08 , 136.79 , 129.01 , 128.20 , 126.21 , 84.38 84.0 , 81.39 , 79.62 74.82 , 73.96 , 66.17 , 58.09 , 54.23 , 53.31 , 52.52 , 29.97 , 26.07 , 13.52 , 9.84 ppm ; ir ( neat ) : = 3453 , 2929 , 2852 , 1725 , 1594 , 1492 , 1446 , 1381 , 1255 , 1179 , 1156 , 1122 , 1071 , 1021 , 804 , 778 , 740 , 699 cm ; hrms ( esi ) : m / z calcd for c43h44con2o2pd : 785.1780 [ m+h ] ; found : 785.1773 . ratio of isomers obtained from integration of signals at 4.40 ( t , j=2.4 hz , 1 h , ( s , sp)-cp - h ) and 4.42 ppm ( t , j=2.4 hz , 1 h , ( s , rp)-cp - h ) . synthesis of di--chlorobis[(-(sp)-(1-pyrrolidinyl)methylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 28 ) : a solution of ( r)-n - acetylphenylalanine ( 0.251 g , 1.21 mmol ) and naoh ( 0.039 g , 0.98 mmol ) in water ( 15 ml ) was added to a solution of na2pd2cl4 ( 0.263 g , 0.89 mmol ) in meoh ( 50 ml ) . the ph of the mixture was adjusted to 8.0 using either aqueous naoh or hcl as required and the mixture was allowed to stir for 20 min . a solution of 21 b ( 0.500 g , 0.89 mmol ) in 5 : 1 meoh / ch2cl2 ( 90 ml ) was then added in portions over 5 min . , the reaction mixture was diluted with ch2cl2 ( 150 ml ) and washed with brine ( 2100 ml ) . the organic phase was dried over mgso4 , filtered and the solvent was removed in vacuo . purification by column chromatography ( sio2 , 4:1 hexanes / etoac ) gave the product ( sp)-28 as an orange solid ( 0.270 g , 43 % ) , ee > 98 % as determined by formation of the proline adducts m.p . > 200 c ( decomp ) ; [ ]=266 ( c=0.5 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) 1:1 mixture of isomers : =7.727.63 ( m , 32 h , ar - h ) , 7.287.16 ( m , 48 h , ar - h ) , 4.534.50 ( m , 2 h , cp - h ) , 4.384.35 ( m , 2 h , cp - h ) , 4.31 ( d , j=1.5 hz , 2 h , cp - h ) , 4.26 ( d , j=1.5 hz , 2 h , cp - h ) , 4.23 ( t , j=2.4 hz , 2 h , cp - h ) , 4.154.09 ( m , 2 h , cp - h ) , 3.443.32 ( m , 4 h , cpchhn ) , 3.082.81 ( m , 16 h , cpchhn and nch2 ) , 2.322.23 ( m , 2 h , nch2 ) , 2.082.03 ( m , 2 h , nch2 ) , 1.81 ( ddd , j=9.3 , 6.2 , 3.5 hz , 4 h , nch2ch2 ) , 1.761.60 ( m , 8 h , nch2ch2 ) , 1.531.45 ppm ( m , 4 h , nch2ch2 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.75 , 136.68 , 129.34 , 129.28 , 127.94 , 125.79 , 125.72 , 103.20 , 102.85 , 102.75 , 102.31 , 84.84 , 82.99 , 80.55 , 79.96 , 77.60 , 76.71 , 75.25 , 74.68 , 74.66 , 60.46 , 60.38 , 60.28 , 60.22 , 59.67 , 30.95 , 22.00 , 21.65 , 21.55 , 21.30 ppm ; ir ( neat ) : = 3056 , 2966 , 1596 , 1498 , 1443 , 909 , 734 , 695 cm ; elemental analysis calcd ( % ) for c76h66cl2co2n2pd2 : c 64.79 , h 4.72 , n 1.98 ; found c 64.81 , h 4.60 , n 2.07 . synthesis of proline adduct ( s , sp)-31 : a solution of ( sp)-28 ( 0.020 g , 0.014 mmol ) in acetone ( 2 ml ) was added to a solution of nahco3 ( 0.003 g , 0.04 mmol ) and ( s)-proline ( 0.003 g , 0.03 mmol ) in water ( 1 ml ) . during the addition the reaction was then vigorously stirred for 16 h at rt and then diluted with ch2cl2 ( 5 ml ) . the phases were separated and the aqueous phase was washed with further portions of ch2cl2 ( 22 ml ) . the organic phases were combined , dried over mgso4 , filtered and the solvent was removed in vacuo yielding the product as an orange solid ( 0.020 g , 90 % ) . 206208 c ; [ ]=37 ( c=1.1 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , [ d6]dmso ) : =7.527.46 ( m , 8 h , ar - h ) , 7.287.20 ( m , 12 h , ar - h ) , 5.525.44 ( m , 1 h , nh ) , 4.36 ( s , 2 h , cp - h ) , 4.25 ( s , 1 h , cp - h ) , 3.56 ( q , j=7.9 hz , 1 h , nhch ) , 3.193.01 ( m , 2 h , nhchh and cpchhn ) , 3.012.94 ( m , 1 h , nhchh ) , 2.922.84 ( m , 1 h , nch2 ) , 2.79 ( d , j=14.5 hz , 1 h , cpchhn ) , 2.352.21 ( m , 2 h , ch2 ) , 2.142.04 ( m , 1 h , chh ) , 1.821.61 ( m , 4 h , ch2 ) , 1.441.33 ( m , 1 h , chh ) , 1.241.16 ppm ( m , 2 h , ch2 ) ; c nmr : not obtained due to poor solubility in cdcl3 and [ d6]dmso ; ir ( neat ) : = 3444 , 2925 , 2855 , 1733 , 1623 , 1590 , 1497 , 1459 , 1378 , 1259 , 1170 , 1070 , 1023 , 926 , 782 , 745 , 702 cm ; hrms ( esi ) : m / z calcd for c43h42con2o2pd : 783.1623 [ m+h ] ; found : 783.1615 . synthesis of proline adduct ( r , sp)-32 : prepared in the same way as ( s , sp)-31 from ( sp)-28 ( 0.020 g , 0.014 mmol ) and ( r)-proline ( 0.003 mg , 0.03 mmol ) to give the product as an orange solid ( 0.011 g , 50 % ) . 206208 c ; [ ]=+72 ( c=0.5 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) : =7.69 ( d , j=6.4 hz , 8 h , ar - h ) , 7.297.26 ( m , 12 h , ar - h ) , 4.45 ( t , j=2.3 hz , 1 h , cp - h ) , 4.33 ( d , j=2.6 hz , 1 h , cp - h ) , 4.20 ( d , j=2.5 hz , 1 h , cp - h ) , 3.44 ( dt , j=11.5 , 7.6 hz , 1 h , nhch ) , 3.34 ( ddt , j=13.5 , 9.1 , 4.9 hz , 2 h , 2nhchh ) , 3.132.91 ( m , 4 h , ch2 ) , 2.712.61 ( m , 1 h , chh ) , 2.472.35 ( m , 1 h , chh ) , 2.182.10 ( m , 1 h , chh ) , 2.051.95 ( m , 2 h , ch2 . ) , 1.941.89 ( m , 2 h , ch2 ) , 1.881.79 ( m , 1 h , chh ) , 1.781.72 ( m , 1 h , chh ) , 1.64 ( br s , 1 h , nh ) , 1.511.38 ppm ( m , 1 h , chh ) ; c nmr ( 126 mhz , cdcl3 ) : =180.32 , 136.76 , 128.87 , 128.26 , 126.09 , 104.56 , 97.53 , 84.31 , 83.78 , 79.33 , 73.73 , 66.13 , 59.97 , 59.65 , 59.13 , 52.98 , 29.60 , 25.44 , 22.06 , 21.95 ppm ; ir ( neat ) : = 3444 , 2925 , 2855 , 1733 , 1623 , 1590 , 1497 , 1459 , 1378 , 1259 , 1170 , 1070 , 1023 , 926 , 782 , 745 , 702 cm ; hrms ( esi ) : m / z calcd for c43h42con2o2pd : 783.1623 [ m+h ] ; found : 783.1614 . synthesis of chloro[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]2-(diphenylphosphino)phenylferrocene - palladium ( 37 ) : 2-(diphenylphosphino)phenylferrocene ( 0.007 g , 0.016 mmol ) was added to a solution of ( sp)-11 ( 0.010 g , 0.015 mmol ) in ch2cl2 ( 1 ml ) and the solution was stirred for 16 h. the solvent was removed in vacuo and the product was purified by column chromatography ( sio2 , 49:1 ch2cl2/meoh ) to give the product ( sp)-35 as a red / orange solid ( 0.015 g , 88 % ) . crystals suitable for x - ray crystallography were obtained by slow diffusion of hexane into a ch2cl2 solution ( 50:1 hexane / ch2cl2 ) . 171 c ; [ ]=+26 ( c=0.5 mg ml in ch2cl2 ) ; h nmr ( 500 mhz , cdcl3 ) : =7.907.80 ( m , 2 h , ar - h ) , 7.487.10 ( m , 28 h , ar - h ) , 7.00 ( t , j=7.6 hz , 1 h , ar - h ) , 6.90 ( t , j=6.8 hz , 2 h , ar - h ) , 6.76 ( dd , j=11.1 , 7.5 hz , 1 h , ar - h ) , 4.48 ( br s , 1 h , cp - h ) , 4.36 ( br s , 1 h , cp - h ) , 4.12 ( s , 1 h , cp - h ) , 4.07 ( s , 1 h , cp - h ) , 4.00 ( s , 1 h , cp - h ) , 3.93 ( s , 5 h , cp - h ) , 3.84 ( s , 1 h , cp - h ) , 3.22 ( s , 1 h , cp - h ) , 3.03 ( d , j=14.1 hz , 1 h , chhnme2 ) , 2.92 ( dd , j=14.0 , 2.5 hz , 1 h , chhnme2 ) , 2.65 ( s , 3 h , ch3 ) , 2.38 ppm ( d , j=2.4 hz , 3 h , ch3 ) ; c nmr ( 126 mhz , cdcl3 ) : =136.66 , 135.99 , 132.84 , 130.20 , 129.42 , 129.06 , 128.05 , 127.99 , 127.95 , 127.91 , 127.52 , 127.44 , 125.89 , 120.35 , 80.82 , 77.60 , 77.29 , 77.03 , 76.78 , 76.24 , 73.82 , 69.68 , 61.90 , 52.06 ppm ; p nmr ( 202 mhz , cdcl3 ) : =32.22 ppm ; ir ( neat ) : = 3056 , 2923 , 1732 , 1596 , 1497 , 1436 , 1194 , 911 , 732 , 695 , 559 cm ; elemental analysis calcd ( % ) for c64h54clcofenppd : c 68.34 , h 4.85 , n 1.25 ; found c 66.35 , h 5.05 , n 1.46 . general procedure for transcyclopalladation : a mixture of ( sp)-14 ( 0.030 g , 0.02 mmol ) and either 33 ( 0.019 g , 0.04 mmol ) or 34 ( 0.020 g , 0.04 mmol ) were heated in toluene ( 0.5 ml ) under nitrogen for 24 h. after being cooled the solvent was evaporated in vacuo and the residue was re - dissolved in 2:1 acetone / water and sodium acetylacetonate ( 0.005 g , 0.04 mmol ) was added to it . after being stirred at room temperature for 16 h the mixture was diluted with ch2cl2 , washed with water , dried ( mgso4 ) , filtered and the solvent was removed in vacuo . the ee and absolute configuration of 35 and 36 were determined by hplc ( chiracel od - h , 99.7:0.3 n - hexane / ipa , 0.8 ml min ) . general procedure for catalysis : the required amount of imidate stock solution was added to a flask charged with catalyst . if silver salts and/or proton sponge was used this was subsequently added . the solution was protected from light then heated to the desired temperature for the allotted time . on completion , the solution was passed through a short pad of celite and the solvent was removed in vacuo . purification by flash chromatography , eluting with 24:1 hexanes / etoac , yielded the product amide 2,2,2-trifluoro - n-(4-methoxyphenyl - n-(1-propylallyl)acetamide ( 43 ) as a pale yellow oil . chiral hplc analysis was used to determine enantiomeric excess after cleavage of the trifluoroacetate group[5a ] ( chiracel od - h , 99.8:0.2 n - hexane / ipa , 0.8 ml min ) . in the same manner amides 44 a d were isolated as colourless oils and chiral hplc analysis was used to determine the enantiomeric excess ( chiracel od - h , 99.5:0.5 n - hexane / ipa , 0.8 ml min).[5b , d ] ccdc-931363 http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi((s,sp)-12 ) , 931364 http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi(15 ) and 931365 http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi((sp)-37 ) contain the supplementary crystallographic data for this paper . these data can be obtained free of charge from the cambridge crystallographic data centre via http://www.ccdc.cam.ac.uk/data_request/cif . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

the reaction of ( 5-(n , n - dimethylaminomethyl)cyclopentadien - yl)(4-tetraphenylcyclobutadiene)cobalt with sodium tetrachloropalladate and ( r)-n - acetylphenylalanine gave planar chiral palladacycle di--chloridebis[(5-(sp)-2-(n , n - dimethylaminomethyl)cyclopentadienyl,1-c,3-n)(4-tetraphenylcyclobutadiene)cobalt]dipalladium [ ( sp)-me2-cap - cl ] in 92 % ee and 64 % yield . enantiopurity ( > 98 % ee ) was achieved by purification of the monomeric ( r)-proline adducts and conversion back to the chloride dimer . treatment with agoac gave ( sp)-me2-cap - oac which was applied to asymmetric transcyclopalladation ( up to 78 % ee ) . the ( r)-n - acetylphenylalanine mediated palladation methodology was applicable also to the corresponding n , n - diethyl ( 82 % ee , 39 % yield ) and pyrrolidinyl ( > 98 % ee , 43 % yield ) cobalt sandwich complexes . a combination of 5 mol % of the latter [ ( sp)-pyrr - cap - cl ] and agno3 ( 3.8 equiv ) is a catalyst for the allylic imidate rearrangement of an ( e)-n - aryltrifluoroacetimidate ( up to 83 % ee ) , and this catalyst system is also applicable to the rearrangement of a range of ( e)-trichloroacetimidates ( up to 99 % ee ) . this asymmetric efficiency combined with the simplicity of catalyst synthesis provides accessible solutions to the generation of non - racemic allylic amine derivatives .

Introduction Results and Discussion Conclusions Experimental Section Supporting Information

conspicuous success has been achieved in catalysis of the asymmetric allylic imidate rearrangement and closely related reactions using planar chiral palladacycles based upon bulky cobalt sandwich complexes ( e.g. diastereoselective synthesis of cobalt oxazoline palladacycle 1 [ ( s , rp)-cop - oac ] and transformation into chloride - bridged dimer 2 [ ( s , rp)-cop - cl ] . [2b , 11 ] there are far fewer examples of planar chiral palladacycles derived from enantioselective c non - racemic planar chiral palladacycles have been generated from a limited number of enantioselective transcyclopalladation reactions , and in 1979 sokolov reported the n - acetyl amino acid mediated enantioselective palladation of n , n - dimethylaminomethylferrocene ( 3 ) . recent re - investigation of this latter chemistry within our group identified conditions for the synthesis of palladacycle ( sp)-4 in 96 % ee using ( r)-n - acetylphenylalanine ( 5 ) , and an extension of the methodology to the kinetic resolution of [ 2.2]paracyclophane ( 6 ) gave palladacycle ( sp)-7 in more than 99 % ee ( scheme 2 ) . in this study we report on the application of enantioselective palladation to the facile synthesis of non - racemic amine palladacycles containing a cobalt sandwich complex , and on the application of these to asymmetric transcyclopalladation and asymmetric allylic imidate rearrangement . application of the room temperature conditions optimised previously for the enantioselective palladation of 3 , with a reaction time of 16 h , resulted in a new chloride bridged palladacycle 11 in 64 % yield ( scheme 4 ) . the high enantioselectivity observed in the palladation reaction points to the involvement of a palladium intermediate containing a coordinated carboxylate ligand obtained by deprotonation of ( r)-n - acetylphenylalanine . repetition of the na2pdcl4/n - acetyl amino acid palladation conditions , with n - acetylglycine in place of ( r)-n - acetylphenylalanine , followed by ligand substitution with sodium hexafluoroacetylacetonate , gave monomeric palladacycle d-(rac)-15/h-(rac)-15 ( 64 % deuterium incorporation ) . all of these values are consistent with a carboxylate ligand accelerated cmd pathway , with the reactions containing ( r)-n - acetylphenylalanine resulting in the preferential formation of the ( sp)-palladacycle . this is based on the dft calculated mechanism of dimethylbenzylamine cyclometalation by palladium acetate , and a suggested extension of this process to the n - acetylphenylalanine mediated enantioselective palladation of phosphines containing a 2-phenylferrocene substituent . variation of the ee of the ( r)-n - acetylphenylalanine employed in cyclometallation resulted in a small positive non - linear effect with respect to the ee of metallacycle ( sp)-11 , an outcome compatible with coordination of a second -carboxylate 18 and chirality matched rate accelerated cyclometallation ( figure 2 ) . conversion back to the cobalt amine palladacycle ( sp)-11 [ ( sp)-me2-cap - cl ] was carried out by stirring , overnight , a biphasic mixture of ( r , sp)-13 in ch2cl2 and aqueous 0.5 m hcl ( scheme 8) . the enantiopure chloride - bridged dimer is formed in good yield as an approximately 1:1 mixture of isomers with respect to the cis / trans arrangements of the two bridged c addition of silver acetate to ( sp)-11 resulted in the clean formation of acetate bridged dimer ( sp)-14 [ ( sp)-me2-cap - oac ] , and in common with other examples of planar chiral acetate - bridged palladacycles this is a single , presumably trans , stereoisomer . treatment of ( sp)-11 with sodium hexafluoroacetylacetonate [ na(hfacac ) ] gave ( sp)-15 [ ( sp)-me2-cap - hfacac ] , although attempts to synthesise this directly from proline adduct ( r , sp)-13 were unsuccessful . a number of amines related to the n , n - dimethylamino containing substrate 8 were synthesised to examine further the asymmetric palladation methodology . application to these new amines of the standard asymmetric palladation conditions resulted in only two new palladacycles , ( sp)-27 and ( sp)-28 derived from the n , n - diethylamine 21 a and pyrrolidinyl complex 21 b , respectively ( scheme 10 ) . the absolute configuration of these new palladacycles was assigned initially by the sign of the specific rotation [ ( sp)=ve , ( rp)=+ve ] , and confirmed by the correspondence between the cd spectra of ( sp)-27 and ( sp)-28 with that of ( sp)-11 ( see the supporting information ) . the n , n - dimethyl , n , n - diethyl and pyrrolidinyl substrates appear to have the correct balance of nitrogen basicity and steric accessibility to permit palladation . following a demonstration of asymmetric transcyclopalladation using palladacycles derived from ( r)-3-amino-3-phenyl-2,2-dimethylpropane,[12a ] one of us reported that the reaction between ( s , rp)-cop - oac ( 1 ) and prochiral phosphines 33 or 34 resulted in the clean formation of phosphapalladacycles 35 and 36 in up to 95 % ee ( r = cy). the triarylphosphine ligand displays an induced p configuration , and the tetraphenylcyclobutadiene moiety is m. this latter configuration is also displayed in the solid state structure of ( rac)-15 ( relative to sp ) , but ( s , sp)-12 and ( r , sp)-13 are p , revealing no correlation between the planar and induced propeller chirality of the -tetraphenylcyclobutadiene group . kinetic and molecular modelling studies on the cop - cl catalysed allylic imidate rearrangement revealed that the planar chirality is also the key factor in controlling the facial selectivity of nitrogen addition to the alkene moiety , this being bound trans to the oxazoline nitrogen in the rate and enantioselectivity determining anti - amino palladation step. [5e ] given the similarity of the coordination site trans to the amine nitrogen , it was anticipated that cap catalysis of the allylic imidate rearrangement by this pathway would result in usable levels of enantioselectivity , and a predictable correspondence between the configurations of planar and product chirality ( sp gives r ) . this was investigated first with the allylic imidate rearrangement of representative ( e)- and ( z)-n-(para - methoxyphenyl)trifluoroacetimidate substrates 42 ( scheme 13 , table 1 ) . the reactions were performed first with 5 mol % of ( sp)-11 at room temperature , which resulted in modest conversions for the formation of ( r)-43 ( 75 % ee ) and ( s)-43 ( 20 % ee ) from e and z substrates , respectively ( entries 1 and 2 ) . under similar conditions 5 mol % of ( s , rp)-cop - cl ( 1 ) resulted in essentially complete conversion , and up to 92 % ee. [5a ] use of ( s)-cap - cl in catalysis of the rearrangement of ( e)- and ( z)-n-(para - methoxyphenyl)trifluoroacetimidates ( 42 ) . use of ( sp)-cap - cl in catalysis of the rearrangement of ( e)- and ( z)-n-(para - methoxyphenyl)trifluoroacetimidates ( 42 ) [ a ] 0.6 m 42 in ch2cl2 , reaction time 60 h at rt or 24 h at 38 c . related chloride - bridged ferrocene imidazoline palladacycles are essentially inactive as catalysts for the allylic imidate rearrangement due the electron - donating properties of the iron containing metallocene . addition of 3.8 equivalents with respect to ( sp)-28 of agno3 prior to the introduction of the substrate ( e)-42 resulted in complete conversion to give ( r)-43 in 81 % ee ( entry 6 ) . essentially the same outcome was obtained on addition of proton sponge ( entry 7 ) , though a tenfold reduction in catalyst loading to 0.5 mol % led to the erosion of enantioselectivity and yield ( entry 8) . encouraged by these results we applied the cap catalysts to the rearrangement of ( e)-trichloroacetimidates 44 ( scheme 14 ) . initial experiments with 1 , 2 and 5 mol % of ( sp)-11 gave a higher ee value with each increase in catalyst loading ( entries 13 ) , and use of 5 mol % of ( sp)-28 further increased the ee to 86 % ( table 2 ) . this gave complete conversion and an ee of 73 % ( entry 5 ) , which on repetition in the presence of proton sponge increased to 99 % ee ( entry 6 ) . decreasing the catalyst loading to 0.5 mol % again eroded significantly the ee and yield ( entry 7 ) , and so a small range of additional substrates were examined at 5 mol % loading ( entries 810 ) . these results are comparable to ( s , rp)-cop - cl catalysed rearrangement of trichloroacetimidates,[5b , d ] but now using a catalyst available readily from highly enantioselective palladation of a simple prochiral substrate . use of ( sp)-cap - cl in catalysis of the rearrangement of ( e)-trichloroacetimidates 44 a d . use of ( sp)-cap - cl in catalysis of the rearrangement of ( e)-trichloroacetimidates ( 44 ) [ a ] 0.6 m 44 in ch2cl2 , reaction time 39 h at 38 c . enantioselective palladation of n , n - dimethylaminomethyl - appended cobalt sandwich complex 8 with na2pdcl4 mediated by ( r)-n - acetylphenylalanine gave the chloride - bridged palladacycle ( sp)-11 in 92 % ee . the enantiopurity of ( sp)-11 may be increased to more than 98 % ee by chromatographic separation of the minor diastereoisomer formed on addition of ( r)-proline followed by treatment of the major diastereoisomer with aqueous hcl . a number of related aminomethyl - substituted cobalt complexes were synthesised readily , but this enantioselective palladation protocol is limited to n , n - diethyl- ( 82 % ee ) and pyrrolidinyl ( > 98 % ee ) substituents . the activity and enantioselectivity of a cobalt amine palladacycle catalyst for the allylic imidate rearrangement is increased significantly following the addition of 3.8 equivalents of silver nitrate . the catalyst generated from ( sp)-28 results in the rearrangement of ( e)-trichloroacetimidates with high enantioselectivity ( up to 99 % ee ) . combined with the simple highly enantioselective generation of ( sp)-28 , this methodology enables the straight - forward generation of highly scalemic allylic amine derivatives for application in organic synthesis . alternative synthesis of ( -n , n - dimethylaminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 8) : a flask was charged with 10 ( 0.986 g , 1.78 mmol ) and it was then dissolved in thf ( 20 ml ) . synthesis of di--chlorobis[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 11 ) : a solution of ( r)-n - acetylphenylalanine ( 0.740 g , 3.57 mmol ) and naoh ( 0.066 g , 1.65 mmol ) in water ( 15 ml ) was added to a solution of na2pd2cl4 ( 0.439 g , 1.49 mmol ) in meoh ( 50 ml ) . synthesis of proline adduct ( s , sp)-12 : a solution of ( sp)-11 ( 0.050 g , 0.04 mmol ) in acetone ( 1 ml ) was added to a solution of nahco3 ( 0.031 g , 0.37 mmol ) and ( s)-proline ( 0.043 g , 0.37 mmol ) in water ( 0.5 ml ) . synthesis of proline adduct ( r , sp)-13 : a solution of ( sp)-11 ( 0.050 g , 0.04 mmol ) in acetone ( 10 ml ) was added to a solution of nahco3 ( 0.088 g , 1.04 mmol ) and ( r)-proline ( 0.085 g , 0.74 mmol ) in water ( 5 ml ) . conversion of ( r , sp)-13 into ( sp)-11 : dilute hydrochloric acid ( 0.64 ml of a 0.5 m solution ) was added to a solution of ( r , sp)-13 ( 0.100 g , 0.13 mmol ) in ch2cl2 ( 2 ml ) and the mixture was stirred vigorously for 16 h. the solution was diluted with ch2cl2 ( 5 ml ) and washed with brine ( 35 ml ) . synthesis of di--acetatobis[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 14 ) : silver acetate ( 0.005 g , 0.03 mmol ) was added to a solution of ( sp)-11 ( 0.020 g , 0.02 mmol ) in acetone ( 1 ml ) . synthesis of hexafluoroacetylacetonate[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]palladium ( 15 ) : sodium hexafluoroacetylacetonate ( 0.007 g , 0.03 mmol ) was added to a solution of ( sp)-11 ( 0.020 g , 0.02 mmol ) in acetone / water ( 2:1 ml ) . synthesis of ( -n , n - diethylaminomethylcyclopentadienyl)(-tetraphenylcyclobutadiene)cobalt ( 21 a ) : a solution of 20 ( 0.100 g , 0.20 mmol ) and pph3 ( 0.051 g , 0.20 mmol ) in thf ( 3 ml ) was cooled to 20 c and nbs ( 0.035 g , 0.30 mmol ) was added in one portion . synthesis of di--chlorobis[(-(sp)-n , n - diethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 27 ) : a solution of ( r)-n - acetylphenylalanine ( 0.250 g , 1.21 mmol ) and naoh ( 0.039 g , 0.98 mmol ) in water ( 15 ml ) was added to a solution of na2pd2cl4 ( 0.263 g , 0.89 mmol ) in meoh ( 50 ml ) . synthesis of proline adducts ( s , sp)-29 and ( s , rp)-30 : a solution of ( sp)-27 ( 0.020 g , 0.014 mmol ) in acetone ( 1 ml ) was added to a solution of nahco3 ( 0.003 g , 0.04 mmol ) and ( s)-proline ( 0.003 g , 0.03 mmol ) in water ( 0.5 ml ) . synthesis of di--chlorobis[(-(sp)-(1-pyrrolidinyl)methylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]dipalladium ( 28 ) : a solution of ( r)-n - acetylphenylalanine ( 0.251 g , 1.21 mmol ) and naoh ( 0.039 g , 0.98 mmol ) in water ( 15 ml ) was added to a solution of na2pd2cl4 ( 0.263 g , 0.89 mmol ) in meoh ( 50 ml ) . purification by column chromatography ( sio2 , 4:1 hexanes / etoac ) gave the product ( sp)-28 as an orange solid ( 0.270 g , 43 % ) , ee > 98 % as determined by formation of the proline adducts m.p . synthesis of proline adduct ( s , sp)-31 : a solution of ( sp)-28 ( 0.020 g , 0.014 mmol ) in acetone ( 2 ml ) was added to a solution of nahco3 ( 0.003 g , 0.04 mmol ) and ( s)-proline ( 0.003 g , 0.03 mmol ) in water ( 1 ml ) . synthesis of chloro[(-(sp)-n , n - dimethylaminomethylcyclopentadienyl,1-c,3-n)(-tetraphenylcyclobutadiene)cobalt]2-(diphenylphosphino)phenylferrocene - palladium ( 37 ) : 2-(diphenylphosphino)phenylferrocene ( 0.007 g , 0.016 mmol ) was added to a solution of ( sp)-11 ( 0.010 g , 0.015 mmol ) in ch2cl2 ( 1 ml ) and the solution was stirred for 16 h. the solvent was removed in vacuo and the product was purified by column chromatography ( sio2 , 49:1 ch2cl2/meoh ) to give the product ( sp)-35 as a red / orange solid ( 0.015 g , 88 % ) .

angiotensin ii ( ang ii ) is the active end - product of the renin angiotensin system ( ras ) . in the classical view , ang ii is produced from angiotensinogen through a series of proteolytic cleavage events , conducted successively by renin , followed by angiotensin - converting enzyme ( for review see de gasparo et al . however , in addition to this classical ras , several alternative pathways have been identified , for which description is out of the scope of this review , but recently reviewed by de kloet et al . ang ii is a peripheral hormone , as well as a neuropeptide , which plays a major role in the central regulation of blood pressure and in the stress response . indeed , since the pioneer studies of mendelsohn et al . ( 1988 ) , the existence of a ras in the brain is now well established . the various components ( angiotensinogen , renin , angiotensin - converting enzyme , ang ii , and ang ii receptors ) are found in areas of the brain involved in the regulation of fluid and electrolyte balance , in the regulation of arterial pressure and in structures involved in cognition , behavior , and locomotion ( for review see phillips and de oliveira , 2008 ; horiuchi et al . , 2010 ) . angiotensin ii binds two major receptors : the ang ii type 1 receptor ( at1r ) and the type 2 receptor ( at2r ) . since ang ii modulates blood pressure and the stress response by binding the at1r , at1r blockers ( arbs ) have been widely used as antihypertensive drugs . there is also evidence that arb treatment attenuates learning and memory deficits , increases cerebral blood flow , and helps protect against brain ischemia and inflammation ( li et al . , 2005 ; zhou et al . , 2006 ; phillips and de oliveira , 2008 ; mogi and horiuchi , 2009 ; sakata et al . , 2009 ; horiuchi et al . , 2010 ) . in the presence of arbs , which selectively block the at1r , ang ii binds to the less - abundant at2r . several studies suggest that the therapeutic effects of arbs may reflect this unopposed activation of the at2r as well as the inhibition of the at1r ( li et al . , , 2007 , 2009 ; arganaraz et al . , 2008 ; gao et al . , 2008 ; mccarthy et al . , 2009 ) . within the context of alzheimer s disease ( ad ) , modulation of at2r signaling could improve cognitive performance not only through its action on blood flow / brain microcirculation but also through more specific effects on neurons . this review summarizes the current state of knowledge and potential therapeutic relevance of central actions of this enigmatic receptor . one of the most striking features of the at2r is its high level of expression in most fetal tissues , including the brain , and the dramatic increase in the at1/at2 receptor ratio after birth ( millan et al . , 1991 ; tsutsumi and saavedra , 1991 ) . this pattern of expression strongly implicates the at2r in fetal development . in the adult , the at2r is predominantly expressed in the locus coeruleus , ventral and dorsal parts of lateral septum , superior colliculus and subthalamic nucleus , many nuclei of the thalamus , and nuclei of the inferior olive . the cingulate cortex , the molecular layer of the cerebellar cortex , the superior colliculus and paraventricular nuclei contain both at1 and at2 receptors ( millan et al . , 1991 ; tsutsumi and saavedra , 1991 ; lenkei et al . , 1996 , 1997 ) . more recent studies have also identified at2r rna and protein in the substantia nigra pars compacta ( grammatopoulos et al . , 2007 ) and in the hippocampus ( arganaraz et al . thus , in the adult , the at2r are concentrated in areas involved in control and learning of motor activity , sensory areas , and selected limbic system structures . at the cellular level , the at2r is localized in neurons but not in astrocytes ( bottari et al . , 1992 ; it is well - accepted that at2r stimulation counteracts most at1r - mediated actions , promoting vasodilation , inhibiting growth , decreasing the expression of pro - inflammatory cytokines , and increasing expression of anti - inflammatory cytokines , both in the brain and in the cardiovascular system ( for reviews see gendron et al . , 2003b ; mogi et al . , 2007a , 2008 ; arganaraz et al . , 2008 ; mogi and horiuchi , 2009 ; porrello et al . , 2009 ; sakata et al . , 2009 ; summary of the properties and main effects targeted by at1 and at2 receptors of angiotensin ii in the brain . amyloid- ( a ) peptide deposition in senile plaques and the presence of neurofibrillary tangles ( nfts ) are the main pathological hallmarks of ad . however , other structural and functional alterations , including inflammation , increased oxidative stress , and vascular damage / ischemia , are also associated with ad ; these alterations may contribute to neuronal and synaptic dysfunction and loss as well as the ensuing cognitive deficits and dementia of this disorder ( iadecola , 2004 ; zlokovic , 2005 ; laferla et al . , 2007 ; boissonneault et al . , 2009 ; mucke , 2009 ; nelson et al . , 2009 ) . in addition , since the development of the amyloid hypothesis of hardy and selkoe ( 2002 ) , evidence strongly suggests that soluble oligomers of a may cause early cognitive impairment , even in the absence of overt cell death ( review in mucke et al . , 2000 ; , 2006 ; haass and selkoe , 2007 ; selkoe , 2008 ; wray and noble , 2009 ) . the abnormal hyperphosphorylation and altered conformation of the microtubule - associated protein ( map ) tau precedes its assembly into paired helical filaments and its accumulation in nfts ( buee et al . , 2000 ; tau is a substrate for several protein kinases , such as glycogen synthase kinase-3 ( gsk-3 ) and cyclin - dependent kinase ( cdk5 ) , and for phosphatases such as protein phosphatase-2a ( pp2a ) ; pp2a activity is down - regulated in the ad brain ( buee et al . , 2000 ; andorfer et al . , 2003 ; gallo et al . , 2007 ; hernandez and avila , 2008 ; hanger et al . , 2009 ; iqbal et al . , 2009 ; hernndez et al . , moreover , experimental evidence suggests that cerebral perfusion is decreased in ad ( farkas and luiten , 2001 ; de la torre , 2004 ; zlokovic , 2008 ; liu et al . , 2009 ) . indeed , the structural and functional integrity of the brain depends on the delicate balance between substrate delivery through blood flow and energy demands imposed by neural activity . neurons , astrocytes and vascular cells seemingly constitute a functional unit , the primary purpose of which is to maintain the homeostasis of the brain s microenvironment . the emerging view is that cerebrovascular dysregulation is a feature not only of cerebrovascular pathologies such as stroke , but also of neurodegenerative conditions such as ad ( iadecola et al . , 2009 ) . since studies suggest that a has deleterious actions both on neurons and cerebral blood vessels , the neuronal and vascular actions of this peptide may act synergistically to induce brain dysfunction in ad ( iadecola , 2004 ; zlokovic , 2005 ) . recent studies have revealed that aging , hypertension , and ad trigger common signaling pathways that lead to deleterious effects on the regulation of the cerebral circulation . these findings reinforce the notion that cerebrovascular dysfunction plays a key role in the cognitive impairment associated with these conditions ( iadecola et al . , 2009 ) . in the entire trademark dysfunctions associated with ad mentioned above , there is several indirect lines of evidence suggesting that at2 receptor activation may have a beneficial effect ( de la torre , 2004 ) . all of the components of the ras are found in the brain , where they actively modulate functions such as stress ( saavedra et al . , 2005 ; saavedra and benicky , 2007 ) , exploratory behavior , anxiety , learning , and memory acquisition ( wright et al . , 2002 ; phillips and de oliveira , 2008 ) . both the at1r and the at2r have been detected in brain areas responsible for these functions , including the amygdala , hippocampus , lateral septum , and frontal cortex ( song et al . , 1991 , 1992 ; lenkei et al . , 1996 ; phillips and sumners , 1998 ; arganaraz et al . , 2008 ; abdalla et al . , 2009 ) . the initial studies indicating a role of at2r in cognitive improvement arise from observations in at2r - deficient mice . the targeted disruption of the agtr2 gene ( which codes for the at2r ) resulted not only in a significant increase in blood pressure , but also in attenuated exploratory behavior and impaired performance in a spatial memory task ( hein et al . , 1995 ; ichiki et al . , 1995 ; okuyama et al . , 1999 ; several recent studies have indicated a beneficial role for arbs in the cognitive impairment associated with vascular diseases , ad , and other neurodegenerative diseases ( phillips and de oliveira , 2008 ; fujita et al . , 2009 ; mogi and horiuchi , 2009 ) . for instance , treatment with the arb valsartan attenuates oligomerization of a peptides into high molecular weight oligomeric peptides and reduces cognitive deterioration in tg2576 mice , a model of ad - type neuropathology that expresses a pathogenic mutant of the amyloid precursor protein ( app ; wang et al . , 2007 ) . on the other hand , other studies with the same model ( tg2576 mice ) have shown that a induces the formation of cross - linked at2r oligomers in the hippocampus that disrupt ang ii signaling . conversely , stereotactic inhibition of at2r oligomers by rna interference delayed tau phosphorylation in tg2576 ( abdalla et al . , 2009 ) . numerous studies suggest that the beneficial cellular effects of the at2r result in improved physiological parameters relevant to ad patients : ang ii type 2 receptor activation promotes vasodilation and the anti - inflammatory process considerable evidence suggests that at1r blockade and increased at2r stimulation improve cerebral blood flow , thereby helping to protect against brain ischemia and inflammation ( iwai et al . , 2004 ; li et al . 2009 ) , and , moreover , that at2r activation improves the microcirculation ( for reviews , see phillips and de oliveira , 2008 ; horiuchi et al . , 2010 ) . numerous recent studies conducted in rodents treated with arbs suggest that at2r protects against cerebral ischemia - induced neuronal injury ( grammatopoulos et al . , 2004 ; , 2007 , 2009 ; mccarthy et al . , 2009 ) , and altered dendritic and neuronal spine morphology ( maul et al . , 2008 ; for review see mogi and horiuchi , 2009 ) . confirming these observations , it has been reported that at2r stimulation supports neuronal survival and neurite outgrowth in response to ischemia - induced neuronal injury ( li et al . , 2005 ; sakata et al . , are observations from models of nerve injury which have elegantly shown that the at2r has regenerative capabilities associated with restored behavioral function and anatomic innervation after sciatic nerve crush and optical axotomy ( gallinat et al . , 1998 ; lucius et al . , 1998 ; ( 2008 ) , neurotrophic factors [ such as nerve growth factor ( ngf ) and brain - derived neurotrophic factor ] are key regulators not only for development , maintenance and survival but also for cognition and storage of memory . they activate various cell signaling pathways acting through the tropomyosin - related kinase or tyrosine receptor kinase family ( trk ) . most neurodegenerative dementias are linked to failures in axonal transport of neurotrophic factors from the cell body ( where they are synthesized ) to their sites of action . for example , in the absence of ngf , morphology and functions of cholinergic neurons are impaired , resulting in a decrease in cholinergic transmission . in this context , we have shown that at2r - mediated effects in vitro are modulated by the presence of growth factors in the culture medium , and are mediated by growth factor - related signaling pathways . in particular , the signaling mechanisms leading to neurite outgrowth in ng108 - 15 cells involves the trka - mediated activation of rap1/b - raf , which in turn , activates mek to induce a delayed but sustained activation of p42/p44 ( gendron et al . alzheimer s disease as a consequence of hypertension and metabolic syndrome : a role for at2r ? several recent studies indicate that arbs may protect against the cognitive impairments associated with vascular disease , ad , and other neurodegenerative diseases ( mogi and horiuchi , 2009 ) . for example , pretreatment with a non - hypotensive dose of telmisartan significantly inhibits the cognitive decline induced by intracerebroventricular ( i.c.v . ) 2008 ) , and also prevents a deposition in ad models ( mogi et al . , 2006 , 2007b , 2008 ; tsukuda et al . , 2007 , 2009 ) figure 2 provides a general overview of the functional changes of ad and how at2r could help recover some of these changes . type 2 diabetes mellitus ( t2 dm ) , hypertension and metabolic syndrome ( which is defined as a cluster of obesity , high blood pressure , hyperglycemia , and insulin resistance ) are associated with an increased risk of dementia ( both ad and vascular dementia ) ( yaffe et al . , 2004a , b ; biessels and kappelle , 2005 ; qiu et al . , 2005 ; , 2006 ; de la monte , 2009 ; mogi and horiuchi , 2009 ) . in t2 dm patients , a major clinical study ( study on cognition and prognosis in the elderly , scope ; lithell et al . , 2003 ) and a clinical double - blind study ( tedesco et al . , 1999 ) have indicated that arbs have a further therapeutic effect on impaired cognitive function beyond their antihypertensive effects compared with other antihypertensive drugs . in this context , tsukuda et al . have demonstrated that candesartan improves the impaired cognitive function induced by t2 dm , with multiple beneficial effects ( tsukuda et al . alzheimer s disease : targets of functional disruptions and proposed protective functions associated with the activation of the at2 receptor of angiotensin ii . during the past 5 years , significant progress has been achieved in elucidating some of the puzzling elements of the at2r - signaling pathway proteins ( gendron et al . , 2003a ; mogi and horiuchi , 2009 ; porrello et al . , 2009 ; steckelings et al . , 2010a ; figure 3 ) some of these elements may be linked to improvement of impaired signaling functions as observed in ad : main signaling pathways for the at2 receptor of angiotensin ii in the brain . ang ii type 2 receptor may improve synaptic plasticity through effects on ionic channel activity , since at2r activation decreases t - type calcium channel activity , increases k channel activity ( kang et al . , 1992 , 1993 ; buisson et al . , 1995 ) , and alters actin cytoskeleton dynamics ( kilian et al . , 2008 ) . indeed , several studies have reported that at2r activates pp2a phosphatase ( huang et al . , 1995 , 1996a,2008 ; kilian et al . , 2008 ) . pp2a is markedly deficient in ad , and responsible for a sustained increase in erk1/erk2 , one of the kinases involved in glycogen synthase kinase-3 ( gsk-3 ) inactivation . we have also shown that activity of fyn , a src - family kinase member , is required for at2r - induced neurite outgrowth ( guimond et al . , 2010a ) . since tau is a substrate for both pp2a phosphatases , gsk-3 and fyn , at2r activation may control the equilibrium between tau phosphorylation and dephosphorylation ( hernandez and avila , 2008 ; hanger et al . ang ii type 2 receptor may also improve neurite architecture , through effects on maps , as shown in neuronal cell lines ( laflamme et al . , 1996 ; meffert et al . , 1996 ; ct et al . , 1999 ; through methyl methanesulfonate sensitive 2 ( mms2 ; mogi et al . , 2007a ) and peroxisome proliferator - activated receptor ( ppar ; mogi et al . , 2008 ) , the at2r improves cognitive function and the decrease in hippocampal neurogenesis observed in amyloid--injection - induced cognitive decline ( mogi et al . , 2008 ) or in at2r - deficient mice ( mogi et al . , 2007a ; for review , see mogi and horiuchi , 2009 ; horiuchi et al . , 2010 ) . ang ii type 2 receptor activation may counteract vasoconstriction , and favor vasodilation / vasorelaxation , through an increase in nitric oxide ( no)cgmp production and a decrease in superoxide production , nadph oxidase superoxide production , and nadph oxidase ( reviewed in volpe et al . ang ii type 2 receptor activation by cgp42112 increases neuronal survival and minimizes experimental post - stroke injury ( mccarthy et al . , 2009 ) , indicating that centrally administered cgp42112 exhibits a neuroprotective effect . such protective effects may be consecutive to an increase in nitric oxide ( no)cgmp production and a decrease in superoxide production and nadph oxidase superoxide production and nadph oxidase ( de la torre , 2004 ; iadecola et al . , 2009 ) or to decreased inflammation . indeed , at2r attenuates chemical hypoxia - induced caspase-3 activation in primary cortical neuronal cultures ( grammatopoulos et al . , 2004 ) . as recently reviewed ( rompe et al . , 2010 ; stegbauer and coffman , 2011 ) , at2r activation , as in other inflammatory models , may decrease tumor necrosis factor - alpha ( tnf- ) and nf - kb ( nuclear factor kappa - light - chain - enhancer of activated b cells ) activity , resulting in decreased production of interleukin 6 ( il-6 ) . this effect is initiated through increased activation of protein phosphatases and increased synthesis of epoxyeicosatrienoic acid ( rompe et al . , 2010 ) . nevertheless , certain contradictory studies suggest that at2r expression and conformation may change with age and may be associated with some of the deleterious changes in ad ( kerr et al . , 2005 ; these conflicting hypotheses have been difficult to reconcile because of experimental limitations , particularly the lack of an orally active , selective ligand for the at2r , as discussed in the next section . moreover , the earliest events associated with activation of at2r and the contribution of at2r signaling to cognitive decline remains unclear . other advances in the field of at2r signaling include the identification of direct intracellular partners , including the phosphatase shp-1 ( cui et al . , 2001 ; feng et al . , li et al . , 2007b ) , the transcription factor promyelocytic zinc finger protein , plzf ( senbonmatsu et al . , 2003 ) and the at2 receptor - interacting protein ( atip ) , also called at2r binding protein of 50 kda ( atbp50 ; nouet et al . , 2004 ; wruck et al . , 2005 ; reviewed in mogi et al . , 2007a ; funke - kaiser et al . , 2010 ; rodrigues - ferreira and nahmias , 2010 ; moreover , recent studies have identified intracellular crosstalk pathways between the at1r and the at2r at the gene expression level . indeed , at1r activation enhances at2r mrna degradation , but at2r activation increases at2r mrna transcription ( shibata et al . , 1997 ) . as previously mentioned , the identification of at2r - specific actions has been hampered by the absence of appropriate selective ligands . until recently , cgp42112a was the only at2r agonist available , but it also acted as an antagonist at high concentrations ( dubey et al . , 1998 ; martineau et al . , 1999 ; ruiz - ortega et al . , 2000 ; fabiani et al . , furthermore , due to its peptidic nature , cgp42112a could not be used readily in in vivo studies . ( 2010a , b ) and unger and dahlof ( 2010 ) , have characterized the properties of several non - peptidic compounds derived from the prototype non - selective at1/at2 receptor agonist l-162,313 ( wan et al . , 2004 ; georgsson et al . , 2005 , 2006 one of these ligands , the m24 compound ( originally called c21 ; wan et al . , 2004 ; georgsson et al . , 2007 ) , exhibits high affinity for the at2r ( 0.4 nm ) , but very low affinity for the at1r ( > 10,000 nm ) and acts as an at2r agonist ( wan et al . , 2004 ) . using a neuronal / glioma cell line ( a variant of ng108 - 15 cells expressing only the at2r ) , we found that c21/m24 stimulates neurite outgrowth through sustained activation of p42/p44 , as observed with ang ii or cgp42112a ( wan et al . , 2004 ) . in addition , c21/m24 also decreases cell proliferation in ng108 - 15 cells , as does cgp42112a . in addition to our results , others have found that c21/m24 lowered mean arterial blood pressure in hypertensive rats ( wan et al . , 2004 ; gelosa et al . , 2009 ; bosnyak et al . , 2010 ) , improved ventricular function in a model of rat myocardial infarction ( kaschina et al . , 2009 ) , and corrected several intracellular perturbations and pro - inflammatory conditions ( kaschina et al . thus , c21/m24 is the most selective at2r agonist available to date and represents a unique tool to delineate the specific roles of at2r in different cellular and animal models ( steckelings et al . , 2010a ; unger and dahlof , 2010 ; recently reviewed in steckelings et al . , the next challenge is now to verify whether at2r activation by c21/m24 could rescue or improve cognitive performance . to answer this question , we have induced learning deficiency by a 2-week treatment with intracerebral injection of amyloid- ( a ) . key findings from our preliminary experiments are that selective at2r activation by c21/m24 attenuates the learning disturbance in the y - maze and water - maze tasks more efficiently than at1r blockade by losartan ( guimond et al . , 2010b and unpublished results ) . it is indeed well documented that a treatment significantly induces a significant learning disturbance in the y - maze and water - maze tasks , in addition to resulting in moderate neuronal loss and promoting amyloid deposition in the cortex and hippocampus ( yamaguchi and kawashima , 2001 ; tajima et al . , 2005 ; mogi et al . , 2006 , 2008 ; liu et al . , 2009 ; klyubin et al . , 2011 ; srivareerat et al . , 2011 ) . from the previous results demonstrating that at2r stimulation modulates phosphorylation of maps , including map2 and map1b or tau , as well as modulates interactions between maps and microtubules ( laflamme et al . , 1996 ; meffert et al . , 1996 ; ct et al . , 1999 ; , it appears therefore that elucidating the signaling mechanisms linking at2r activation and cytoskeletal remodeling is key to understanding the cognitive roles of the at2r in hippocampal neurons . based on the current paradigms of at1r / at2r function , one aspect of at1r / at2r regulation is particularly intriguing . could the age - related shift in the relative expression of the at1r and at2r , in which at1r expression increases and at2r expression decreases , explain some cellular aspects of aging , especially those relating to altered cell number ( von bohlen und halbach et al . , 2001 ) ? poor cognitive performance in ad significantly impairs social interaction and the quality of life of patients . therefore any treatment aimed at improving cognitive functions is likely to slow down symptoms and improve quality of life . an estimated 33 million elderly persons worldwide suffer from dementia , and this number is expected to reach 81.1 million by 2040 ( ferri et al . , 2005 ; source : rising tide : the impact of dementia on canadian society , a report of the alzheimer s society of canada ) . life style - related disorders , such as hypertension , diabetes mellitus , and obesity have moreover been implicated as risk factors for dementia ( yaffe et al . , 2004a , b ; biessels and kappelle , 2005 ; qiu et al . , 2005 ; whitmer et al . , 2005 ; , 2006 ; de la monte , 2009 ; mogi and horiuchi , 2009 ) . as described in the previous sections , at2r activation may act at several locations in the cascade of alterations leading to cognitive impairment and neuronal dysfunction observed in ad . in particular , at2r may act not only at the neuronal level , but also on vasculature and on inflammation associated with alzheimer s . as outlined in this review , an increasing number of studies suggest that the protective effects of angiotensin ii ( at1 ) receptor blockers on brain damage and cognition may result not only from the inhibition of at1r effects , but also from the beneficial effect due to unopposed activation of at2r . in addition , the relationship between impaired energy metabolism / obesity / insulin resistance and the increased risk of dementia ( both ad and vascular dementia ; yaffe et al . , 2004a , b ; qiu et al . , 2005 ; whitmer et al . , 2005 ; mogi and horiuchi , 2009 ) emphasizes that all the mechanisms by which at2r acts may have a beneficial protective effect . if further research confirms the promising early results , the neuroprotective effect of central at2r stimulation with the recently developed c21/m24 , a non - peptide , selective at2r agonist , may thus represent a new pharmacological tool in ad and others neurological cognitive disorders . in addition , unraveling the underlying effects of the at2r on neuronal plasticity may lead to the development of even more selective therapies . 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 .

amyloid- peptide deposition , abnormal hyperphosphorylation of tau , as well as inflammation and vascular damage , are associated with the development of alzheimer s disease ( ad ) . angiotensin ii ( ang ii ) is a peripheral hormone , as well as a neuropeptide , which binds two major receptors , namely the ang ii type 1 receptor ( at1r ) and the type 2 receptor ( at2r ) . activation of the at2r counteracts most of the at1r - mediated actions , promoting vasodilation , decreasing the expression of pro - inflammatory cytokines , both in the brain and in the cardiovascular system . there is evidence that treatment with at1r blockers ( arbs ) attenuates learning and memory deficits . studies suggest that the therapeutic effects of arbs may reflect this unopposed activation of the at2r in addition to the inhibition of the at1r . within the context of ad , modulation of at2r signaling could improve cognitive performance not only through its action on blood flow / brain microcirculation but also through more specific effects on neurons . this review summarizes the current state of knowledge and potential therapeutic relevance of central actions of this enigmatic receptor . in particular , we highlight the possibility that selective at2r activation by non - peptide and highly selective agonists , acting on neuronal plasticity , could represent new pharmacological tools that may help improve impaired cognitive performance in ad and other neurological cognitive disorders .

Introduction Expression and Roles of the AT2R in the Brain Is There a Link between AT2R Activation and AD? Linking Signaling and Function: Always a Difficult Challenge! The AT2R: Previous Limitations and New Perspectives Conclusion Relevance to Alzheimers Disease and Related Dementias Conflict of Interest Statement

angiotensin ii ( ang ii ) is the active end - product of the renin angiotensin system ( ras ) . in the classical view , ang ii is produced from angiotensinogen through a series of proteolytic cleavage events , conducted successively by renin , followed by angiotensin - converting enzyme ( for review see de gasparo et al . however , in addition to this classical ras , several alternative pathways have been identified , for which description is out of the scope of this review , but recently reviewed by de kloet et al . ang ii is a peripheral hormone , as well as a neuropeptide , which plays a major role in the central regulation of blood pressure and in the stress response . ( 1988 ) , the existence of a ras in the brain is now well established . the various components ( angiotensinogen , renin , angiotensin - converting enzyme , ang ii , and ang ii receptors ) are found in areas of the brain involved in the regulation of fluid and electrolyte balance , in the regulation of arterial pressure and in structures involved in cognition , behavior , and locomotion ( for review see phillips and de oliveira , 2008 ; horiuchi et al . angiotensin ii binds two major receptors : the ang ii type 1 receptor ( at1r ) and the type 2 receptor ( at2r ) . since ang ii modulates blood pressure and the stress response by binding the at1r , at1r blockers ( arbs ) have been widely used as antihypertensive drugs . there is also evidence that arb treatment attenuates learning and memory deficits , increases cerebral blood flow , and helps protect against brain ischemia and inflammation ( li et al . in the presence of arbs , which selectively block the at1r , ang ii binds to the less - abundant at2r . several studies suggest that the therapeutic effects of arbs may reflect this unopposed activation of the at2r as well as the inhibition of the at1r ( li et al . within the context of alzheimer s disease ( ad ) , modulation of at2r signaling could improve cognitive performance not only through its action on blood flow / brain microcirculation but also through more specific effects on neurons . this review summarizes the current state of knowledge and potential therapeutic relevance of central actions of this enigmatic receptor . one of the most striking features of the at2r is its high level of expression in most fetal tissues , including the brain , and the dramatic increase in the at1/at2 receptor ratio after birth ( millan et al . this pattern of expression strongly implicates the at2r in fetal development . in the adult , the at2r is predominantly expressed in the locus coeruleus , ventral and dorsal parts of lateral septum , superior colliculus and subthalamic nucleus , many nuclei of the thalamus , and nuclei of the inferior olive . more recent studies have also identified at2r rna and protein in the substantia nigra pars compacta ( grammatopoulos et al . , 2007 ) and in the hippocampus ( arganaraz et al . thus , in the adult , the at2r are concentrated in areas involved in control and learning of motor activity , sensory areas , and selected limbic system structures . , 1992 ; it is well - accepted that at2r stimulation counteracts most at1r - mediated actions , promoting vasodilation , inhibiting growth , decreasing the expression of pro - inflammatory cytokines , and increasing expression of anti - inflammatory cytokines , both in the brain and in the cardiovascular system ( for reviews see gendron et al . , 2009 ; summary of the properties and main effects targeted by at1 and at2 receptors of angiotensin ii in the brain . amyloid- ( a ) peptide deposition in senile plaques and the presence of neurofibrillary tangles ( nfts ) are the main pathological hallmarks of ad . however , other structural and functional alterations , including inflammation , increased oxidative stress , and vascular damage / ischemia , are also associated with ad ; these alterations may contribute to neuronal and synaptic dysfunction and loss as well as the ensuing cognitive deficits and dementia of this disorder ( iadecola , 2004 ; zlokovic , 2005 ; laferla et al . in addition , since the development of the amyloid hypothesis of hardy and selkoe ( 2002 ) , evidence strongly suggests that soluble oligomers of a may cause early cognitive impairment , even in the absence of overt cell death ( review in mucke et al . the abnormal hyperphosphorylation and altered conformation of the microtubule - associated protein ( map ) tau precedes its assembly into paired helical filaments and its accumulation in nfts ( buee et al . , 2000 ; tau is a substrate for several protein kinases , such as glycogen synthase kinase-3 ( gsk-3 ) and cyclin - dependent kinase ( cdk5 ) , and for phosphatases such as protein phosphatase-2a ( pp2a ) ; pp2a activity is down - regulated in the ad brain ( buee et al . , moreover , experimental evidence suggests that cerebral perfusion is decreased in ad ( farkas and luiten , 2001 ; de la torre , 2004 ; zlokovic , 2008 ; liu et al . indeed , the structural and functional integrity of the brain depends on the delicate balance between substrate delivery through blood flow and energy demands imposed by neural activity . neurons , astrocytes and vascular cells seemingly constitute a functional unit , the primary purpose of which is to maintain the homeostasis of the brain s microenvironment . the emerging view is that cerebrovascular dysregulation is a feature not only of cerebrovascular pathologies such as stroke , but also of neurodegenerative conditions such as ad ( iadecola et al . since studies suggest that a has deleterious actions both on neurons and cerebral blood vessels , the neuronal and vascular actions of this peptide may act synergistically to induce brain dysfunction in ad ( iadecola , 2004 ; zlokovic , 2005 ) . recent studies have revealed that aging , hypertension , and ad trigger common signaling pathways that lead to deleterious effects on the regulation of the cerebral circulation . these findings reinforce the notion that cerebrovascular dysfunction plays a key role in the cognitive impairment associated with these conditions ( iadecola et al . in the entire trademark dysfunctions associated with ad mentioned above , there is several indirect lines of evidence suggesting that at2 receptor activation may have a beneficial effect ( de la torre , 2004 ) . all of the components of the ras are found in the brain , where they actively modulate functions such as stress ( saavedra et al . both the at1r and the at2r have been detected in brain areas responsible for these functions , including the amygdala , hippocampus , lateral septum , and frontal cortex ( song et al . the initial studies indicating a role of at2r in cognitive improvement arise from observations in at2r - deficient mice . the targeted disruption of the agtr2 gene ( which codes for the at2r ) resulted not only in a significant increase in blood pressure , but also in attenuated exploratory behavior and impaired performance in a spatial memory task ( hein et al . , 1999 ; several recent studies have indicated a beneficial role for arbs in the cognitive impairment associated with vascular diseases , ad , and other neurodegenerative diseases ( phillips and de oliveira , 2008 ; fujita et al . for instance , treatment with the arb valsartan attenuates oligomerization of a peptides into high molecular weight oligomeric peptides and reduces cognitive deterioration in tg2576 mice , a model of ad - type neuropathology that expresses a pathogenic mutant of the amyloid precursor protein ( app ; wang et al . on the other hand , other studies with the same model ( tg2576 mice ) have shown that a induces the formation of cross - linked at2r oligomers in the hippocampus that disrupt ang ii signaling . conversely , stereotactic inhibition of at2r oligomers by rna interference delayed tau phosphorylation in tg2576 ( abdalla et al . numerous studies suggest that the beneficial cellular effects of the at2r result in improved physiological parameters relevant to ad patients : ang ii type 2 receptor activation promotes vasodilation and the anti - inflammatory process considerable evidence suggests that at1r blockade and increased at2r stimulation improve cerebral blood flow , thereby helping to protect against brain ischemia and inflammation ( iwai et al . numerous recent studies conducted in rodents treated with arbs suggest that at2r protects against cerebral ischemia - induced neuronal injury ( grammatopoulos et al . , are observations from models of nerve injury which have elegantly shown that the at2r has regenerative capabilities associated with restored behavioral function and anatomic innervation after sciatic nerve crush and optical axotomy ( gallinat et al . , 1998 ; ( 2008 ) , neurotrophic factors [ such as nerve growth factor ( ngf ) and brain - derived neurotrophic factor ] are key regulators not only for development , maintenance and survival but also for cognition and storage of memory . for example , in the absence of ngf , morphology and functions of cholinergic neurons are impaired , resulting in a decrease in cholinergic transmission . in this context , we have shown that at2r - mediated effects in vitro are modulated by the presence of growth factors in the culture medium , and are mediated by growth factor - related signaling pathways . in particular , the signaling mechanisms leading to neurite outgrowth in ng108 - 15 cells involves the trka - mediated activation of rap1/b - raf , which in turn , activates mek to induce a delayed but sustained activation of p42/p44 ( gendron et al . alzheimer s disease as a consequence of hypertension and metabolic syndrome : a role for at2r ? several recent studies indicate that arbs may protect against the cognitive impairments associated with vascular disease , ad , and other neurodegenerative diseases ( mogi and horiuchi , 2009 ) . 2008 ) , and also prevents a deposition in ad models ( mogi et al . , 2007 , 2009 ) figure 2 provides a general overview of the functional changes of ad and how at2r could help recover some of these changes . type 2 diabetes mellitus ( t2 dm ) , hypertension and metabolic syndrome ( which is defined as a cluster of obesity , high blood pressure , hyperglycemia , and insulin resistance ) are associated with an increased risk of dementia ( both ad and vascular dementia ) ( yaffe et al . in t2 dm patients , a major clinical study ( study on cognition and prognosis in the elderly , scope ; lithell et al . , 1999 ) have indicated that arbs have a further therapeutic effect on impaired cognitive function beyond their antihypertensive effects compared with other antihypertensive drugs . alzheimer s disease : targets of functional disruptions and proposed protective functions associated with the activation of the at2 receptor of angiotensin ii . during the past 5 years , significant progress has been achieved in elucidating some of the puzzling elements of the at2r - signaling pathway proteins ( gendron et al . , 2010a ; figure 3 ) some of these elements may be linked to improvement of impaired signaling functions as observed in ad : main signaling pathways for the at2 receptor of angiotensin ii in the brain . ang ii type 2 receptor may improve synaptic plasticity through effects on ionic channel activity , since at2r activation decreases t - type calcium channel activity , increases k channel activity ( kang et al . pp2a is markedly deficient in ad , and responsible for a sustained increase in erk1/erk2 , one of the kinases involved in glycogen synthase kinase-3 ( gsk-3 ) inactivation . since tau is a substrate for both pp2a phosphatases , gsk-3 and fyn , at2r activation may control the equilibrium between tau phosphorylation and dephosphorylation ( hernandez and avila , 2008 ; hanger et al . ang ii type 2 receptor may also improve neurite architecture , through effects on maps , as shown in neuronal cell lines ( laflamme et al . , 2007a ) and peroxisome proliferator - activated receptor ( ppar ; mogi et al . , 2008 ) , the at2r improves cognitive function and the decrease in hippocampal neurogenesis observed in amyloid--injection - induced cognitive decline ( mogi et al . ang ii type 2 receptor activation may counteract vasoconstriction , and favor vasodilation / vasorelaxation , through an increase in nitric oxide ( no)cgmp production and a decrease in superoxide production , nadph oxidase superoxide production , and nadph oxidase ( reviewed in volpe et al . ang ii type 2 receptor activation by cgp42112 increases neuronal survival and minimizes experimental post - stroke injury ( mccarthy et al . , 2010 ; stegbauer and coffman , 2011 ) , at2r activation , as in other inflammatory models , may decrease tumor necrosis factor - alpha ( tnf- ) and nf - kb ( nuclear factor kappa - light - chain - enhancer of activated b cells ) activity , resulting in decreased production of interleukin 6 ( il-6 ) . nevertheless , certain contradictory studies suggest that at2r expression and conformation may change with age and may be associated with some of the deleterious changes in ad ( kerr et al . , 2005 ; these conflicting hypotheses have been difficult to reconcile because of experimental limitations , particularly the lack of an orally active , selective ligand for the at2r , as discussed in the next section . moreover , the earliest events associated with activation of at2r and the contribution of at2r signaling to cognitive decline remains unclear . other advances in the field of at2r signaling include the identification of direct intracellular partners , including the phosphatase shp-1 ( cui et al . , 2003 ) and the at2 receptor - interacting protein ( atip ) , also called at2r binding protein of 50 kda ( atbp50 ; nouet et al . , 2010 ; rodrigues - ferreira and nahmias , 2010 ; moreover , recent studies have identified intracellular crosstalk pathways between the at1r and the at2r at the gene expression level . as previously mentioned , the identification of at2r - specific actions has been hampered by the absence of appropriate selective ligands . ( 2010a , b ) and unger and dahlof ( 2010 ) , have characterized the properties of several non - peptidic compounds derived from the prototype non - selective at1/at2 receptor agonist l-162,313 ( wan et al . , 2007 ) , exhibits high affinity for the at2r ( 0.4 nm ) , but very low affinity for the at1r ( > 10,000 nm ) and acts as an at2r agonist ( wan et al . using a neuronal / glioma cell line ( a variant of ng108 - 15 cells expressing only the at2r ) , we found that c21/m24 stimulates neurite outgrowth through sustained activation of p42/p44 , as observed with ang ii or cgp42112a ( wan et al . in addition , c21/m24 also decreases cell proliferation in ng108 - 15 cells , as does cgp42112a . in addition to our results , others have found that c21/m24 lowered mean arterial blood pressure in hypertensive rats ( wan et al . , 2009 ) , and corrected several intracellular perturbations and pro - inflammatory conditions ( kaschina et al . thus , c21/m24 is the most selective at2r agonist available to date and represents a unique tool to delineate the specific roles of at2r in different cellular and animal models ( steckelings et al . , the next challenge is now to verify whether at2r activation by c21/m24 could rescue or improve cognitive performance . to answer this question , we have induced learning deficiency by a 2-week treatment with intracerebral injection of amyloid- ( a ) . key findings from our preliminary experiments are that selective at2r activation by c21/m24 attenuates the learning disturbance in the y - maze and water - maze tasks more efficiently than at1r blockade by losartan ( guimond et al . it is indeed well documented that a treatment significantly induces a significant learning disturbance in the y - maze and water - maze tasks , in addition to resulting in moderate neuronal loss and promoting amyloid deposition in the cortex and hippocampus ( yamaguchi and kawashima , 2001 ; tajima et al . from the previous results demonstrating that at2r stimulation modulates phosphorylation of maps , including map2 and map1b or tau , as well as modulates interactions between maps and microtubules ( laflamme et al . , 1999 ; , it appears therefore that elucidating the signaling mechanisms linking at2r activation and cytoskeletal remodeling is key to understanding the cognitive roles of the at2r in hippocampal neurons . based on the current paradigms of at1r / at2r function , one aspect of at1r / at2r regulation is particularly intriguing . could the age - related shift in the relative expression of the at1r and at2r , in which at1r expression increases and at2r expression decreases , explain some cellular aspects of aging , especially those relating to altered cell number ( von bohlen und halbach et al . poor cognitive performance in ad significantly impairs social interaction and the quality of life of patients . , 2005 ; source : rising tide : the impact of dementia on canadian society , a report of the alzheimer s society of canada ) . as described in the previous sections , at2r activation may act at several locations in the cascade of alterations leading to cognitive impairment and neuronal dysfunction observed in ad . in particular , at2r may act not only at the neuronal level , but also on vasculature and on inflammation associated with alzheimer s . as outlined in this review , an increasing number of studies suggest that the protective effects of angiotensin ii ( at1 ) receptor blockers on brain damage and cognition may result not only from the inhibition of at1r effects , but also from the beneficial effect due to unopposed activation of at2r . in addition , the relationship between impaired energy metabolism / obesity / insulin resistance and the increased risk of dementia ( both ad and vascular dementia ; yaffe et al . if further research confirms the promising early results , the neuroprotective effect of central at2r stimulation with the recently developed c21/m24 , a non - peptide , selective at2r agonist , may thus represent a new pharmacological tool in ad and others neurological cognitive disorders . in addition , unraveling the underlying effects of the at2r on neuronal plasticity may lead to the development of even more selective therapies . 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 .

surfactants normally adsorb to an airwater interface at rates that decrease as the surface tension ( ) falls [ sodium dodecyl sulfate ( sds ) in figure 1 ] . insertion into a more densely occupied surface occurs more slowly as approaches its equilibrium value ( e ) . the adsorption isotherms , however , can reach an inflection point , and during the final approach to e , the slope can become substantially steeper ( figure 1 ) . adsorption is likely to be particularly important in this range of . in the lungs , where pulmonary surfactant exerts its biological function , when the alveolar airwater interface expands during inhalation , the rise of above e is limited . the surfactant vesicles therefore most likely adsorb in the alveolus to an airwater interface that has the at which the acceleration occurs . adsorption of clse to a constant interfacial area . a wilhelmy plate measured the at 37 c after aspirating the surface of a subphase containing vesicles dispersed by sonication in hsc with a phospholipid concentration of 100 m . a control experiment with 7 mm sds at 23 c shows the behavior expected for standard surfactants . the superimposed dashed lines give the smoothed fit to the experimental data that provided the basis for differentiation . ( b ) instantaneous slopes of the adsorption isotherms , found by differentiating the smoothed curves , plotted against . despite the likely importance of the unexpected accelerated decrease in , remarkably little information is available concerning its basic characteristics . the experiments here test a series of hypotheses concerning the basis of the late acceleration . our studies emphasize in particular two fundamental issues : whether the late acceleration reflects a maturation of adsorbing material or changes in the characteristics of the interfacial film ; and the extent to which the hydrophobic proteins , which greatly facilitate the adsorption of the surfactant lipids at all , are necessary and sufficient to cause the late acceleration . dioleoyl phosphatidylcholine ( dopc ) and dioleoyl phosphatidylethanolamine ( dope ) were obtained from avanti polar lipids ( alabaster , al ) and used without further characterization or purification . the following reagents were obtained from commercial sources : sds ( invitrogen , carlsbad , ca ) ; n-2-hydroxyethylpiperazine - n-2-ethanesulfonic acid ( hepes ) ( sigma , st . louis , mo ) ; nacl , cacl2 ; chloroform ; and methanol ( mallinckrodt , hazelwood , mo ) . water was filtered and photo - oxidized with ultraviolet ( uv ) light using a nanopure diamond toc - uv water - purification system ( barnstead / thermolyne , dubuque , la ) . the following buffers were used routinely : 10 mm hepes at ph 7.0 and 150 mm nacl ( hs ) ; and 10 mm hepes at ph 7.0 , 150 mm nacl , and 1.5 mm cacl2 ( hsc ) . the hydrophobic constituents of calf surfactant [ calf lung surfactant extract ( clse ) ] , obtained by extracting(14 ) the constituents of phospholipid aggregates collected by centrifugation from material lavaged from freshly excised calf lungs,(15 ) were provided by dr . subfractions of the constituents in clse were obtained by gel - permeation chromatography using a mobile phase of acidified chloroform / methanol [ 1:1:0.05 ( v / v / v ) chloroform / methanol/0.1 n hcl ] , which separates the hydrophobic surfactant proteins ( sp - b and sp - c ) , the phospholipids , and the neutral lipids ( cholesterol ) into distinct peaks . pooling the appropriate eluted fractions provides preparations containing the complete set of surfactant proteins and phospholipids ( sp&pl ) or the combined neutral and phospholipids ( n&pl ) . previous studies have shown that the phospholipids obtained by this procedure contain the same headgroups(18 ) and acyl constituents(20 ) as the original clse . if the eluting solvent contains no acid , the anionic phospholipids stick to the matrix , allowing collection of a fraction that includes the proteins with modified phospholipids that lack the anionic compounds ( sp&mpl).(18 ) the proteins used in samples with dopc were collected using the neutral solvent . films were formed on the bottom of test tubes by mixing the different constituents in chloroform or chloroform / methanol [ 1:1 ( v / v ) ] and drying the films under a stream of nitrogen before evaporating the remaining solvent at reduced pressures . initial studies concerning the role of the different constituents in clse used vesicles dispersed in aqueous media by probe - sonication of samples on ice . subsequent experiments used material that was suspended initially by gentle vortexing after prolonged hydration , subjected to repeated cycles of freezing and thawing , and then extruded 11 times through a polycarbonate filter with pores of 100 nm ( whatman , maidstone , u.k . ) . extrusions were performed at 45 c for clse and n&pl , and at room temperature ( 23 1 c ) for samples containing purchased lipids . dynamic light scattering ( protein solutions dynapro , wyatt , santa barbara , ca ) determined the hydrodynamic radii of the dispersed vesicles . extruded vesicles of clse had a hydrodynamic radius of 93.8 5.8 nm , while n&pl vesicles were smaller at 49.5 3.9 nm . dope / dopc vesicles had a hydrodynamic radius of 105 32.2 nm , with a greater polydispersity than for the vesicles containing the surfactant constituents . the shape of the bubble viewed along the horizontal axis provides its surface area and .(21 ) the bubbles provided the significant advantage that their surface area could be varied , allowing measurements of adsorption during which was held constant . because the bubbles float against an agarose ceiling , the bubbles had the theoretical disadvantage that some of the airwater interface might be inaccessible to adsorbing vesicles , and that the inaccessible fraction of the interface could change during adsorption when the variation of alters the shape of the bubble . the comparable results obtained with bubbles and with adsorption to an open interface ( see below ) suggested that any shift in accessible surface area made no contribution to our findings . the previously described apparatus(22 ) for containing and monitoring the captive bubble was built from templates provided by dr . jon goerke ( university of california san francisco , san francisco , ca ) . the instrument uses a modification(23 ) of the original instrument constructed by schrch(21 ) and readily allows access for spreading of films at the surface of a 7080 l bubble.(24 ) adsorption was initiated either by injecting small aliquots of concentrated vesicles prepared in hs , in which the absence of ca minimized fusion among vesicles , into a stirred subphase of hsc , or by formation of a bubble in a subphase already containing hsc and vesicles . experiments with preformed monolayers first deposited < 0.05 l of sample in 1:1 ( v / v ) methanol / chloroform to produce a film with the desired . the subphase was then washed with hsc to remove the spreading solvent before injection of vesicular material.(24 ) images obtained along the horizontal axis of the bubble provided its height and diameter , which were used to calculate and surface area using a semi - empirical approach based on the equation of young and laplace.(25 ) programs written in labview ( national instruments , austin , tx ) performed real - time calculations of these variables , which could then be manipulated using a computer - controlled syringe pump to change the size of the bubble by infusing and withdrawing buffer from the enclosed subphase . to simplify the manipulations , most experiments used ambient temperatures ( 23 1 c ) . experiments at 37 c maintained temperature with heating pads ( minco , minneapolis , mn ) applied along the side of the chamber and regulated by a temperature - controller ( cole - palmer , vernon hills , il ) . experiments concerning the different preparations obtained from clse measured adsorption to the airwater interface above a subphase in a teflon cup enclosed within a temperature - regulated chamber containing open water to maintain full humidification . these experiments used a wilhelmy plate composed of filter paper monitored by a force transducer ( riegler and kirstein gmbh , potsdam , germany ) to determine . measurements were calibrated with the moistened plate in air and minimally immersed in the subphase with a clean interface . data were recorded to a computer using programs constructed with the graphical user interface labview . experiments were initiated by aspirating the surface layer to create a clean interface above a subphase containing vesicles dispersed by sonication . the slopes of the isotherms were obtained using the program igor ( wavemetrics , lake oswego , or ) first to smooth the curves using a fourth - order savitskygolay algorithm , followed by differentiation . vesicles of extracted calf surfactant ( clse ) at 37 c lowered at rates that initially decreased in magnitude as adsorption progressed ( figure 1 ) . at 40 mn / m , however , the adsorption isotherms reached an inflection point and the slopes became steeper . control experiments with sds demonstrated the behavior expected for standard surfactants , with falling at progressively slower rates until reaching a constant value ( figure 1 ) . although present in many prior studies of adsorption , the absence of the late acceleration in some previously published reports raised the possibility that the phenomenon reflected some feature of the manner in which the experiments were conducted . we therefore tested the dependence of the late acceleration on the following aspects of our protocol.(a)measurement of : experiments obtained comparable results for adsorption to a captive bubble , the shape of which provided , and to the surface of a subphase in an open cup , using a wilhelmy plate to monitor .(b)initiation of adsorption : with the captive bubble , experiments initiated adsorption by injecting aliquots of concentrated clse into the subphase or by replacing a subphase of buffered electrolyte with a dispersion of clse . with the subphase in an open cup monitored with a wilhelmy plate , adsorption began with either the injection of clse into the subphase or aspiration of the initial film from the surface above homogeneously dispersed clse.(26 ) in each case , the steeper slope was present at the end of adsorption.(c)stirring : in comparison to adsorption from an unstirred subphase , stirring shortened the time to reach any and increased the variation among different experiments . below 35 mn / m , however , the isotherms in the presence and absence of stirring became parallel , with steeper slopes in both cases.(d)dispersion : samples dispersed by sonication or extrusion , both of which yielded vesicles with a hydrodynamic radius of 100 nm for clse , produced comparable results . dispersion by cyclic freezing and thawing , which should produce multilamellar vesicles ( mlvs),(27 ) generated particles with the least evidence of a late acceleration . the adsorption isotherms for mlvs frequently included an inflection point at low , but the subsequent steepening of the slopes was always minimal . the late acceleration therefore most clearly represented a phenomenon of a single bilayer , which presumably is the initial step in the adsorption of a mlv . measurement of : experiments obtained comparable results for adsorption to a captive bubble , the shape of which provided , and to the surface of a subphase in an open cup , using a wilhelmy plate to monitor . initiation of adsorption : with the captive bubble , experiments initiated adsorption by injecting aliquots of concentrated clse into the subphase or by replacing a subphase of buffered electrolyte with a dispersion of clse . with the subphase in an open cup monitored with a wilhelmy plate , adsorption began with either the injection of clse into the subphase or aspiration of the initial film from the surface above homogeneously dispersed clse.(26 ) in each case , the steeper slope was present at the end of adsorption . stirring : in comparison to adsorption from an unstirred subphase , stirring shortened the time to reach any and increased the variation among different experiments . below 35 mn / m , however , the isotherms in the presence and absence of stirring became parallel , with steeper slopes in both cases . dispersion : samples dispersed by sonication or extrusion , both of which yielded vesicles with a hydrodynamic radius of 100 nm for clse , produced comparable results . dispersion by cyclic freezing and thawing , which should produce multilamellar vesicles ( mlvs),(27 ) generated particles with the least evidence of a late acceleration . the adsorption isotherms for mlvs frequently included an inflection point at low , but the subsequent steepening of the slopes was always minimal . the late acceleration therefore most clearly represented a phenomenon of a single bilayer , which presumably is the initial step in the adsorption of a mlv . the steeper slopes at the end of the adsorption isotherm could reflect the relationship between the surface concentration ( ) and rather than a true increase in the rate of adsorption . if the slope of the isotherm became steeper close to e , then at low values , would fall more quickly , despite adsorption at a constant rate . increasing produced by compression , however , showed only the previously reported,(28 ) roughly linear decrease in from shortly after lift - off to the onset of collapse ( figure 2 ) . assuming that had the same relationship to during compression of a monolayer and during adsorption , the late accelerated fall in indicated a faster increase in and more rapid adsorption . monomolecular films of clse were spread on a 0.88 0.04 cm captive bubble to a molecular area just beyond the point at which changes from the value for the clean interface , heated to 37 c , and compressed by decreasing the volume of the bubble at 3 l / min . surface tensions were plotted in terms of both relative surface area ( bottom axis ) and relative surface concentration ( top axis ) . symbols give the mean standard deviation ( sd ) at specific points for five experiments . the late acceleration could reflect changes in the film , achieved at a critical , or changes in the adsorbing material . although the vesicles dispersed in the bulk of the subphase should remain unchanged by the introduction of an interface , material adjacent to the surface might undergo a kinetic transformation that would allow faster adsorption . the two possibilities of changes in the film or in the adsorbing material predicted opposite dependence on and the duration of adsorption . accelerated adsorption that occurred because of changes in the film should depend exclusively upon , regardless of the time needed to reach the critical value . a late acceleration that instead reflected changes in the adsorbing material should become apparent in measurements of sufficient duration , independent of the . adsorption isobars distinguished between kinetic dependence on time and . isobars , obtained at constant , measured rates of adsorption from the change in area , a , necessary to maintain constant.(29 ) assuming that uniquely determined , such that , for n constituents confined within a , where the variable c had a single value at any particular , then for adsorption at constant , the fractional rate of change in area would equal the fractional rate at which constituents adsorb to the interface : if the steeper slopes at the end of the adsorption isotherms reflected a maturation of adsorbing material and the passage of time , then adsorption isobars should all become steeper at later times , regardless of the that was held constant . if instead the steeper isotherms occurred because of the lower , then the semi - logarithmic isobars should have slopes that remain constant . these isobaric slopes should show the same dependence on as the slope of the adsorption isotherms . the isobaric measurements extended to times at which adsorption to a constant surface area demonstrated the late acceleration ( figure 3a ) . changes in the adsorbing material that might have caused the late acceleration in the experiments with a constant surface area should have occurred during the isobaric measurements . after the chamber reached its minimum hydrostatic pressure , which prevented further expansion to maintain isobaric conditions , the experiments measured the fall in during adsorption to a constant area ( figure 3a ) . the isotherms after the isobaric adsorption paralleled the curves obtained during adsorption to an area that was fixed throughout the entire experiment , demonstrating the same late increase in slope . both the constant isobaric rates and the parallel final segments of the isotherms , despite interruption of the fall in by a prolonged isobaric interval , argued that the late acceleration occurred when the films reached a critical rather than when time achieved a critical duration required for maturation of the adsorbing material . extruded vesicles of clse in hs were injected into a stirred subphase of hsc below captive bubbles at 23 1 c to achieve a final concentration of 250 m phospholipid . the surface area was held constant , either for the duration of the experiment ( continuous isotherm ) or until reached 37 mn / m , which was then held constant ( with isobaric segment ) using simple feedback to control the syringe pump that infused or withdrew buffer from the subphase . isobaric adsorption terminated when the hydrostatic pressure in the chamber fell below 0.5 atm , after which further expansion of the bubble was no longer possible , so that adsorption continued to a surface with a constant area . curves give results from representative experiments to illustrate features that can be obscured during averaging . symbols give the mean sd of time to reach selected ( n = 5 for continuous isotherms and 3 for curves with isobaric segments ) . ( b ) semi - logarithmic plot of the area versus time after beginning the isobaric segments of the adsorption isotherms . the solid black trace gives results for a representative experiment , with the least - squares linear fit indicated by the superimposed white line . the steeper slopes close to e could still reflect changes in the adsorbing material if the isobaric expansion altered the concentration of constituents immediately adjacent to the surface as well as within the interface . the classical model of adsorption developed by ward and tordai(30 ) postulates equilibrium between the film and an unstirred layer immediately adjacent to the interface . if the key step leading to the accelerated adsorption was the maturation of material in the unstirred layer , then isobaric expansion during adsorption might delay that process . therefore , experiments also tested whether prespread films , which would greatly shorten the time required to reach a hypothetical critical , would advance the onset of the late acceleration . conversely , adsorption that required a kinetic process adjacent to the interface should be unaffected by the artificially lowered . the pre - existing films greatly shortened the time required for adsorption to reach e ( figure 4 ) . from the initial value of 3034 mn / m for the prespread films , fell during subsequent adsorption along isotherms at least as steep as for vesicles adsorbing to an initially clean interface ( figure 4 ) . the kinetics of adsorption to spread and adsorbed films at the same were comparable ( figure 4 ) . the steeper slopes occurred without delay , arguing that maturation of the adsorbing material was unnecessary , and that the onset of the late acceleration depended instead on reaching a critical . adsorption of clse vesicles to prespread films of clse . solutions of clse in 1:1 ( v / v ) methanol / chloroform were deposited on the surface of captive bubbles to < 35 mn / m . after the spreading solvent was removed by exchange of the subphase with 20 ml of fresh buffer , aliquots of dispersed clse were injected into the subphase to achieve a final concentration of 250 m phospholipid . a control experiment , indicated by , gives the response to vesicles injected below a bubble without a pre - existing film . for adsorption to the pre - existing films , indicated by , the black curves give the actual data ; gray curves give the same data offset in time , so that the initial coincided with the isotherm for adsorption to a clean interface . isobaric measurements at different directly tested how the rate of adsorption depended on . to minimize the effects of variation among samples , we measured isobaric adsorption at sequentially lower surface tensions for individual samples , alternatively holding or area constant , during the course of a single experiment ( figure 5a ) . during the isobaric segments , , the rates of isobaric adsorption initially fell at progressively lower , but then increased just above e ( figure 5b ) . the sequential isobars provided further support that determined the onset of the accelerated late adsorption . sequential isobaric adsorption . during adsorption of extruded clse at 250 m phospholipid to the surface of a captive bubble at 23 1 c , surface area was alternatively held constant or manipulated to maintain constant . ( a ) variation of ( left axis ) and ln a ( right axis ) during the course of an individual experiment . ( b ) rates of adsorption , obtained from the fractional rate of expansion , ( da / a)/dt , at specific surface tensions for eight individual experiments , distinguished by different symbols and lines . the y axis is split to emphasize how rates varied toward the end of adsorption . to test how the different components of pulmonary surfactant contributed to the late acceleration , collection of appropriate fractions eluted from a gel - permeation column separated clse into preparations containing the surfactant proteins and phospholipids ( sp&pl ) , without cholesterol ; the surfactant proteins and modified phospholipids ( sp&mpl ) , lacking cholesterol and the anionic phospholipids ; and the neutral and phospholipids ( n&pl ) , without the proteins . comparing the adsorption of these different preparations indicated the role played in the complete biological mixture by the omitted constituents . the isotherms for clse and sp&pl , which differ only in their content of cholesterol , were similar . at 35 mn / m , the curves for both preparations became steeper , with the slope achieving greater magnitude for sp&pl than for clse ( figure 6b ) . the additional removal of the anionic phospholipids to yield sp&mpl slowed adsorption generally ( figure 6a ) , but the late acceleration persisted , with relatively little change in the steeper slopes at 30 mn / m ( figure 6b ) . removal of only the proteins slowed adsorption further and prevented n&pl from reaching below 50 mn / m ( figure 6a ) . our experiments were not designed to distinguish whether further adsorption without the proteins ceased completely or simply slowed to an undetectable rate , but during measurements extended to 6 h , n&pl produced no further change in . compositional dependence for adsorption of pulmonary surfactant . preparations obtained from clse contained the complete set of neutral and phospholipids ( n&pl ) , without the proteins ; the surfactant proteins and phospholipids ( sp&pl ) , without cholesterol ; or the surfactant proteins with the phospholipids modified by the removal of the anionic compounds ( sp&mpl ) . a wilhelmy plate measured at 37 c after aspirating the interface of a subphase containing vesicles dispersed by sonication in hsc with a concentration of 100 m phospholipid . averaged data for clse were obtained from the individual curves in figure 1 . for each graph , the curves give results from a representative experiment . slopes were obtained by the procedure used in figure 1 , differentiating the smoothed experimental isotherms . the absence of the proteins might prevent from ever reaching a critical value , but have no effect on subsequent adsorption if the film artificially achieved that . to test whether vesicles could adsorb rapidly at lower without the proteins , experiments measured adsorption of vesicles with and without the proteins to monolayers of n&pl prespread to 30 mn / m ( figure 7 ) . at these low , injection of the n&pl vesicles produced an initial drop of below the value for the pre - existing film by < 2 mn / m but no further decrease in ( figure 7 ) . control experiments confirmed that vesicles of clse , with the proteins , adsorbed readily to the films of n&pl . at above 50 mn / m , insertion into the interface could occur slowly for the lipids alone ( figure 6 ) , but adsorption at lower , including for the final accelerated approach to e , required the proteins . films of n&pl were spread on the surface of captive bubbles to of 32.5 1.5 mn / m by depositing appropriate volumes of solutions in 1:1 ( v / v ) methanol / chloroform . concentrated dispersions of n&pl or clse were then injected into the subphase to initiate adsorption . was measured at 23 c during adsorption to a surface area held constant by simple feedback . curves give the mean of ( n = 4 for clse and 5 for n&pl ) . the importance of the proteins suggested that their concentration within the interface might explain the late acceleration . prior studies have shown that the proteins increase the rate of adsorption , whether they are located in the vesicles or at the airwater interface . if the contents of a vesicle insert collectively , then during adsorption , the composition of the nascent film and its fractional content of protein would remain constant . the increased availability of protein at the point of contact between the vesicles and the film , contributed from both sides of the junction , might cause adsorption to accelerate at above a critical value . experiments with vesicles of clse adsorbing to pre - existing films of n&pl tested that possibility . during adsorption of clse to initial films of n&pl , the of protein at any would be lower than for clse adsorbed to an initially clean interface . the pre - existing film of n&pl would delay the point at which the adsorbing clse would reach the hypothetical critical of protein , and the onset of the late acceleration would shift to a lower . the films of n&pl produced the predicted slowing of the initial adsorption . despite starting at higher , vesicles adsorbing to an initially clean interface quickly reached lower than adsorption to a pre - existing film ( figure 8) . the onset of the late acceleration , however , occurred at the same , unaffected by the of n&pl in pre - existing films ( figure 8) . the equilibrium isotherm ( figure 2 ) indicated that , at e , the of protein in films formed by adsorption to n&pl with 50 mn / m would be 30% of the value for clse adsorption to a clean interface . the inflection point of the kinetic isotherms nonetheless occurred at a that was unchanged ( figure 8) . adsorption of clse vesicles to pre - existing films of n&pl with different . after different amounts of n&pl were spread on the surface of captive bubbles , concentrated aliquots of clse were injected into the subphase at 23 c to achieve a phospholipid concentration of 250 m . previous reports have suggested that the structural discontinuity between coexisting phases within a leaflet might represent an important focus for the initiation of fusion between two bilayers.(33 ) to test whether the accelerated late adsorption required the presence of an interfacial boundary within the lipids , in either the vesicular bilayer or the interfacial film , we measured during adsorption of vesicles containing the proteins with a single phospholipid present as a single phase at temperatures well above its ll transition.(34 ) vesicles of the sps with dopc produced adsorption isotherms similar in shape to traces for clse , including the inflection point and the steeper slopes just above e ( figure 9 ) . curves give representative traces for adsorption of dopc + 1% ( w / w ) sp ( 500 m phospholipid ) and dopc alone ( 1 mm phospholipid ) to the surface of a captive bubble at 23 c . symbols give the mean sd of the time at specific for spdopc ( n = 3 ) and the mean sd for at specific times for dopc alone ( n = 5 ) . the dependence of the accelerated late adsorption on both and the presence of the proteins suggested that , at a certain , the proteins , the phospholipids , or both achieved a configuration that allowed more favorable interaction of the nascent film with the adsorbing vesicles . to test whether the proteins or the phospholipids represented the more important component , we used other lipids , unrelated to pulmonary surfactant , to determine if vesicles without proteins would also demonstrate the late acceleration . compounds that induce lipid leaflets to adopt negative curvature , with a concave hydrophobic face , promote faster adsorption . the phosphatidylethanolamines , which under appropriate conditions form the negatively curved inverse hexagonal ( hii ) phase , provide a well - documented example of this effect . our experiments used mixtures of dope / dopc ( 8:2 , mol / mol ) at ambient temperatures . small - angle x - ray scattering ( saxs ) , which at higher temperatures detected coexisting l and hii phases , showed only lamellar structures below 48 c ( figure 10 ) . adsorption should involve the insertion of vesicles , directly comparable to the process for clse . saxs from dope / dopc ( 8:2 , mol / mol ) at different temperatures . intensities are plotted as a function of q = 4 sin / , where is the wavelength of the x - ray and is half the angle between the incident and diffracted beams . labels indicate diffraction from lamellar structures ( l ) , which occurs at values of q with ratios of 1:2 : ... , and from hexagonal structures ( hii ) at 1:(3):2 : ... the kinetics of adsorption for dope / dopc varied more than for the vesicles with the sps . for seven experiments at 1.0 mm phospholipid , the isotherms , however , were similar in shape to each other and to the curves for clse . if normalized to the total time required to reach e , the adsorption isotherms were reproducible ( figure 11 ) . after an initial acceleration , fell at rates that decreased until reaching an inflection point at 40 mn / m , after which the slopes became steeper . the late acceleration of adsorption for vesicles that lacked the proteins suggested that the critical structural change at low occurred in the lipids rather than the proteins . dopc , either alone or mixed with dope ( xdope = 0.8 ) , was dispersed into hsc buffer and introduced into the subphase below a captive bubble at 1 mm phospholipid . was monitored at ambient temperature ( 23 1 c ) while the surface area was maintained constant using simple feedback . time of adsorption for the dopc / dope vesicles ( lower axis ) is expressed relative to the interval required to reach a constant . time for the vesicles of dopc alone ( upper axis ) is expressed in unnormalized units . the slowest adsorption for dopc / dope vesicles required 48 min to reach a that remained constant . symbols give the mean sd for dopc ( n = 5 ) and dope / dopc ( n = 7 ) . the faster rate at which pulmonary surfactant lowers close to e results from faster adsorption . our results rule out the alternative explanation that the steeper slopes reflect the relationship between and . the adsorption of dimyristoyl phosphatidylcholine ( dmpc ) provides an example of that other possibility.(37 ) during the first - order transition between coexisting phases within the monolayer , increasing the of dmpc produces little change in . after completing the transition , the slope of the equilibrium isotherm becomes steeper . consequently during adsorption , can fall more rapidly after coexistence without a faster increase in . for spread monolayers of clse and spdopc , the isotherms include no isobaric plateaus ( figure 2 ) . the equation of state that relates and provides no explanation for the accelerated drop in , whether from phase coexistence or any other mechanism . the kinetics by which surfactants adsorb to an interface have been considered most commonly in terms of sequential steps . surfactants first diffuse across an unstirred layer to reach the surface and then insert into the interface . insertion may require nothing more than arrival at the interface , or a major reconfiguration may be necessary , involving significant energy of activation . surfactants can be classified according to whether rates of adsorption depend on diffusion , the actual insertion into the interface , or both processes.(41 ) for adsorption determined strictly by the rate of diffusion , expressions that describe the accumulation of adsorbed material simplify considerably if the reverse process of desorption can be ignored,(30 ) which is reasonable for the insoluble compounds considered here . the rate of adsorption dominated by diffusion should then depend on ( time).(30 ) that dependence indicates that diffusion can not explain the kinetics observed here . these changes in adsorption could result from alterations in either the absorbing material or the interfacial film . material adjacent to the interface might undergo the sort of maturation that occurs during adsorption of vesicles to solid supports . vesicles attach to the solid support only if they exceed a critical size , for which the favorable energy of adhesion is greater than the unfavorable energy of bending at the perimeter of the flattened vesicle . the subsequent step , in which the attached vesicle disrupts to form disks , is restricted to vesicles above a larger size by the line tension between the perimeter of the disk and the surrounding aqueous environment . an analogous maturation of vesicles adjacent to the airwater interface represents one process that might explain the late increase in rates of adsorption . our results show , however , that the late acceleration is a characteristic of the films rather than the adsorbing material . instead of requiring a critical duration during which the adjacent material undergoes some functional change , prespread films , used to advance the reduction in , produce an earlier onset of the late acceleration ( figure 4 ) . adsorption to these prespread films occurs at approximately the same rate as to adsorbed films at the same ( figure 4 ) . interruptions of the fall in by intervals of isobaric adsorption have no effect on the subsequent fall in ( figure 3 ) . prolonged isobaric adsorption occurs at a constant rate , with no evidence for a time - dependent change ( figure 3b ) . isobaric experiments show the same dependence of adsorption rates on as the slope of the adsorption isotherm , including the increase in rates at low ( figure 5 ) . in each of these several experimental approaches , the rate of adsorption reflects only rather than time , with rates that are faster rather than slower below a critical . this distinction argues that the late acceleration results from changes in the films rather than a maturation of the adsorbing material . our results suggest that the late acceleration may represent a characteristic of adsorption by fusion between the inserting vesicle and the nascent film . the sps and dope , which both induce the late acceleration ( figures 6 and 11 ) , both also promote the fusion of vesicles . to the best of our knowledge , an accelerated fall in has not been reported for adsorption of micelle - forming surfactants that insert into an interface as individual monomers . fusion between vesicles begins with the outer leaflets merging to form a single continuous structure , in which lipids diffuse freely over the otherwise intact vesicles.(49 ) adsorption would proceed by a comparable process , in which the outer leaflet of the adsorbing vesicle would first merge with the nascent film before the constituents would insert into the interface as a collective unit . the mixing of lipids between the adsorbing vesicle and the interfacial film , comparable to mixing between the outer leaflets of fusing vesicles , remains unconfirmed . compounds that form structures with spontaneous curvature similarly promote or inhibit both adsorption and the fusion of vesicles . constituents that affect both adsorption and fusion have similar effects when added to either of the leaflets that will merge . mlvs , which are large enough to allow microscopic visualization , adsorb as intact structures . adsorption would occur by merger of the vesicles outer leaflet with the nascent film , followed by insertion of its constituents as a collective unit . the late acceleration would then reflect a greater susceptibility of the film to fusion with the adsorbing vesicle below a critical . our results reject two possible explanations suggested by previous reports that would reflect local characteristics of the film caused by the protein or the lipids . because the proteins can promote adsorption at the interface as well as in vesicles , the lower might reflect the higher of the proteins and the greater likelihood that a protein , contributed from either of the fusing structures , would be present to stabilize the initial merged structure . for adsorption of clse , however , pre - existing films that change the relationship between the of the proteins and have no effect on the at which the late acceleration begins ( figure 8) . with the lipids , the separation of phases within the film , which varies with , provides a structural discontinuity that might facilitate the initial merger between two leaflets . vesicles of the sps with dopc occur as a single phase under the conditions used here(34 ) yet undergo a well - defined late acceleration ( figure 9 ) . phase separation in the lipids and the of the proteins are both unlikely to explain the late acceleration . the kinetics of adsorption at different most likely reflect structural changes in the film . those changes could occur either in the small amount of protein that greatly facilitates adsorption or in the lipids that constitute most of the film . our results with dope / dopc , which show the same late acceleration in the absence of the proteins ( figure 11 ) , suggest that the crucial change occurs in the lipids . our experiments provide no information on the structure of the proteins and can not rule out the prior suggestion that lower causes a functionally important conformational change in the proteins.(6 ) the late acceleration with samples containing only lipids , however , supports the paradoxical initial impression that interfacial lipids more rapidly accommodate the insertion of additional material when they are more densely packed . the specific changes in the structure of the lipids that allow faster adsorption at lower remain uncertain . the dramatic acceleration of adsorption at a specific suggests a qualitative change in the structure of the film , such as occurs at a percolation threshold . the alteration that seems most likely to allow better interaction with the outer leaflet of the adsorbing vesicle is the tilt of the acyl chains away from the plane of the interface , which increases at higher .(54 ) the hydrophobic thickness of the merging leaflets would then be similar . approaches that would vary the tilt of the lipids without changing unfortunately are unavailable . exactly which structural changes in the films cause adsorption to accelerate remains the object of speculation . in summary , our findings show that the more rapid fall in close to e observed for vesicles of pulmonary surfactant reflects faster adsorption . the faster rates most likely reflect the greater susceptibility of the lipid monolayer to fusion with the adsorbing vesicle because of structural changes at lower .

adsorption of pulmonary surfactant to an airwater interface lowers surface tension ( ) at rates that initially decrease progressively , but which then accelerate close to the equilibrium . the studies here tested a series of hypotheses concerning mechanisms that might cause the late accelerated drop in . experiments used captive bubbles and a wilhelmy plate to measure during adsorption of vesicles containing constituents from extracted calf surfactant . the faster fall in reflects faster adsorption rather than any feature of the equation of state that relates to surface concentration ( ) . adsorption accelerates when reaches a critical value rather than after an interval required to reach that . the hydrophobic surfactant proteins ( sps ) represent key constituents , both for reaching the at which the acceleration occurs and for producing the acceleration itself . the at which rates of adsorption increase , however , is unaffected by the of protein in the films . in the absence of the proteins , a phosphatidylethanolamine , which , like the sps , induces fusion of the vesicles with the interfacial film , also causes adsorption to accelerate . our results suggest that the late acceleration is characteristic of adsorption by fusion of vesicles with the nascent film , which proceeds more favorably when the of the lipids exceeds a critical value .

Introduction Materials and Methods Results Discussion

surfactants normally adsorb to an airwater interface at rates that decrease as the surface tension ( ) falls [ sodium dodecyl sulfate ( sds ) in figure 1 ] . in the lungs , where pulmonary surfactant exerts its biological function , when the alveolar airwater interface expands during inhalation , the rise of above e is limited . the surfactant vesicles therefore most likely adsorb in the alveolus to an airwater interface that has the at which the acceleration occurs . the experiments here test a series of hypotheses concerning the basis of the late acceleration . our studies emphasize in particular two fundamental issues : whether the late acceleration reflects a maturation of adsorbing material or changes in the characteristics of the interfacial film ; and the extent to which the hydrophobic proteins , which greatly facilitate the adsorption of the surfactant lipids at all , are necessary and sufficient to cause the late acceleration . subfractions of the constituents in clse were obtained by gel - permeation chromatography using a mobile phase of acidified chloroform / methanol [ 1:1:0.05 ( v / v / v ) chloroform / methanol/0.1 n hcl ] , which separates the hydrophobic surfactant proteins ( sp - b and sp - c ) , the phospholipids , and the neutral lipids ( cholesterol ) into distinct peaks . because the bubbles float against an agarose ceiling , the bubbles had the theoretical disadvantage that some of the airwater interface might be inaccessible to adsorbing vesicles , and that the inaccessible fraction of the interface could change during adsorption when the variation of alters the shape of the bubble . the comparable results obtained with bubbles and with adsorption to an open interface ( see below ) suggested that any shift in accessible surface area made no contribution to our findings . (24 ) adsorption was initiated either by injecting small aliquots of concentrated vesicles prepared in hs , in which the absence of ca minimized fusion among vesicles , into a stirred subphase of hsc , or by formation of a bubble in a subphase already containing hsc and vesicles . (24 ) images obtained along the horizontal axis of the bubble provided its height and diameter , which were used to calculate and surface area using a semi - empirical approach based on the equation of young and laplace. experiments concerning the different preparations obtained from clse measured adsorption to the airwater interface above a subphase in a teflon cup enclosed within a temperature - regulated chamber containing open water to maintain full humidification . these experiments used a wilhelmy plate composed of filter paper monitored by a force transducer ( riegler and kirstein gmbh , potsdam , germany ) to determine . vesicles of extracted calf surfactant ( clse ) at 37 c lowered at rates that initially decreased in magnitude as adsorption progressed ( figure 1 ) . although present in many prior studies of adsorption , the absence of the late acceleration in some previously published reports raised the possibility that the phenomenon reflected some feature of the manner in which the experiments were conducted . we therefore tested the dependence of the late acceleration on the following aspects of our protocol. (a)measurement of : experiments obtained comparable results for adsorption to a captive bubble , the shape of which provided , and to the surface of a subphase in an open cup , using a wilhelmy plate to monitor . (b)initiation of adsorption : with the captive bubble , experiments initiated adsorption by injecting aliquots of concentrated clse into the subphase or by replacing a subphase of buffered electrolyte with a dispersion of clse . with the subphase in an open cup monitored with a wilhelmy plate , adsorption began with either the injection of clse into the subphase or aspiration of the initial film from the surface above homogeneously dispersed clse. below 35 mn / m , however , the isotherms in the presence and absence of stirring became parallel , with steeper slopes in both cases. dispersion by cyclic freezing and thawing , which should produce multilamellar vesicles ( mlvs),(27 ) generated particles with the least evidence of a late acceleration . the late acceleration therefore most clearly represented a phenomenon of a single bilayer , which presumably is the initial step in the adsorption of a mlv . measurement of : experiments obtained comparable results for adsorption to a captive bubble , the shape of which provided , and to the surface of a subphase in an open cup , using a wilhelmy plate to monitor . with the subphase in an open cup monitored with a wilhelmy plate , adsorption began with either the injection of clse into the subphase or aspiration of the initial film from the surface above homogeneously dispersed clse. below 35 mn / m , however , the isotherms in the presence and absence of stirring became parallel , with steeper slopes in both cases . dispersion by cyclic freezing and thawing , which should produce multilamellar vesicles ( mlvs),(27 ) generated particles with the least evidence of a late acceleration . the late acceleration therefore most clearly represented a phenomenon of a single bilayer , which presumably is the initial step in the adsorption of a mlv . the steeper slopes at the end of the adsorption isotherm could reflect the relationship between the surface concentration ( ) and rather than a true increase in the rate of adsorption . increasing produced by compression , however , showed only the previously reported,(28 ) roughly linear decrease in from shortly after lift - off to the onset of collapse ( figure 2 ) . assuming that had the same relationship to during compression of a monolayer and during adsorption , the late accelerated fall in indicated a faster increase in and more rapid adsorption . the late acceleration could reflect changes in the film , achieved at a critical , or changes in the adsorbing material . although the vesicles dispersed in the bulk of the subphase should remain unchanged by the introduction of an interface , material adjacent to the surface might undergo a kinetic transformation that would allow faster adsorption . accelerated adsorption that occurred because of changes in the film should depend exclusively upon , regardless of the time needed to reach the critical value . isobars , obtained at constant , measured rates of adsorption from the change in area , a , necessary to maintain constant. (29 ) assuming that uniquely determined , such that , for n constituents confined within a , where the variable c had a single value at any particular , then for adsorption at constant , the fractional rate of change in area would equal the fractional rate at which constituents adsorb to the interface : if the steeper slopes at the end of the adsorption isotherms reflected a maturation of adsorbing material and the passage of time , then adsorption isobars should all become steeper at later times , regardless of the that was held constant . the isobaric measurements extended to times at which adsorption to a constant surface area demonstrated the late acceleration ( figure 3a ) . changes in the adsorbing material that might have caused the late acceleration in the experiments with a constant surface area should have occurred during the isobaric measurements . after the chamber reached its minimum hydrostatic pressure , which prevented further expansion to maintain isobaric conditions , the experiments measured the fall in during adsorption to a constant area ( figure 3a ) . the isotherms after the isobaric adsorption paralleled the curves obtained during adsorption to an area that was fixed throughout the entire experiment , demonstrating the same late increase in slope . both the constant isobaric rates and the parallel final segments of the isotherms , despite interruption of the fall in by a prolonged isobaric interval , argued that the late acceleration occurred when the films reached a critical rather than when time achieved a critical duration required for maturation of the adsorbing material . isobaric adsorption terminated when the hydrostatic pressure in the chamber fell below 0.5 atm , after which further expansion of the bubble was no longer possible , so that adsorption continued to a surface with a constant area . the steeper slopes close to e could still reflect changes in the adsorbing material if the isobaric expansion altered the concentration of constituents immediately adjacent to the surface as well as within the interface . if the key step leading to the accelerated adsorption was the maturation of material in the unstirred layer , then isobaric expansion during adsorption might delay that process . therefore , experiments also tested whether prespread films , which would greatly shorten the time required to reach a hypothetical critical , would advance the onset of the late acceleration . conversely , adsorption that required a kinetic process adjacent to the interface should be unaffected by the artificially lowered . the steeper slopes occurred without delay , arguing that maturation of the adsorbing material was unnecessary , and that the onset of the late acceleration depended instead on reaching a critical . for adsorption to the pre - existing films , indicated by , the black curves give the actual data ; gray curves give the same data offset in time , so that the initial coincided with the isotherm for adsorption to a clean interface . during adsorption of extruded clse at 250 m phospholipid to the surface of a captive bubble at 23 1 c , surface area was alternatively held constant or manipulated to maintain constant . to test how the different components of pulmonary surfactant contributed to the late acceleration , collection of appropriate fractions eluted from a gel - permeation column separated clse into preparations containing the surfactant proteins and phospholipids ( sp&pl ) , without cholesterol ; the surfactant proteins and modified phospholipids ( sp&mpl ) , lacking cholesterol and the anionic phospholipids ; and the neutral and phospholipids ( n&pl ) , without the proteins . comparing the adsorption of these different preparations indicated the role played in the complete biological mixture by the omitted constituents . the additional removal of the anionic phospholipids to yield sp&mpl slowed adsorption generally ( figure 6a ) , but the late acceleration persisted , with relatively little change in the steeper slopes at 30 mn / m ( figure 6b ) . our experiments were not designed to distinguish whether further adsorption without the proteins ceased completely or simply slowed to an undetectable rate , but during measurements extended to 6 h , n&pl produced no further change in . compositional dependence for adsorption of pulmonary surfactant . preparations obtained from clse contained the complete set of neutral and phospholipids ( n&pl ) , without the proteins ; the surfactant proteins and phospholipids ( sp&pl ) , without cholesterol ; or the surfactant proteins with the phospholipids modified by the removal of the anionic compounds ( sp&mpl ) . the absence of the proteins might prevent from ever reaching a critical value , but have no effect on subsequent adsorption if the film artificially achieved that . to test whether vesicles could adsorb rapidly at lower without the proteins , experiments measured adsorption of vesicles with and without the proteins to monolayers of n&pl prespread to 30 mn / m ( figure 7 ) . control experiments confirmed that vesicles of clse , with the proteins , adsorbed readily to the films of n&pl . at above 50 mn / m , insertion into the interface could occur slowly for the lipids alone ( figure 6 ) , but adsorption at lower , including for the final accelerated approach to e , required the proteins . the importance of the proteins suggested that their concentration within the interface might explain the late acceleration . prior studies have shown that the proteins increase the rate of adsorption , whether they are located in the vesicles or at the airwater interface . if the contents of a vesicle insert collectively , then during adsorption , the composition of the nascent film and its fractional content of protein would remain constant . the increased availability of protein at the point of contact between the vesicles and the film , contributed from both sides of the junction , might cause adsorption to accelerate at above a critical value . during adsorption of clse to initial films of n&pl , the of protein at any would be lower than for clse adsorbed to an initially clean interface . the pre - existing film of n&pl would delay the point at which the adsorbing clse would reach the hypothetical critical of protein , and the onset of the late acceleration would shift to a lower . the onset of the late acceleration , however , occurred at the same , unaffected by the of n&pl in pre - existing films ( figure 8) . the equilibrium isotherm ( figure 2 ) indicated that , at e , the of protein in films formed by adsorption to n&pl with 50 mn / m would be 30% of the value for clse adsorption to a clean interface . (33 ) to test whether the accelerated late adsorption required the presence of an interfacial boundary within the lipids , in either the vesicular bilayer or the interfacial film , we measured during adsorption of vesicles containing the proteins with a single phospholipid present as a single phase at temperatures well above its ll transition. the dependence of the accelerated late adsorption on both and the presence of the proteins suggested that , at a certain , the proteins , the phospholipids , or both achieved a configuration that allowed more favorable interaction of the nascent film with the adsorbing vesicles . to test whether the proteins or the phospholipids represented the more important component , we used other lipids , unrelated to pulmonary surfactant , to determine if vesicles without proteins would also demonstrate the late acceleration . labels indicate diffraction from lamellar structures ( l ) , which occurs at values of q with ratios of 1:2 : ... , and from hexagonal structures ( hii ) at 1:(3):2 : ... the kinetics of adsorption for dope / dopc varied more than for the vesicles with the sps . for seven experiments at 1.0 mm phospholipid , the isotherms , however , were similar in shape to each other and to the curves for clse . after an initial acceleration , fell at rates that decreased until reaching an inflection point at 40 mn / m , after which the slopes became steeper . the late acceleration of adsorption for vesicles that lacked the proteins suggested that the critical structural change at low occurred in the lipids rather than the proteins . time of adsorption for the dopc / dope vesicles ( lower axis ) is expressed relative to the interval required to reach a constant . the faster rate at which pulmonary surfactant lowers close to e results from faster adsorption . the equation of state that relates and provides no explanation for the accelerated drop in , whether from phase coexistence or any other mechanism . material adjacent to the interface might undergo the sort of maturation that occurs during adsorption of vesicles to solid supports . vesicles attach to the solid support only if they exceed a critical size , for which the favorable energy of adhesion is greater than the unfavorable energy of bending at the perimeter of the flattened vesicle . the subsequent step , in which the attached vesicle disrupts to form disks , is restricted to vesicles above a larger size by the line tension between the perimeter of the disk and the surrounding aqueous environment . an analogous maturation of vesicles adjacent to the airwater interface represents one process that might explain the late increase in rates of adsorption . our results show , however , that the late acceleration is a characteristic of the films rather than the adsorbing material . instead of requiring a critical duration during which the adjacent material undergoes some functional change , prespread films , used to advance the reduction in , produce an earlier onset of the late acceleration ( figure 4 ) . in each of these several experimental approaches , the rate of adsorption reflects only rather than time , with rates that are faster rather than slower below a critical . this distinction argues that the late acceleration results from changes in the films rather than a maturation of the adsorbing material . our results suggest that the late acceleration may represent a characteristic of adsorption by fusion between the inserting vesicle and the nascent film . the sps and dope , which both induce the late acceleration ( figures 6 and 11 ) , both also promote the fusion of vesicles . to the best of our knowledge , an accelerated fall in has not been reported for adsorption of micelle - forming surfactants that insert into an interface as individual monomers . (49 ) adsorption would proceed by a comparable process , in which the outer leaflet of the adsorbing vesicle would first merge with the nascent film before the constituents would insert into the interface as a collective unit . the mixing of lipids between the adsorbing vesicle and the interfacial film , comparable to mixing between the outer leaflets of fusing vesicles , remains unconfirmed . adsorption would occur by merger of the vesicles outer leaflet with the nascent film , followed by insertion of its constituents as a collective unit . the late acceleration would then reflect a greater susceptibility of the film to fusion with the adsorbing vesicle below a critical . our results reject two possible explanations suggested by previous reports that would reflect local characteristics of the film caused by the protein or the lipids . for adsorption of clse , however , pre - existing films that change the relationship between the of the proteins and have no effect on the at which the late acceleration begins ( figure 8) . with the lipids , the separation of phases within the film , which varies with , provides a structural discontinuity that might facilitate the initial merger between two leaflets . phase separation in the lipids and the of the proteins are both unlikely to explain the late acceleration . those changes could occur either in the small amount of protein that greatly facilitates adsorption or in the lipids that constitute most of the film . our results with dope / dopc , which show the same late acceleration in the absence of the proteins ( figure 11 ) , suggest that the crucial change occurs in the lipids . (6 ) the late acceleration with samples containing only lipids , however , supports the paradoxical initial impression that interfacial lipids more rapidly accommodate the insertion of additional material when they are more densely packed . the specific changes in the structure of the lipids that allow faster adsorption at lower remain uncertain . the dramatic acceleration of adsorption at a specific suggests a qualitative change in the structure of the film , such as occurs at a percolation threshold . the alteration that seems most likely to allow better interaction with the outer leaflet of the adsorbing vesicle is the tilt of the acyl chains away from the plane of the interface , which increases at higher . exactly which structural changes in the films cause adsorption to accelerate remains the object of speculation . in summary , our findings show that the more rapid fall in close to e observed for vesicles of pulmonary surfactant reflects faster adsorption .

breast cancer incidence and mortality rates have declined steadily in the us for the past 1015 years [ 17 ] . the relative 5-year survival rate for breast cancer overall has also increased in the past decade to 89% . unfortunately , disparities exist in breast cancer outcomes between racial groups in the us . although survival has increased for both white and black women over time , the survival increase in black women has been smaller . several reasons have been suggested for the survival disparity between black and white women , including racial differences in access to and utilization of screening and treatment [ 1012 ] , risk factors that are differentially distributed by race [ 10 , 1315 ] and socioeconomic status ( ses ) [ 14 , 1620 ] , and biological differences such as tumor aggressiveness [ 21 , 22 ] . the results of research studies assessing the role of ses in racial disparities in breast cancer survival are mixed ; some studies reported that racial differences in survival disappear after adjusting for ses [ 17 , 19 , 20 , 23 , 24 ] , while other studies show survival differences persisting after adjustment for ses [ 13 , 14 , 17 , 20 , 2527 ] . the conflicting results may be due to the lack of ses information in cancer registries in the us , resulting in insufficient characterization of socioeconomic levels of patients [ 11 , 20 , 25 , 28 ] . in addition to ses , differential levels of healthcare resources at the neighborhood level have also been examined in previous studies as potentially contributing to racial disparities in cancer outcomes [ 2936 ] . however , the same limitations exist as with studies of ses effect on racial disparities in breast cancer survival lack of consistency in which measure of health care resources to use and questions about which geographic level neighborhood differences are measured . defining healthcare access is complicated , mainly because the construct encompasses dimensions of healthcare availability , affordability , acceptability , and accessibility . to ensure that valid inferences are drawn , it is necessary to specify which aspect of healthcare access is being measured . for this study , we focused on the availability of healthcare resources at the county level as our measure of healthcare access . to our knowledge , no other research study has developed a specific measure of neighborhood healthcare access as a predictor of breast cancer survival among blacks and whites in the us , while controlling for individual and neighborhood level ses . other studies attempting to examine the impact of neighborhood effects on breast cancer survival have done so without individual level ses data or did not simultaneously control for healthcare resource availability . the aim of this study was to assess the impact of neighborhood health care resources on white - black disparities in breast cancer survival by adjusting for individual and neighborhood ses . this study utilized the national cancer institute 's surveillance epidemiology and end results data linked to the us census bureau 's national longitudinal mortality study database ( nlms ) . the linked dataset is referred to in this study as ( seer - nlms ) . detailed methodology regarding this dataset has been published elsewhere [ 13 , 37 , 38 ] . in brief , seer data on cancer incidence , prevalence and survival from 15 participating registries , covering about 25% of the us population was linked with the nlms data to capture demographic , socioeconomic , and occupation attributes from the current population surveys and the annual social and economic supplement . a full description of the nlms is available at the census website ( http:/www.census.gov / nlms/ ) . data on county level ses , health care facilities , and health care personnel were obtained from the 2009 - 2010 area resource file which contains over 6,000 variables relating to healthcare , socioeconomic , and environmental characteristics for each county in the us . data on the number of certified mammography facilities was obtained through a freedom of information ( foi ) request to the food and drug administration ( fda ) , the agency in charge of certifications . a list and addresses of all mammography facilities certified in the year 2000 the dataset used in the final analysis was restricted to non - hispanic black and non - hispanic white first primary breast cancer cases among women with ages 40 years and older , diagnosed between 1973 and 2003 . the analysis was restricted to non - hispanic black and white women to facilitate the comparison of two racial groups in the us that have shown sustained and consistently high disparities in breast cancer survival . we focused the main analysis on women residing in counties that contained at least one black and one white breast cancer patient to ensure that the neighborhood level predictors represented the actual experiences of both groups of women . a total of 1796 women met this criterion : 1580 whites and 216 blacks residing in 60 counties in the us . a flowchart describing the sample selection is available as a supplemental figure in supplementary materials available at http://dx.doi.org/10.1155/2013/490472 . counties were identified using the federal information processing standard ( fips ) code to link county variables with cases ' county of residence at diagnosis as recorded in the seer dataset . marital status was categorized as married , widowed , divorced / separated , and never married . employment status was categorized as women in the labor force and women not in the labor force . education level was categorized as less than high school ( < 12 years ) , high school graduate ( 12 years ) , and at least some college ( 13 years ) . age at diagnosis and income were analyzed as continuous variables ; income was analyzed as inflation - adjusted annual household income ( 1990 standard ) . clinical variables . stage at presentation was categorized as in situ / localized , regional , and distant / unstaged . surgical treatment was categorized as surgical treatment received , not received due to medical reasons , and not received due to nonmedical reasons . radiation treatment was also categorized as received , not received due to medical reasons , and not received due to nonmedical reasons . surgical or radiation treatment not received due to nonmedical reasons included those that were recommended but not performed , recommended and it was unknown if performed , or treatment information was unknown due to death certificate or autopsy diagnosis . survival . survival time was calculated as the number of months between diagnosis and either date of death , date last known to be alive , or december 31 , 2003 . we created two censoring variables based on seer cause of death variable : one that indicated if a person had died with breast cancer as the underlying cause , and the other that indicated if a person had died of any cause . patients who died of other causes or were alive at the date of last followup were censored in the first censoring variable , and patients who were alive at the date of last followup were censored in the second censoring variable . health care access variables . health care access was defined as the linear combination of measures of density of health care resources in the county . we assessed specific variables related to health care resources at the county level and adjusted for differences in population size by dividing the counts per 10,000 people in each county . the derived variables were subject to principal component analysis ( pca ) on count per 10,000 of number of hospitals , number of medical doctors , number of medical doctors with obstetrics and gynecology specialty , number of osteopathic doctors ( do ) , number of dos with obstetrics and gynecology specialty , number of nurse practitioners , and number of mammography facilities . sas proc factor was used to generate scores using an eigenequation based on our input variables . the scores were categorized into tertiles : poorest , middle , and highest . county level ses was defined using the index of concentration in the extremes ( ice ) . this index was chosen over other approaches ( such as the proportion of county residents below poverty level ) because it allows for conceptualizing the concentration of affluence and disadvantage as falling along a single continuum . the index theoretically ranges from 1 ( where all households are disadvantaged ) through 0 ( where there is equal proportion of affluent and disadvantaged households ) to + 1 ( where all households are affluent ) . ice - income = ( ( # households with household income , $ 100,000 + ) ( # households with families below poverty level)/total number of households ) . ice - education = ( ( % persons 25 years with 4 yrs college ) ( % persons 25 years with < 9 yrs sch)/100 ) . for easy interpretation , these measures were also categorized into tertiles . to control for other county level variables that may be associated with income , we included county level proportion of blacks and percent non - english speaking . all county level variables were obtained from the area resource file for the year 2000 because it had the most complete year of data and straddled the years included in the seer dataset . we compared scores for 1990 and 2000 and found them to be highly correlated ( ice income correlation coefficient = 0.90 , p < 0.05 and ice education correlation coefficient = 0.94 , p < 0.05 ) . there were 3141 counties available for the county level analysis of health care access and ses . the first two components of the pca had eigenvalues greater than 1 , and the scree test also showed a clear break after the second component ( figure not shown ) . the two components together accounted for 50.5% of the total variance ; components that had a factor loading value of greater than 0.4 were said to have loaded on a specific component . based on this criterion , mds per 10,000 , mds in ob - gyn per 10,000 , nurse practitioners per 10,000 , and dos per 10,000 were loaded on the first component , hereafter named personnel . number of hospitals per 10,000 and number of mammography facilities per 10,000 were loaded on the second component , hereafter named facilities . all statistical analyses were performed using sas statistical software ( sas , version 9.2 ) . multivariable proportional hazards regression was used to model the hazards of breast cancer and all - cause mortality in three separate models . the first model adjusted for demographic variables , the second model included clinical variables regarding stage of presentation and treatment , and the third model included county level variables . robust sandwich estimates for the covariance matrix were used to account for the clustering of cases within counties , and the county fips code was specified as the clustering variable . table 1 provides information on the distribution of county variables relating to healthcare resources , socioeconomic status , and other controls across the us . table 2 illustrates the distribution of demographic , clinical , and county level variables . on average , black patients were about 2 years younger compared with white patients , had a lower annual household income ( $ 25,000 for blacks versus $ 44,700 for whites ) , were more likely to be single ( 19.1% of blacks versus 7.4% of whites ) and divorced ( 19.7% of blacks versus 9.8% of whites ) , and were more likely to have less than a high school education ( 23.6% of blacks versus 9.5% of whites ) . the distribution of stage at diagnosis was similar between blacks and whites ; about 53% of blacks were diagnosed at in situ / localized stage compared with 54.6% of white cases , and 14.2% of blacks black patients were also less likely to have received surgical ( 91% of blacks versus 95% of whites ) and radiation treatment ( 31% of blacks versus 39% of whites ) compared with white patients . at the county level , black patients were more likely than white patients to live in counties that had a higher proportion of households in poverty ( 36.9% of blacks versus 16.8% of whites ) , higher proportion of adults with less than 9 years of education ( 43.2% of blacks versus 26.9% of whites ) , and higher proportion of blacks ( 87.5% of blacks versus 67.5% of whites ) . whites were more likely to reside in counties with the poorest healthcare facilities ( 38.1% of whites versus 32.7 of blacks ) and a higher proportion of non - english speaking residents ( 77.6% of whites versus 59.7% of blacks ) . all of these differences were statistically significant at the p < 0.05 level . in unadjusted analysis ( tables 3 and 4 ) , black women had a 53% higher likelihood of dying of breast cancer ( p = 0.008 ) and 32% higher likelihood of dying of any cause ( p = 0.02 ) compared with white women . having less than high school education increased the likelihood of breast cancer mortality by 68% , and all - cause mortality by 95% . furthermore , being diagnosed at a distant stage and not receiving surgical or radiation treatment was also associated with significantly higher likelihood of death . table 3 also presents the results of three sequential cox proportional hazards multivariable models assessing the determinants of breast cancer survival . model 1 assessed the effect of race on breast cancer mortality after adjusting for demographic variables . race was no longer a statistically significant predictor of breast cancer death after adjusting for the individual variables ( hazard ratio , 1.40 ; 95% ci , 0.991.97 ) . model 2 adjusted additionally for stage at presentation and treatment , and the effect of race remained nonsignificant . model 3 presents the effect of county level variables adjusting for individual demographic and clinical variables on breast cancer mortality . residing in counties with a higher proportion of households in poverty increased the likelihood of breast cancer deaths compared with counties with a higher proportion of affluent households . the hazard ratio of ice - income comparing the poorest versus highest group was 1.29 ( 95% ci , 0.822.05 ) and comparing the middle versus highest group was 1.49 ( 95% ci , 1.121.99 ) . on the other hand , residing in counties with a higher proportion of residents with less than 9 years of education appeared to reduce the likelihood of breast cancer deaths . the hazard ratio of ice - education comparing the poorest versus highest group was 0.55 ( 95% ci , 0.310.98 ) and comparing the middle versus highest group was 0.65 ( 95% ci , 0.440.96 ) . furthermore , residing in a county with a higher proportion of black residents ( 6% ) significantly increased the likelihood of breast cancer death ( hazard ratio , 1.74 ; 95% ci , 1.212.48 ) . facilities and personnel variables did not appear to have an independent significant effect on the likelihood of breast cancer death after adjusting for other variables in the model . table 4 presents the results of the cox proportional hazards models assessing the determinants of all - cause mortality . in contrast to the model predicting breast cancer survival ( table 3 ) , race remained a statistically significant predictor of higher mortality among blacks compared to whites even after adjusting for individual demographic and clinical variables . in model 1 which adjusted for demographic variables , being black was associated with a 38% increase in the likelihood of death due to any cause compared with being white ( 95% ci , 1.081.76 ) . in model 2 , the hazard ratio associated with being black was 1.33 ( 95% ci , 1.041.70 ) after adjusting for demographic and clinical variables . after adjusting for county level variables in model 3 , the effect of race was attenuated and became nonsignificant ( hazard ratio , 1.27 ; 95% ci , 0.951.71 ) . personnel , facilities , county proportion of blacks , and proportion of non - english speaking residents were not significantly associated with all - cause mortality . in this study of black and white women with breast cancer , the effect of race on breast cancer mortality became nonsignificant after adjusting for individual demographic variables . for all - cause mortality , race was a significant predictor even after adjusting for demographic and clinical variables such as stage of presentation and treatment . we observed sustained differences in the receipt of surgical and radiation treatment between black and white patients in this study . this is particularly troubling given that these treatment variables remained the most significant predictors of breast cancer mortality and all - cause mortality even after adjusting for individual and neighborhood level variables . it has been suggested that the disparity in treatment receipt among black women may be due to cultural factors which make them more likely to refuse treatment . however , in this study we were able to distinguish between patients who did not receive treatment due to medical reasons or nonmedical reasons . among women who did not receive treatment due to medical reasons ( implying that a medical personnel made the decision not to treat ) , the proportion of black women was still much higher . more research is urgently needed in this area to better understand the factors that determine who gets treatment . upon adjusting for county level healthcare access variables , race was no longer significantly associated with all - cause mortality . facilities and personnel variables did not appear to have a significant independent effect on the likelihood of breast cancer or all - cause mortality after adjusting for other variables in the model . furthermore , residing in counties with a higher proportion of households in poverty increased the likelihood of breast cancer mortality and all - cause mortality compared with counties with a higher proportion of affluent households . our findings suggest that neighborhood poverty and lack of healthcare resources to care might explain part of the black - white disparity in breast cancer survival especially if examined from both individual and neighborhood levels . many studies have sought to explain the causes of racial disparities in breast cancer survival [ 1013 , 17 , 23 , 4554 ] , with varying results . however , none of these studies assessed the role of neighborhood healthcare resources in breast cancer or all - cause survival although other studies have been published about the impact of neighborhood healthcare resources on stage at diagnosis [ 12 , 33 , 55 ] . our study suggests that stage at presentation is an important predictor of breast cancer survival ; late stage at diagnosis was associated with a fourfold increase in the hazard of breast cancer mortality after adjusting for other variables . studies that assessed the role of county level healthcare access on late stage diagnosis of cancer found that women residing in counties with fewer physicians and poor access to mammography facilities were more likely to have late stage cancer diagnosis . other studies have suggested that important predictors of mammography use are having a primary care physician , travel times , and public transportation hassles [ 5564 ] . these are all factors which may be compounded if there are inadequate healthcare facilities and personnel within a county . we anticipated that these county characteristics , number of physicians , and mammography facilities would also be associated with breast cancer survival through the availability of early diagnosis and adequate treatment . however , our measures of healthcare access did not independently predict breast cancer survival , even though the initial racial disparity in survival disappeared . this finding is consistent with a recent publication which did not find an association between the availability of medical resources and breast cancer mortality at the county level . it is likely that some heterogeneity in exposure ( i.e. , county ses and healthcare access ) is lost by aggregating neighborhood measures to the county level as opposed to the zip code or census tract level . however , due to patient privacy concerns , the seer dataset does not routinely disclose patient geographic location at levels smaller than the county . however , the concept of healthcare access is very complex and multidimensional , incorporating aspects of availability such as the presence of medical facilities and personnel as well as aspects of accessibility such as distance , affordability , and cultural barriers . this study serves as a first step in understanding the role of one aspect of healthcare access on breast cancer survival , and future studies may build on this research to further improve understanding of other aspects . third , other neighborhood level factors such as racial or economic segregation may also be important in understanding racial differences in breast cancer survival and need to be examined . for instance , we found that women residing in counties with a higher proportion of blacks had significantly higher hazards of breast cancer mortality . this is especially relevant to this study of racial disparities because studies have shown that due to lower income levels on average , blacks are more likely to reside in poor counties [ 26 , 32 ] . however , due to established social and family networks , even blacks that belong to higher ses groups are more likely to continue to reside in these poor counties . this has implications for the understanding of the impact of socioeconomic status and breast cancer survival among blacks , because black women earning higher incomes may not benefit as much from their socioeconomic status as white women earning similar wages but residing in high ses counties . we observed a significant increase hazard of breast cancer and all - cause mortality due to neighborhood income . women residing in counties with a higher proportion of low income residents compared with high income residents had higher hazards . this is consistent with other studies using other definitions of county ses [ 20 , 23 , 66 , 67 ] . however , we also observed significant reduction in the hazard of breast cancer and all - cause mortality for counties with a higher proportion of less educated residents . this observation was unexpected especially since higher individual education level was found to be protective in this study as well as in others [ 24 , 37 , 66 , 68 ] . one explanation for this finding may be the small sample size in counties with a higher proportion of less educated residents . another potential explanation is the high proportion of immigrants who may be less educated but more likely to have close - knit social networks that has been found to be protective against adverse health outcomes . the major strength of this study was the availability of individual ses information for cancer patients . this allowed for better control of potential confounding of the association between neighborhood characteristics and survival by individual ses . another strength is the development of a specific measure of neighborhood healthcare access using the availability of healthcare resources including mammography facilities to better capture factors that may affect early detection which is a major determinant of survival . first , there is a possibility of exposure misclassification bias because measures of individual ses ( household income , education , and employment ) were not obtained at the time of diagnosis with cancer . these measures were obtained through surveys which may have been administered before or after cancer diagnosis . however , our analysis was restricted to breast cancer cases ages 40 and older , reducing the likelihood of dramatic changes in ses through the study period . secondly , our measure of healthcare access at the county level is an indicator of availability , not necessarily accessibility . several other factors may determine if a person actually benefits from living in a county with good healthcare facilities such as language or cultural barriers , mistrust of the medical system , and lack of health insurance . thirdly , for analytical reasons , the study sample was restricted to women residing in counties that had at least one black and one white breast cancer patients . this implies that the population may be more urban compared with the rest of the us . however , the results of this analysis may still be applicable to semi - urban or rural areas where the impact of county ses and healthcare access on breast cancer survival may be even more pronounced . we performed a sensitivity analysis using only white women ( n = 1580 ) and found similar results ; the neighborhood variables did not significantly predict breast cancer survival among white women . this supports our earlier conclusion that the county level variables may influence breast cancer survival ( for both white and black women ) through the availability of screening and timely treatment . the differential impact of these county variables which may contribute to the racial disparities in breast cancer survival between black and white women ( effect measure modification ) was assessed through interaction terms in the final model ; however there were no significant interactions possibly due to the low sample size of black women within each group . finally , we performed a post hoc power analysis to assess the statistical power of the study to detect a statistically significant difference between breast cancer survival in black and white women . given the sample size of 216 black and 1580 white patients , type 1 error rate of 0.05 , and a followup of 30 years , the study had a power level of 85% . this implies that if a difference in races existed , we had an 85% chance of detecting it . in summary , our study adds an important component to the existing evidence on survival disparities by conceptualizing healthcare access at the county level as a potential determinant of health outcomes and as a potential modifier of the association between race and mortality . further studies may focus on defining healthcare access at smaller geographic levels , for example , zip code or census tracts which may be more homogenous in the distribution of healthcare facilities and personnel . furthermore , while our study focused on the quantity of healthcare facilities and personnel , other studies may attempt to include a measure of healthcare quality as another measure of healthcare access and a potential determinant of survival .

background . breast cancer survival has improved significantly in the us in the past 1015 years . however , disparities exist in breast cancer survival between black and white women . purpose . to investigate the effect of county healthcare resources and ses as well as individual ses status on breast cancer survival disparities between black and white women . methods . data from 1,796 breast cancer cases were obtained from the surveillance epidemiology and end results and the national longitudinal mortality study dataset . cox proportional hazards models were constructed accounting for clustering within counties . three sequential cox models were fit for each outcome including demographic variables ; demographic and clinical variables ; and finally demographic , clinical , and county - level variables . results . in unadjusted analysis , black women had a 53% higher likelihood of dying of breast cancer and 32% higher likelihood of dying of any cause ( p < 0.05 ) compared with white women . adjusting for demographic variables explained away the effect of race on breast cancer survival ( hr , 1.40 ; 95% ci , 0.991.97 ) , but not on all - cause mortality . the racial difference in all - cause survival disappeared only after adjusting for county - level variables ( hr , 1.27 ; ci , 0.951.71 ) . conclusions . improving equitable access to healthcare for all women in the us may help eliminate survival disparities between racial and socioeconomic groups .

1. Introduction 2. Methods 3. Statistical Analysis 4. Results 5. Discussion

breast cancer incidence and mortality rates have declined steadily in the us for the past 1015 years [ 17 ] . the relative 5-year survival rate for breast cancer overall has also increased in the past decade to 89% . unfortunately , disparities exist in breast cancer outcomes between racial groups in the us . although survival has increased for both white and black women over time , the survival increase in black women has been smaller . several reasons have been suggested for the survival disparity between black and white women , including racial differences in access to and utilization of screening and treatment [ 1012 ] , risk factors that are differentially distributed by race [ 10 , 1315 ] and socioeconomic status ( ses ) [ 14 , 1620 ] , and biological differences such as tumor aggressiveness [ 21 , 22 ] . the results of research studies assessing the role of ses in racial disparities in breast cancer survival are mixed ; some studies reported that racial differences in survival disappear after adjusting for ses [ 17 , 19 , 20 , 23 , 24 ] , while other studies show survival differences persisting after adjustment for ses [ 13 , 14 , 17 , 20 , 2527 ] . the conflicting results may be due to the lack of ses information in cancer registries in the us , resulting in insufficient characterization of socioeconomic levels of patients [ 11 , 20 , 25 , 28 ] . however , the same limitations exist as with studies of ses effect on racial disparities in breast cancer survival lack of consistency in which measure of health care resources to use and questions about which geographic level neighborhood differences are measured . defining healthcare access is complicated , mainly because the construct encompasses dimensions of healthcare availability , affordability , acceptability , and accessibility . to our knowledge , no other research study has developed a specific measure of neighborhood healthcare access as a predictor of breast cancer survival among blacks and whites in the us , while controlling for individual and neighborhood level ses . other studies attempting to examine the impact of neighborhood effects on breast cancer survival have done so without individual level ses data or did not simultaneously control for healthcare resource availability . the aim of this study was to assess the impact of neighborhood health care resources on white - black disparities in breast cancer survival by adjusting for individual and neighborhood ses . this study utilized the national cancer institute 's surveillance epidemiology and end results data linked to the us census bureau 's national longitudinal mortality study database ( nlms ) . in brief , seer data on cancer incidence , prevalence and survival from 15 participating registries , covering about 25% of the us population was linked with the nlms data to capture demographic , socioeconomic , and occupation attributes from the current population surveys and the annual social and economic supplement . data on county level ses , health care facilities , and health care personnel were obtained from the 2009 - 2010 area resource file which contains over 6,000 variables relating to healthcare , socioeconomic , and environmental characteristics for each county in the us . data on the number of certified mammography facilities was obtained through a freedom of information ( foi ) request to the food and drug administration ( fda ) , the agency in charge of certifications . a list and addresses of all mammography facilities certified in the year 2000 the dataset used in the final analysis was restricted to non - hispanic black and non - hispanic white first primary breast cancer cases among women with ages 40 years and older , diagnosed between 1973 and 2003 . the analysis was restricted to non - hispanic black and white women to facilitate the comparison of two racial groups in the us that have shown sustained and consistently high disparities in breast cancer survival . we focused the main analysis on women residing in counties that contained at least one black and one white breast cancer patient to ensure that the neighborhood level predictors represented the actual experiences of both groups of women . a total of 1796 women met this criterion : 1580 whites and 216 blacks residing in 60 counties in the us . counties were identified using the federal information processing standard ( fips ) code to link county variables with cases ' county of residence at diagnosis as recorded in the seer dataset . marital status was categorized as married , widowed , divorced / separated , and never married . employment status was categorized as women in the labor force and women not in the labor force . education level was categorized as less than high school ( < 12 years ) , high school graduate ( 12 years ) , and at least some college ( 13 years ) . age at diagnosis and income were analyzed as continuous variables ; income was analyzed as inflation - adjusted annual household income ( 1990 standard ) . clinical variables . surgical treatment was categorized as surgical treatment received , not received due to medical reasons , and not received due to nonmedical reasons . radiation treatment was also categorized as received , not received due to medical reasons , and not received due to nonmedical reasons . surgical or radiation treatment not received due to nonmedical reasons included those that were recommended but not performed , recommended and it was unknown if performed , or treatment information was unknown due to death certificate or autopsy diagnosis . we created two censoring variables based on seer cause of death variable : one that indicated if a person had died with breast cancer as the underlying cause , and the other that indicated if a person had died of any cause . patients who died of other causes or were alive at the date of last followup were censored in the first censoring variable , and patients who were alive at the date of last followup were censored in the second censoring variable . health care access was defined as the linear combination of measures of density of health care resources in the county . the derived variables were subject to principal component analysis ( pca ) on count per 10,000 of number of hospitals , number of medical doctors , number of medical doctors with obstetrics and gynecology specialty , number of osteopathic doctors ( do ) , number of dos with obstetrics and gynecology specialty , number of nurse practitioners , and number of mammography facilities . the scores were categorized into tertiles : poorest , middle , and highest . county level ses was defined using the index of concentration in the extremes ( ice ) . this index was chosen over other approaches ( such as the proportion of county residents below poverty level ) because it allows for conceptualizing the concentration of affluence and disadvantage as falling along a single continuum . all county level variables were obtained from the area resource file for the year 2000 because it had the most complete year of data and straddled the years included in the seer dataset . we compared scores for 1990 and 2000 and found them to be highly correlated ( ice income correlation coefficient = 0.90 , p < 0.05 and ice education correlation coefficient = 0.94 , p < 0.05 ) . there were 3141 counties available for the county level analysis of health care access and ses . the first two components of the pca had eigenvalues greater than 1 , and the scree test also showed a clear break after the second component ( figure not shown ) . the two components together accounted for 50.5% of the total variance ; components that had a factor loading value of greater than 0.4 were said to have loaded on a specific component . based on this criterion , mds per 10,000 , mds in ob - gyn per 10,000 , nurse practitioners per 10,000 , and dos per 10,000 were loaded on the first component , hereafter named personnel . multivariable proportional hazards regression was used to model the hazards of breast cancer and all - cause mortality in three separate models . the first model adjusted for demographic variables , the second model included clinical variables regarding stage of presentation and treatment , and the third model included county level variables . robust sandwich estimates for the covariance matrix were used to account for the clustering of cases within counties , and the county fips code was specified as the clustering variable . table 1 provides information on the distribution of county variables relating to healthcare resources , socioeconomic status , and other controls across the us . table 2 illustrates the distribution of demographic , clinical , and county level variables . on average , black patients were about 2 years younger compared with white patients , had a lower annual household income ( $ 25,000 for blacks versus $ 44,700 for whites ) , were more likely to be single ( 19.1% of blacks versus 7.4% of whites ) and divorced ( 19.7% of blacks versus 9.8% of whites ) , and were more likely to have less than a high school education ( 23.6% of blacks versus 9.5% of whites ) . the distribution of stage at diagnosis was similar between blacks and whites ; about 53% of blacks were diagnosed at in situ / localized stage compared with 54.6% of white cases , and 14.2% of blacks black patients were also less likely to have received surgical ( 91% of blacks versus 95% of whites ) and radiation treatment ( 31% of blacks versus 39% of whites ) compared with white patients . at the county level , black patients were more likely than white patients to live in counties that had a higher proportion of households in poverty ( 36.9% of blacks versus 16.8% of whites ) , higher proportion of adults with less than 9 years of education ( 43.2% of blacks versus 26.9% of whites ) , and higher proportion of blacks ( 87.5% of blacks versus 67.5% of whites ) . all of these differences were statistically significant at the p < 0.05 level . in unadjusted analysis ( tables 3 and 4 ) , black women had a 53% higher likelihood of dying of breast cancer ( p = 0.008 ) and 32% higher likelihood of dying of any cause ( p = 0.02 ) compared with white women . having less than high school education increased the likelihood of breast cancer mortality by 68% , and all - cause mortality by 95% . furthermore , being diagnosed at a distant stage and not receiving surgical or radiation treatment was also associated with significantly higher likelihood of death . table 3 also presents the results of three sequential cox proportional hazards multivariable models assessing the determinants of breast cancer survival . model 1 assessed the effect of race on breast cancer mortality after adjusting for demographic variables . race was no longer a statistically significant predictor of breast cancer death after adjusting for the individual variables ( hazard ratio , 1.40 ; 95% ci , 0.991.97 ) . model 2 adjusted additionally for stage at presentation and treatment , and the effect of race remained nonsignificant . model 3 presents the effect of county level variables adjusting for individual demographic and clinical variables on breast cancer mortality . residing in counties with a higher proportion of households in poverty increased the likelihood of breast cancer deaths compared with counties with a higher proportion of affluent households . the hazard ratio of ice - income comparing the poorest versus highest group was 1.29 ( 95% ci , 0.822.05 ) and comparing the middle versus highest group was 1.49 ( 95% ci , 1.121.99 ) . on the other hand , residing in counties with a higher proportion of residents with less than 9 years of education appeared to reduce the likelihood of breast cancer deaths . the hazard ratio of ice - education comparing the poorest versus highest group was 0.55 ( 95% ci , 0.310.98 ) and comparing the middle versus highest group was 0.65 ( 95% ci , 0.440.96 ) . furthermore , residing in a county with a higher proportion of black residents ( 6% ) significantly increased the likelihood of breast cancer death ( hazard ratio , 1.74 ; 95% ci , 1.212.48 ) . facilities and personnel variables did not appear to have an independent significant effect on the likelihood of breast cancer death after adjusting for other variables in the model . table 4 presents the results of the cox proportional hazards models assessing the determinants of all - cause mortality . in contrast to the model predicting breast cancer survival ( table 3 ) , race remained a statistically significant predictor of higher mortality among blacks compared to whites even after adjusting for individual demographic and clinical variables . in model 1 which adjusted for demographic variables , being black was associated with a 38% increase in the likelihood of death due to any cause compared with being white ( 95% ci , 1.081.76 ) . in model 2 , the hazard ratio associated with being black was 1.33 ( 95% ci , 1.041.70 ) after adjusting for demographic and clinical variables . after adjusting for county level variables in model 3 , the effect of race was attenuated and became nonsignificant ( hazard ratio , 1.27 ; 95% ci , 0.951.71 ) . personnel , facilities , county proportion of blacks , and proportion of non - english speaking residents were not significantly associated with all - cause mortality . in this study of black and white women with breast cancer , the effect of race on breast cancer mortality became nonsignificant after adjusting for individual demographic variables . for all - cause mortality , race was a significant predictor even after adjusting for demographic and clinical variables such as stage of presentation and treatment . we observed sustained differences in the receipt of surgical and radiation treatment between black and white patients in this study . this is particularly troubling given that these treatment variables remained the most significant predictors of breast cancer mortality and all - cause mortality even after adjusting for individual and neighborhood level variables . it has been suggested that the disparity in treatment receipt among black women may be due to cultural factors which make them more likely to refuse treatment . however , in this study we were able to distinguish between patients who did not receive treatment due to medical reasons or nonmedical reasons . among women who did not receive treatment due to medical reasons ( implying that a medical personnel made the decision not to treat ) , the proportion of black women was still much higher . upon adjusting for county level healthcare access variables , race was no longer significantly associated with all - cause mortality . facilities and personnel variables did not appear to have a significant independent effect on the likelihood of breast cancer or all - cause mortality after adjusting for other variables in the model . furthermore , residing in counties with a higher proportion of households in poverty increased the likelihood of breast cancer mortality and all - cause mortality compared with counties with a higher proportion of affluent households . our findings suggest that neighborhood poverty and lack of healthcare resources to care might explain part of the black - white disparity in breast cancer survival especially if examined from both individual and neighborhood levels . many studies have sought to explain the causes of racial disparities in breast cancer survival [ 1013 , 17 , 23 , 4554 ] , with varying results . however , none of these studies assessed the role of neighborhood healthcare resources in breast cancer or all - cause survival although other studies have been published about the impact of neighborhood healthcare resources on stage at diagnosis [ 12 , 33 , 55 ] . our study suggests that stage at presentation is an important predictor of breast cancer survival ; late stage at diagnosis was associated with a fourfold increase in the hazard of breast cancer mortality after adjusting for other variables . studies that assessed the role of county level healthcare access on late stage diagnosis of cancer found that women residing in counties with fewer physicians and poor access to mammography facilities were more likely to have late stage cancer diagnosis . other studies have suggested that important predictors of mammography use are having a primary care physician , travel times , and public transportation hassles [ 5564 ] . we anticipated that these county characteristics , number of physicians , and mammography facilities would also be associated with breast cancer survival through the availability of early diagnosis and adequate treatment . however , our measures of healthcare access did not independently predict breast cancer survival , even though the initial racial disparity in survival disappeared . this finding is consistent with a recent publication which did not find an association between the availability of medical resources and breast cancer mortality at the county level . however , due to patient privacy concerns , the seer dataset does not routinely disclose patient geographic location at levels smaller than the county . however , the concept of healthcare access is very complex and multidimensional , incorporating aspects of availability such as the presence of medical facilities and personnel as well as aspects of accessibility such as distance , affordability , and cultural barriers . this study serves as a first step in understanding the role of one aspect of healthcare access on breast cancer survival , and future studies may build on this research to further improve understanding of other aspects . third , other neighborhood level factors such as racial or economic segregation may also be important in understanding racial differences in breast cancer survival and need to be examined . for instance , we found that women residing in counties with a higher proportion of blacks had significantly higher hazards of breast cancer mortality . this has implications for the understanding of the impact of socioeconomic status and breast cancer survival among blacks , because black women earning higher incomes may not benefit as much from their socioeconomic status as white women earning similar wages but residing in high ses counties . we observed a significant increase hazard of breast cancer and all - cause mortality due to neighborhood income . women residing in counties with a higher proportion of low income residents compared with high income residents had higher hazards . this is consistent with other studies using other definitions of county ses [ 20 , 23 , 66 , 67 ] . however , we also observed significant reduction in the hazard of breast cancer and all - cause mortality for counties with a higher proportion of less educated residents . this observation was unexpected especially since higher individual education level was found to be protective in this study as well as in others [ 24 , 37 , 66 , 68 ] . this allowed for better control of potential confounding of the association between neighborhood characteristics and survival by individual ses . another strength is the development of a specific measure of neighborhood healthcare access using the availability of healthcare resources including mammography facilities to better capture factors that may affect early detection which is a major determinant of survival . first , there is a possibility of exposure misclassification bias because measures of individual ses ( household income , education , and employment ) were not obtained at the time of diagnosis with cancer . these measures were obtained through surveys which may have been administered before or after cancer diagnosis . however , our analysis was restricted to breast cancer cases ages 40 and older , reducing the likelihood of dramatic changes in ses through the study period . thirdly , for analytical reasons , the study sample was restricted to women residing in counties that had at least one black and one white breast cancer patients . this implies that the population may be more urban compared with the rest of the us . however , the results of this analysis may still be applicable to semi - urban or rural areas where the impact of county ses and healthcare access on breast cancer survival may be even more pronounced . we performed a sensitivity analysis using only white women ( n = 1580 ) and found similar results ; the neighborhood variables did not significantly predict breast cancer survival among white women . this supports our earlier conclusion that the county level variables may influence breast cancer survival ( for both white and black women ) through the availability of screening and timely treatment . the differential impact of these county variables which may contribute to the racial disparities in breast cancer survival between black and white women ( effect measure modification ) was assessed through interaction terms in the final model ; however there were no significant interactions possibly due to the low sample size of black women within each group . finally , we performed a post hoc power analysis to assess the statistical power of the study to detect a statistically significant difference between breast cancer survival in black and white women . given the sample size of 216 black and 1580 white patients , type 1 error rate of 0.05 , and a followup of 30 years , the study had a power level of 85% . in summary , our study adds an important component to the existing evidence on survival disparities by conceptualizing healthcare access at the county level as a potential determinant of health outcomes and as a potential modifier of the association between race and mortality . further studies may focus on defining healthcare access at smaller geographic levels , for example , zip code or census tracts which may be more homogenous in the distribution of healthcare facilities and personnel .

the secretion of saliva is mediated by the autonomic nervous system , which modifies the protein composition of saliva and triggers fluid secretion . the neuronal release of acetylcholine from parasympathetic nerves plays a central role in inducing salivary fluid secretion from the salivary glands . salivary acinar and ductal functions are regulated by numerous molecular components and mainly involve the activation of ca and cyclic adenosine monophosphate ( camp ) signaling . phosphodiesterase ( pde ) is an important enzyme , responsible for the regulation of intracellular camp and cyclic guanosine monophosphate ( cgmp ) level . it is well established that enhanced camp concentration activates camp - dependent kinase and subsequently triggers exocytosis . pdes are classified into at least 11 families based on affinity , specificity , and amino acid sequences [ 3 , 4 ] . in the submandibular gland ( smg ) , pde isoforms pde1pde5 are expressed in an age - dependent or tissue - specific manner in rodents . pde4 is broadly distributed throughout the body and identified with four gene products and multiple splice variants [ 5 , 6 ] . for example , the involvement of pde4 has been studied in the release of amylase from parotid acinar cells . targeting of pde5 is associated with -adrenergic receptor - stimulated mucin secretion in smg cells . however , in tissues except the salivary tissue , the functional significance of pde isoforms with differential expression and specificity for any species or any tissue is not completely understood . lipopolysaccharides ( lps ) are characteristic components of the bacterial cell wall and stimulate host cells of the innate immune system via the toll - like receptor 4 ( tlr4 ) , a member of the toll - like receptor protein family . tlr signaling is associated with the adaptive immune system and the initiation of inflammatory responses . enhanced tlr4 expression is involved in sjgren 's syndrome ( ss ) and inflammatory intestinal bowel diseases such as crohn 's disease . moreover , we previously provided evidences that tlr4 signaling is critically involved in the proinflammatory cytokine expression in gingival fibroblasts and periodontal ligament fibroblasts [ 11 , 12 ] . recently , dusad et al . demonstrated that repetitive intranasal inhalant exposures to lps resulted in significant bone deterioration . microbial infection and noninfectious stimuli trigger the activation of inflammasome , the protein complex consisting of nod - like receptors ( nlrs ) family , pyrin domain containing-3 ( nlrp3 , known as cryopyrin , cias1 , or nalp3 ) , apoptosis - associated speck - like protein containing a card ( asc ) , and caspase-1 , as the critical components of innate immune response . the activated inflammasome complex leads to the secretion of proinflammatory mediators such as interleukin-1 ( il-1 ) and il-18 , which cause cell damage . it has also been reported that nlrp3 inflammasome - p2x7 receptor complex is involved in the inflammatory response in primary ss patients . however , little is known about nlrp3 inflammasome activation by pathological stimulation in salivary glands . oxidative stress induced by reactive oxygen species ( ros ) is a major risk factor that participates in various cellular functions including salivary gland dysfunction . several studies have demonstrated that ros has significant capacity to mediate cell apoptosis [ 18 , 19 ] . the tlr4 signaling triggered by lps enhances ros production through nadph oxidase [ 20 , 21 ] . the stimulation of tlr4 by lps as an oral pathogen triggers an increase in intracellular ca concentration ( [ ca]i ) and results in inflammatory reaction and enhanced inflammatory cytokine expression as well as nlrp inflammasome activation as mentioned above . pde4 inhibitors have anti - inflammatory and antioxidant effect ; for example , the pde4 inhibitor roflumilast attenuates lps - induced inflammatory mediators in macrophages . another type of pde4 inhibitor rolipram possesses antioxidant potency against the modulation of formyl - methionine - leucine - phenylalanine- ( fmlp- ) induced superoxide anion release in bronchoalveolar lavage cells . the costimulation with -adrenergic agonist isoproterenol and rolipram enhanced amylase secretion in parotid acinar cells . ductal fluid and hco3 secretion are regulated by the luminal and basolateral membrane - associated ion transporters . in addition to the antioxidative effect of rolipram , the enhanced camp level induced by rolipram may be involved in the synergistic stimulation of neuronal input signal and ductal bicarbonate secretion . however , little is known about the expression pattern of pde4 and the inhibitory effect of the pde4 inhibitor rolipram on the inflammatory mediator - induced signals in salivary glands . in addition , the regulatory role of rolipram on neuronal agonist - induced signals and camp - mediated ductal bicarbonate secretion in salivary glands should be clarified . in this study , we investigated the multifunction of rolipram in inflammatory mediators / neuronal agonist - induced signaling and modulation of nlrp3 inflammasome . moreover , we explored whether rolipram modulates camp - dependent chloride / bicarbonate exchange activity in ductal bicarbonate secretion in isolated mouse smg cells . fura-2-acetoxymethyl ester ( fura-2-am ) and 2,7-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein- ( bcecf- ) am were purchased from teflabs ( austin , tx ) . 3-aminobenzamide ( 3-ab ) , histamine , lps from pseudomonas aeruginosa serotype 10 , rolipram , carbamyl choline chloride ( carbachol ) , isoproterenol , hydrogen peroxide , trypsin inhibitor , sodium pyruvate , bovine serum albumin ( bsa ) , -actin antibody , and all other chemicals not mentioned here were purchased from sigma . phosphate - buffered saline ( pbs ) , pluronic f-127 ( 20% in dmso ) , goat serum , e - cadherin antibody , zo-1 antibody , parp-1 antibody , and mqae dye were purchased from invitrogen ( carlsbad , ca ) . pde4 and caspase-1 antibodies were purchased from fabgennix ( frisco , tx ) and abcam ( cambridge , ma ) , respectively . all procedures for maintaining the mice and for the isolation of acini and ducts followed gachon university guidelines and were approved by the animal care and use committee of gachon university . smg isolated from 3035 g balb c wild - type mice were washed and resuspended in physiological salt solution ( pss ) containing 140 mm nacl , 10 mm glucose , 1 mm mgcl2 , 5 mm kcl , 10 mm hepes and 1 mm cacl2 , ph 7.4 , 0.02% soybean - trypsin inhibitor , 0.1% sodium pyruvate , and 0.1% bsa and kept on ice until use . briefly , the minced smg was incubated in physiological salt solution a ( psa ) containing 2.5 mg/10 ml collagenase p ( roche ) for 8 min at 37c . the digest was washed with psa for three times , resuspended in psa , and kept on ice until use . isolated smg cells were transferred onto cover glasses and incubated with 4 m fura-2-am in the presence of 0.05% pluronic f-127 for 45 min for smg cells in pss at room temperature in the dark and then washed for 10 min with pss . changes in [ ca]i were determined by measuring the fluorescence intensities using dual excitation wavelengths , 340 and 380 nm , and an emission wavelength of 510 nm . emitted fluorescence was monitored using a ccd camera ( photometrics , az ) attached to an inverted microscope ( olympus , japan ) and analyzed with a metafluor system ( molecular devices , pa ) . fluorescence images were obtained at 1 sec intervals and the background fluorescence at each excitation wavelength was subtracted from raw signals . ca responses were calculated by dividing the maximum ca peak of agonist in the presence of rolipram by the maximum ca peak of agonist stimulation . phi was measured with bcecf at dual excitation wavelengths of 495 nm and 440 nm . bcecf fluorescence was read at emission wavelengths above 530 nm . isolated smg cells attached onto coverslips were loaded in the chamber with bcecf in the presence of 0.05% pluronic f-127 for 15 min incubation with pss at room temperature with 6 m bcecf - am . after stabilizing the fluorescence , the cells were perfused with pss for at least 5 min before measuring phi at 37c . cbe activity was measured by incubating the cells with co2-saturated hco3-buffered media to acidify the cytosol . cbe activity was initiated by perfusing the cells with cl - free hco3-buffered media containing 140 mm na . the emitted fluorescence was monitored with a ccd camera ( photometrics , tucson , az ) attached to an inverted microscope ( olympus , japan ) and analyzed with a metamorph system ( molecular devices , pa ) . fluorescence images were obtained at 1 sec intervals and the background fluorescence was subtracted from the raw background signals at each wavelength . cbe activity was determined from the slopes and the derivatives of the first 3045 sec of phi increases . experiments were performed with sliced smg tissues and isolated smg acinar and ductal clusters were evaluated for the localization of e - cadherin . isolated submandibular acini and ducts were plated on glass coverslips for 5 min at room temperature prior to fixation with chilled ( 20c ) methanol . after fixation , immunostaining was performed as described previously using 1 : 100 dilution of the e - cadherin and zo-1 antibodies . briefly , the cells were incubated with the primary antibodies overnight at 4c and after washing unbound antibodies with 5% bsa / pbs for three times , the bound antibodies were detected with goat anti - rabbit immunoglobulin g ( igg ) tagged with fluorescein isothiocyanate ( fitc ) ( e - cadherin and zo-1 ) and then washed with pbs for three times . coverslips were mounted on glass slides with fluoromount - gtm including dapi ( electron microscopy sciences , hatfield , pa ) and analyzed using a lsm 700 zeiss confocal microscope ( germany ) with zen software . to determine the normalized intensity of e - cadherin in each image , average intensity of e - images were collected from four to five separate preparations of acinar and ductal cells and results are the averages from all experiments . total rna was extracted from isolated smg cells using trizol extraction system from invitrogen according to the manufacturer 's instructions . total rna was amplified according to the manufacturer 's protocol using topscript one - step rt - pcr kit from enzynomics ( daejeon , south korea ) . the pcr was started with a denaturation step at 95c for 5 min , followed by 35 cycles at 95c for 1 min , an annealing step for 1 min , an extension step at 72c for 1 min , and a final extension step at 72c for 10 min . the pcr products were electrophoresed on 1% agarose gels . to measure the ros production in isolated smg cells , oxiselect intracellular ros assay kit with green fluorescence ( cell biolabs , ca ) was used . the cells were attached to a 96-well plate treated with 0.005% poly - l - lysine ( sigma ) . cells were incubated with 100 l dcfh - da - containing media at 37c for 1 hr and washed with pbs for three times . after discarding last pbs , the cells were treated with 20 g / ml lps , 100 m histamine , and 100 m h2o2 in the presence or absence of 10 g / ml rolipram for 1 hr at room temperature . then , the cells were washed with pbs for three times and lysed with psa solution - containing cell lysis buffer ( cell biolabs ) . after incubation for 5 min , cell lysate was transferred to a new 96-well plate . the dcf fluorescence of the plate was measured with victo3 ( perkinelmer , ma ) at 485 nm/535 nm ( excitation / emission ) wavelengths . to calculate dcf concentrations from plate , a standard curve was plotted as per the manufacturer 's instructions ( cell biolabs ) . the value of absorbance was substituted for the y - variable in the equation of standard curve . the smg ductal cells on the coverslip were loaded with mqae by 30 min of incubation at room temperature in bath solution containing 5 mm mqae and were washed by perfusion with nacl - based solution until stabilization of the baseline signal . the mqae fluorescence was recorded for at least 3 min to obtain the baseline and then switched perfusion solution with 0 mm cl ( 0cl ) . then mqae fluorescence was recorded at an excitation of 360 nm and light emitted at a wavelength higher than 530 nm was collected with a ccd camera ( photometrics ) . cell lysates were prepared in lysis buffer ( containing 20 mm tris , 150 mm nacl , 2 mm edta , 1% tritonx-100 , and a protease inhibitor mixture ) by passing 1518 times through a 27-gauge needle after sonication . the lysates were centrifuged at 11,000 g for 20 min at 4c , and protein concentration in the supernatants was determined . proteins were denatured by heating in sds sample buffer at 37c for 30 min . the 30 g heated protein samples were subjected to sds - page and transferred to methanol - soaked polyvinylidene difluoride ( pvdf ) membranes . transferred proteins on pvdf membranes were visualized with caspase-1 ( abcam ) and -actin ( sigma ) antibodies by enhanced luminescent solution ( thermo scientific ) . to evaluate the inhibitory role of rolipram in inflammatory mediator signaling , rt - pcr was used to assess the expression of pde4 subfamily , tlr4 , and histamine receptors ( hr ) in mouse smg cells . primarily isolated smg acinar cells expressed pde4a through pde4d , tlr4 , and h1r mrna ( figure 1(a ) ) . it will be of particular interest to determine the localization of pde4 , which may regulate camp - dependent cellular functions . thus , we evaluated the protein expression of pde4 in smg tissues and isolated cells . expression of pde4 isoforms was not modulated in the presence of rolipram ( figure 1(a ) ) . to evaluate whether the modulatory effect of rolipram was mediated by tlr4 activation in isolated smg acinar cells , lps - induced [ ca]i measurement was performed in the absence or presence of rolipram . pretreatment of rolipram inhibited lps - induced [ ca]i peak ( n = 4 , figure 1(b ) ) . these results show that lps - triggered [ ca]i response significantly ( p < 0.01 ) decreased in the presence of rolipram . similarly , rolipram inhibited histamine - evoked [ ca]i response ( n = 3 , figures 1(d ) and 1(e ) ) . these results show that rolipram has strong inhibitory effect on the inflammatory mediator - induced [ ca]i signals . since inflammatory mediators can recruit ros - mediated signal , h2o2-evoked [ ca]i mobilization was evaluated in the presence of rolipram in the salivary glands . to examine the antioxidative role of rolipram , smg acinar cells were stimulated with 10 mm h2o2 in the presence or absence of rolipram . the h2o2-induced [ ca]i increase was significantly inhibited by rolipram ( n = 4 , figures 2(a ) and 2(b ) ) . preincubation of human salivary gland ( hsg ) cells with the ploy nad - metabolite adp - ribose polymerase-1 ( parp-1 ) inhibitor 3-ab significantly decreased h2o2-induced [ ca]i increase . similarly , in the present study , the h2o2-induced [ ca]i signaling in mouse smg cells was attenuated by 3-ab ( n = 4 , figures 2(c ) and 2(d ) ) . parp-1 was localized in the cytosol and predominantly in smg cell nuclei ( figure 2(e ) ) . these results suggest that rolipram attenuates h2o2-induced [ ca]i increase in smg acinar cells . to determine whether rolipram inhibits ros production , whole smg cells including ductal cells were stimulated with inflammatory mediators lps , histamine , and h2o2 for 30 min and the dcf fluorescence intensities were measured . rolipram inhibited lps- and histamine - induced ros production as well as the resting ros level ; however , rolipram moderately inhibited h2o2 stimulation ( n = 3 , figure 2(f ) ) . while rolipram significantly inhibited the inflammatory mediator - induced [ ca]i response , its effect on h2o2 stimulation was not marked . accordingly , we compared the effect of rolipram on h2o2-induced [ ca]i response between acini and ductal cells . interestingly , rolipram inhibited h2o2-induced [ ca]i response of acinar cells but showed no effect on that of ductal cells ( figure 3(a ) ) . to further demonstrate the role of rolipram as shown in figure 3(b ) , the cells were stimulated with lps to induce cell membrane damage and stained with the plasma membrane marker e - cadherin . the notable finding was that the smg acini cell membrane was protected by rolipram in the presence of lps ( figures 3(b ) and 3(c ) ) . it was hard to find ductal cells after stimulation with lps ( data not shown ) ; however , in the presence of rolipram , e - cadherin and tight junction marker zo-1 staining could be observed at the plasma and apical membrane , respectively ( figures 3(b)~3(e ) ) . tlr4 activation by lps and subsequent [ ca]i increases are necessary for the activation of the nlrp3 inflammasome . thus , to determine the expression of nlrp3 induced by lps treatment and the regulatory role of rolipram in expressing the nlrp3 inflammasome , smg cells were treated with lps for 30 min in the absence and presence of rolipram . we observed for the first time that isolated smg cells significantly expressed nlrp3 in the presence of lps . as shown in figure 3(f ) , lps enhanced nlrp3 mrna expression , but rolipram attenuated nlrp3 expression . we confirmed that well - established inflammasome component caspase-1 was inhibited by rolipram ( figure 3(g ) ) . in addition to the anti - inflammatory effect of rolipram , the role of rolipram in neurotransmitter inputs such as cholinergic / adrenergic stimulation - induced [ ca]i response was evaluated in smg acinar and ductal cells ; the cells were stimulated with the muscarinic receptor agonist , carbachol , and the -adrenergic agonist , isoproterenol ( iso ) in the absence and presence of rolipram . in figures 4(a)4(d ) , acinar and ductal cells showed carbachol / isoproterenol - induced [ ca]i peak and plateau responses except ductal isoproterenol - induced [ ca]i response ( figure 4(e ) ) . these results indicated that cholinergic / adrenergic stimulation - induced [ ca]i responses were enhanced in the presence of rolipram . the camp signaling pathway stimulates epithelial chloride / bicarbonate exchanger such as luminal solute carrier 26 family member 6 ( slc26a6 ) in the duct . enhanced camp and [ ca]i concentration resulted in the synergistic activation of ion transporters , which induce changes in intracellular ph ( phi ) and directly reflect epithelial fluid secretion [ 25 , 28 ] . to determine effects of rolipram on the modulation of camp - associated ion transporters such as anion exchanger and solute carrier transporter , we measured the chloride / bicarbonate exchanger activity in ductal cells . the chloride / bicarbonate exchanger activity was evaluated by measuring the changes in phi induced by acute chloride removal and subsequent addition of chloride ( figures 5(a ) and 5(b ) ) . the slope of the change in phi effect was measured using chloride - free bicarbonate - buffered solution . we confirmed chloride movement with chloride - sensitive dye mqae in ductal cells ( figure 5(c ) ) . as shown in figure 5 , the slope of phi as chloride / bicarbonate exchanger activity and chloride movement in the presence of rolipram did not show any statistical difference . in this study , we demonstrated the regulatory effect of pde4 inhibitor rolipram on ca signaling and ros production induced by the stimulation of inflammatory mediators in acinar and ductal cells isolated from mouse submandibular salivary glands . additionally , various pde4 subfamilies have been reported to be present in mouse submandibular glands and pde4 was localized in the luminal membrane of acini and ducts . rolipram has been developed and studied as a potent antidepressant , which elevates rat brain camp in vivo [ 29 , 30 ] . pde family members degrade camp , and they are expressed fundamentally in all immune cells including neutrophils , eosinophils , lymphocytes , and macrophages . the anti - inflammatory role of pde inhibitor rolipram on immune cells is well established , whereas the role of rolipram on salivary glands has been addressed in camp - dependent amylase secretion [ 5 , 7 ] . regulation of inflammatory signal and camp - dependent electrolyte secretion by rolipram in salivary gland remains unknown . moreover , there are no previous reports indicating the effect of rolipram on inflammatory mediators or cholinergic / adrenergic - induced signaling and camp - dependent bicarbonate secretion in the salivary glands . in the present study , we focused on the inhibitory effect of rolipram on inflammatory mediator - induced [ ca]i signaling and ros production . in the presence of rolipram , lps or histamine - induced [ ca]i signaling was dramatically attenuated in mouse smg acinar cells . pretreatment of rolipram increased camp concentration and subsequently may activate protein kinase a ( pka ) , leading to reduction of ros production and , moreover , may block the [ ca]i signaling cascade . however , how to acutely attenuate [ ca]i signaling by rolipram and its signaling mechanism should be clarified in coming years . lps - induced oxidative stress was addressed by ros through nadph oxidase ( nox ) . tlr4 recruits the nox4 and then is able to generate ros in the form of h2o2 . although the candidate of ros source was most likely nox4 , we can not rule out the mitochondrial ros source . the tlr signaling results in the production and expression of inflammatory mediators including il-6 and il-8 in the salivary glands . normal human smg cells expressed tlr1 - 10 mrna and salivary glands of patients with ss showed enhanced expression of tlr2 , tlr3 , and tlr4 [ 9 , 33 ] . stimulation of human salivary gland cells with tlr ligands augmented the expression of inflammatory cytokines such as il-6 and cd54 and further cell apoptosis , which means tlr signaling may be involved in pathological process in ss . we provided the first evidence that tlr - mediated [ ca]i signal is associated with the enhanced gene expression of nlrp3 inflammasome , which is attenuated by rolipram in smg cells ( figure 3(f ) ) and cleaved caspase-1 signaling was inhibited ( figure 3(g ) ) . although ligand of nlrp3 inflammasome is unclear , enhanced camp production inhibited the activation of the nlrp3 inflammasome through the direct regulatory role of camp on the nlrp3 complex . however , further studies investigating the precise role of rolipram and the possible involvement of pde in the inflammasome complex such as thioredoxin - interacting protein txnip and adaptor protein asc should be carried out . it is noteworthy that rolipram , which regulates intracellular camp levels and inhibits the inflammatory signaling , may be applied in systemic autoimmune diseases such as ss and acute bacterial infection . in addition to the regulatory role of rolipram on the inflammatory mediators in this study , rolipram enhanced the amylase release by -adrenergic stimulation in parotid glands . fluid and protein secretion from the salivary gland due to signals such as neurotransmitters input are determined by coordinated spatial and temporal modulation of ca signaling mechanism . moreover , the magnitude or spatial and temporal regulation of ca signaling is important for the stimulation and maintenance of fluid secretion . cholinergic/-adrenergic stimulations by carbachol / isoproterenol - induced [ ca]i signaling were enhanced in the presence of rolipram in figure 4 , suggesting that enhanced camp caused by rolipram and [ ca]i concentration by carbachol / isoproterenol results in the synergistic activation for fluid secretion . it has been reported that amylase secretion is regulated by camp and [ ca]i , which mediate the enhanced secretion by combined stimulation . generally , high ca microdomain at luminal membrane is essential to drive protein and fluid secretion . localization of pde4 at apical membrane as shown in figure 1(a ) may ensure the fidelity and rapid activation for protein secretion by regulating the local camp level . ca and camp are debatably the classical second messengers that control various types of cellular homeostasis . neurotransmitter signaling such as cholinergic and muscarinic receptor activation might be integrated with synergism to control the physiological response through camp - pka mechanism , whereas the signaling through histamine or lps did not mediate in this mechanism . in salivary glands , histamine stimulation transfers its signal through h1 receptor , which is independent signaling with pka and the lps signal was inhibited by pka in the pretreatment with rolipram . otherwise , it is possible that the upstream mechanism of pka might be involved . although the differential role of pka in both neuronal input and inflammatory signaling pathway should be elucidated , rolipram is an effective agent , which modulates those two signaling pathways . in the present study , we did not observe significantly altered chloride / bicarbonate exchange activity for bicarbonate secretion in ductal cells , suggesting the effect of rolipram is more associated with acinar cells than ductal cells . the cellular distribution or expression level of pde family including pde4 between smg acinar and ductal cells may be different and should be correlated with their function . although the exact expression of other pde isoforms might be determined in the coming years , the ductal cells were dominantly damaged by oxidative stress and showed no statistical difference in the chloride / bicarbonate exchanger activity with enhanced camp level by rolipram , suggesting that the duct may require other pde family members for fluid secretion or possess more sensitivity to oxidative stress than acini . moreover , the significance of the different pde4 isoforms in the effect of rolipram needs to be clarified . rolipram possessed different inhibitory action on ductal versus acinar cells in the treatment with h2o2 . the antioxidative capacity between ductal and acinar cells not differential inhibitory action of rolipram should be different . the differential identification of ros scavenging system and oxidative - dependent channels should be clarified between ductal and acinar cells . it has been proposed that long - term administration up to 2 months of calcineurin inhibitors decreases antioxidant enzyme activity and alters saliva composition . similarly , the administration of high dose of pde inhibitor ici 153,110 for up to 6 months influenced the epithelial cell proliferation including salivary glands . accordingly , long - term administration of rolipram may result in decreased redox control , as reflected by the enhanced antioxidative role of rolipram . in the current study , although we did not examine the long - term administration of rolipram , the involvement of pde4 was found to modulate acute tlr - mediated [ ca]i signaling and ros production .

exposure to bacterial lipopolysaccharides ( lps ) induces inflammatory signals in salivary glands . we investigated the regulatory role of phosphodiesterase 4 ( pde4 ) inhibitor rolipram on inflammatory mediators and cholinergic / adrenergic stimulation - induced intracellular ca2 + signaling in salivary acinar and ductal cells . submandibular gland ( smg ) expressed pde4a through 4d mrna and pde4 was localized in the luminal membrane of smg . lps induced ca2 + signaling and ros production in smg . treatment with rolipram blocked lps - induced ca2 + increase and ros production . the application of histamine evoked ca2 + signals and ros production , which were attenuated by rolipram in smg cells . moreover , lps - induced nlrp3 inflammasome and cleaved caspase-1 were inhibited by rolipram . the inhibitory role of rolipram in ros - induced ca2 + signaling was mainly observed in acinar cells and not in ductal cells . rolipram also protected smg acinar but not ductal cells from lps - induced cell membrane damage . in the case of cholinergic / adrenergic stimulation , carbachol / isoproterenol - induced ca2 + signals were upregulated by the treatment of rolipram in smg . in the case of camp - dependent ductal bicarbonate secretion by rolipram , no effect was observed on the modulation of ductal chloride / bicarbonate exchange activity . rolipram could suppress the inflammatory signals and could be a potential therapeutic strategy against lps - induced inflammation to protect the salivary gland cells .

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

the secretion of saliva is mediated by the autonomic nervous system , which modifies the protein composition of saliva and triggers fluid secretion . the neuronal release of acetylcholine from parasympathetic nerves plays a central role in inducing salivary fluid secretion from the salivary glands . salivary acinar and ductal functions are regulated by numerous molecular components and mainly involve the activation of ca and cyclic adenosine monophosphate ( camp ) signaling . it is well established that enhanced camp concentration activates camp - dependent kinase and subsequently triggers exocytosis . in the submandibular gland ( smg ) , pde isoforms pde1pde5 are expressed in an age - dependent or tissue - specific manner in rodents . for example , the involvement of pde4 has been studied in the release of amylase from parotid acinar cells . targeting of pde5 is associated with -adrenergic receptor - stimulated mucin secretion in smg cells . lipopolysaccharides ( lps ) are characteristic components of the bacterial cell wall and stimulate host cells of the innate immune system via the toll - like receptor 4 ( tlr4 ) , a member of the toll - like receptor protein family . moreover , we previously provided evidences that tlr4 signaling is critically involved in the proinflammatory cytokine expression in gingival fibroblasts and periodontal ligament fibroblasts [ 11 , 12 ] . it has also been reported that nlrp3 inflammasome - p2x7 receptor complex is involved in the inflammatory response in primary ss patients . however , little is known about nlrp3 inflammasome activation by pathological stimulation in salivary glands . the tlr4 signaling triggered by lps enhances ros production through nadph oxidase [ 20 , 21 ] . pde4 inhibitors have anti - inflammatory and antioxidant effect ; for example , the pde4 inhibitor roflumilast attenuates lps - induced inflammatory mediators in macrophages . another type of pde4 inhibitor rolipram possesses antioxidant potency against the modulation of formyl - methionine - leucine - phenylalanine- ( fmlp- ) induced superoxide anion release in bronchoalveolar lavage cells . the costimulation with -adrenergic agonist isoproterenol and rolipram enhanced amylase secretion in parotid acinar cells . ductal fluid and hco3 secretion are regulated by the luminal and basolateral membrane - associated ion transporters . in addition to the antioxidative effect of rolipram , the enhanced camp level induced by rolipram may be involved in the synergistic stimulation of neuronal input signal and ductal bicarbonate secretion . however , little is known about the expression pattern of pde4 and the inhibitory effect of the pde4 inhibitor rolipram on the inflammatory mediator - induced signals in salivary glands . in addition , the regulatory role of rolipram on neuronal agonist - induced signals and camp - mediated ductal bicarbonate secretion in salivary glands should be clarified . in this study , we investigated the multifunction of rolipram in inflammatory mediators / neuronal agonist - induced signaling and modulation of nlrp3 inflammasome . moreover , we explored whether rolipram modulates camp - dependent chloride / bicarbonate exchange activity in ductal bicarbonate secretion in isolated mouse smg cells . 3-aminobenzamide ( 3-ab ) , histamine , lps from pseudomonas aeruginosa serotype 10 , rolipram , carbamyl choline chloride ( carbachol ) , isoproterenol , hydrogen peroxide , trypsin inhibitor , sodium pyruvate , bovine serum albumin ( bsa ) , -actin antibody , and all other chemicals not mentioned here were purchased from sigma . isolated smg cells were transferred onto cover glasses and incubated with 4 m fura-2-am in the presence of 0.05% pluronic f-127 for 45 min for smg cells in pss at room temperature in the dark and then washed for 10 min with pss . ca responses were calculated by dividing the maximum ca peak of agonist in the presence of rolipram by the maximum ca peak of agonist stimulation . isolated smg cells attached onto coverslips were loaded in the chamber with bcecf in the presence of 0.05% pluronic f-127 for 15 min incubation with pss at room temperature with 6 m bcecf - am . experiments were performed with sliced smg tissues and isolated smg acinar and ductal clusters were evaluated for the localization of e - cadherin . to determine the normalized intensity of e - cadherin in each image , average intensity of e - images were collected from four to five separate preparations of acinar and ductal cells and results are the averages from all experiments . total rna was extracted from isolated smg cells using trizol extraction system from invitrogen according to the manufacturer 's instructions . to measure the ros production in isolated smg cells , oxiselect intracellular ros assay kit with green fluorescence ( cell biolabs , ca ) was used . after discarding last pbs , the cells were treated with 20 g / ml lps , 100 m histamine , and 100 m h2o2 in the presence or absence of 10 g / ml rolipram for 1 hr at room temperature . the smg ductal cells on the coverslip were loaded with mqae by 30 min of incubation at room temperature in bath solution containing 5 mm mqae and were washed by perfusion with nacl - based solution until stabilization of the baseline signal . to evaluate the inhibitory role of rolipram in inflammatory mediator signaling , rt - pcr was used to assess the expression of pde4 subfamily , tlr4 , and histamine receptors ( hr ) in mouse smg cells . primarily isolated smg acinar cells expressed pde4a through pde4d , tlr4 , and h1r mrna ( figure 1(a ) ) . it will be of particular interest to determine the localization of pde4 , which may regulate camp - dependent cellular functions . thus , we evaluated the protein expression of pde4 in smg tissues and isolated cells . expression of pde4 isoforms was not modulated in the presence of rolipram ( figure 1(a ) ) . to evaluate whether the modulatory effect of rolipram was mediated by tlr4 activation in isolated smg acinar cells , lps - induced [ ca]i measurement was performed in the absence or presence of rolipram . pretreatment of rolipram inhibited lps - induced [ ca]i peak ( n = 4 , figure 1(b ) ) . these results show that lps - triggered [ ca]i response significantly ( p < 0.01 ) decreased in the presence of rolipram . these results show that rolipram has strong inhibitory effect on the inflammatory mediator - induced [ ca]i signals . since inflammatory mediators can recruit ros - mediated signal , h2o2-evoked [ ca]i mobilization was evaluated in the presence of rolipram in the salivary glands . to examine the antioxidative role of rolipram , smg acinar cells were stimulated with 10 mm h2o2 in the presence or absence of rolipram . the h2o2-induced [ ca]i increase was significantly inhibited by rolipram ( n = 4 , figures 2(a ) and 2(b ) ) . preincubation of human salivary gland ( hsg ) cells with the ploy nad - metabolite adp - ribose polymerase-1 ( parp-1 ) inhibitor 3-ab significantly decreased h2o2-induced [ ca]i increase . similarly , in the present study , the h2o2-induced [ ca]i signaling in mouse smg cells was attenuated by 3-ab ( n = 4 , figures 2(c ) and 2(d ) ) . parp-1 was localized in the cytosol and predominantly in smg cell nuclei ( figure 2(e ) ) . these results suggest that rolipram attenuates h2o2-induced [ ca]i increase in smg acinar cells . to determine whether rolipram inhibits ros production , whole smg cells including ductal cells were stimulated with inflammatory mediators lps , histamine , and h2o2 for 30 min and the dcf fluorescence intensities were measured . rolipram inhibited lps- and histamine - induced ros production as well as the resting ros level ; however , rolipram moderately inhibited h2o2 stimulation ( n = 3 , figure 2(f ) ) . while rolipram significantly inhibited the inflammatory mediator - induced [ ca]i response , its effect on h2o2 stimulation was not marked . accordingly , we compared the effect of rolipram on h2o2-induced [ ca]i response between acini and ductal cells . interestingly , rolipram inhibited h2o2-induced [ ca]i response of acinar cells but showed no effect on that of ductal cells ( figure 3(a ) ) . to further demonstrate the role of rolipram as shown in figure 3(b ) , the cells were stimulated with lps to induce cell membrane damage and stained with the plasma membrane marker e - cadherin . the notable finding was that the smg acini cell membrane was protected by rolipram in the presence of lps ( figures 3(b ) and 3(c ) ) . it was hard to find ductal cells after stimulation with lps ( data not shown ) ; however , in the presence of rolipram , e - cadherin and tight junction marker zo-1 staining could be observed at the plasma and apical membrane , respectively ( figures 3(b)~3(e ) ) . tlr4 activation by lps and subsequent [ ca]i increases are necessary for the activation of the nlrp3 inflammasome . thus , to determine the expression of nlrp3 induced by lps treatment and the regulatory role of rolipram in expressing the nlrp3 inflammasome , smg cells were treated with lps for 30 min in the absence and presence of rolipram . we observed for the first time that isolated smg cells significantly expressed nlrp3 in the presence of lps . we confirmed that well - established inflammasome component caspase-1 was inhibited by rolipram ( figure 3(g ) ) . in addition to the anti - inflammatory effect of rolipram , the role of rolipram in neurotransmitter inputs such as cholinergic / adrenergic stimulation - induced [ ca]i response was evaluated in smg acinar and ductal cells ; the cells were stimulated with the muscarinic receptor agonist , carbachol , and the -adrenergic agonist , isoproterenol ( iso ) in the absence and presence of rolipram . in figures 4(a)4(d ) , acinar and ductal cells showed carbachol / isoproterenol - induced [ ca]i peak and plateau responses except ductal isoproterenol - induced [ ca]i response ( figure 4(e ) ) . these results indicated that cholinergic / adrenergic stimulation - induced [ ca]i responses were enhanced in the presence of rolipram . the camp signaling pathway stimulates epithelial chloride / bicarbonate exchanger such as luminal solute carrier 26 family member 6 ( slc26a6 ) in the duct . enhanced camp and [ ca]i concentration resulted in the synergistic activation of ion transporters , which induce changes in intracellular ph ( phi ) and directly reflect epithelial fluid secretion [ 25 , 28 ] . to determine effects of rolipram on the modulation of camp - associated ion transporters such as anion exchanger and solute carrier transporter , we measured the chloride / bicarbonate exchanger activity in ductal cells . the chloride / bicarbonate exchanger activity was evaluated by measuring the changes in phi induced by acute chloride removal and subsequent addition of chloride ( figures 5(a ) and 5(b ) ) . we confirmed chloride movement with chloride - sensitive dye mqae in ductal cells ( figure 5(c ) ) . as shown in figure 5 , the slope of phi as chloride / bicarbonate exchanger activity and chloride movement in the presence of rolipram did not show any statistical difference . in this study , we demonstrated the regulatory effect of pde4 inhibitor rolipram on ca signaling and ros production induced by the stimulation of inflammatory mediators in acinar and ductal cells isolated from mouse submandibular salivary glands . additionally , various pde4 subfamilies have been reported to be present in mouse submandibular glands and pde4 was localized in the luminal membrane of acini and ducts . the anti - inflammatory role of pde inhibitor rolipram on immune cells is well established , whereas the role of rolipram on salivary glands has been addressed in camp - dependent amylase secretion [ 5 , 7 ] . regulation of inflammatory signal and camp - dependent electrolyte secretion by rolipram in salivary gland remains unknown . moreover , there are no previous reports indicating the effect of rolipram on inflammatory mediators or cholinergic / adrenergic - induced signaling and camp - dependent bicarbonate secretion in the salivary glands . in the present study , we focused on the inhibitory effect of rolipram on inflammatory mediator - induced [ ca]i signaling and ros production . in the presence of rolipram , lps or histamine - induced [ ca]i signaling was dramatically attenuated in mouse smg acinar cells . pretreatment of rolipram increased camp concentration and subsequently may activate protein kinase a ( pka ) , leading to reduction of ros production and , moreover , may block the [ ca]i signaling cascade . lps - induced oxidative stress was addressed by ros through nadph oxidase ( nox ) . the tlr signaling results in the production and expression of inflammatory mediators including il-6 and il-8 in the salivary glands . normal human smg cells expressed tlr1 - 10 mrna and salivary glands of patients with ss showed enhanced expression of tlr2 , tlr3 , and tlr4 [ 9 , 33 ] . stimulation of human salivary gland cells with tlr ligands augmented the expression of inflammatory cytokines such as il-6 and cd54 and further cell apoptosis , which means tlr signaling may be involved in pathological process in ss . we provided the first evidence that tlr - mediated [ ca]i signal is associated with the enhanced gene expression of nlrp3 inflammasome , which is attenuated by rolipram in smg cells ( figure 3(f ) ) and cleaved caspase-1 signaling was inhibited ( figure 3(g ) ) . although ligand of nlrp3 inflammasome is unclear , enhanced camp production inhibited the activation of the nlrp3 inflammasome through the direct regulatory role of camp on the nlrp3 complex . however , further studies investigating the precise role of rolipram and the possible involvement of pde in the inflammasome complex such as thioredoxin - interacting protein txnip and adaptor protein asc should be carried out . it is noteworthy that rolipram , which regulates intracellular camp levels and inhibits the inflammatory signaling , may be applied in systemic autoimmune diseases such as ss and acute bacterial infection . in addition to the regulatory role of rolipram on the inflammatory mediators in this study , rolipram enhanced the amylase release by -adrenergic stimulation in parotid glands . fluid and protein secretion from the salivary gland due to signals such as neurotransmitters input are determined by coordinated spatial and temporal modulation of ca signaling mechanism . moreover , the magnitude or spatial and temporal regulation of ca signaling is important for the stimulation and maintenance of fluid secretion . cholinergic/-adrenergic stimulations by carbachol / isoproterenol - induced [ ca]i signaling were enhanced in the presence of rolipram in figure 4 , suggesting that enhanced camp caused by rolipram and [ ca]i concentration by carbachol / isoproterenol results in the synergistic activation for fluid secretion . it has been reported that amylase secretion is regulated by camp and [ ca]i , which mediate the enhanced secretion by combined stimulation . generally , high ca microdomain at luminal membrane is essential to drive protein and fluid secretion . localization of pde4 at apical membrane as shown in figure 1(a ) may ensure the fidelity and rapid activation for protein secretion by regulating the local camp level . neurotransmitter signaling such as cholinergic and muscarinic receptor activation might be integrated with synergism to control the physiological response through camp - pka mechanism , whereas the signaling through histamine or lps did not mediate in this mechanism . in salivary glands , histamine stimulation transfers its signal through h1 receptor , which is independent signaling with pka and the lps signal was inhibited by pka in the pretreatment with rolipram . although the differential role of pka in both neuronal input and inflammatory signaling pathway should be elucidated , rolipram is an effective agent , which modulates those two signaling pathways . in the present study , we did not observe significantly altered chloride / bicarbonate exchange activity for bicarbonate secretion in ductal cells , suggesting the effect of rolipram is more associated with acinar cells than ductal cells . the cellular distribution or expression level of pde family including pde4 between smg acinar and ductal cells may be different and should be correlated with their function . although the exact expression of other pde isoforms might be determined in the coming years , the ductal cells were dominantly damaged by oxidative stress and showed no statistical difference in the chloride / bicarbonate exchanger activity with enhanced camp level by rolipram , suggesting that the duct may require other pde family members for fluid secretion or possess more sensitivity to oxidative stress than acini . moreover , the significance of the different pde4 isoforms in the effect of rolipram needs to be clarified . rolipram possessed different inhibitory action on ductal versus acinar cells in the treatment with h2o2 . the antioxidative capacity between ductal and acinar cells not differential inhibitory action of rolipram should be different . the differential identification of ros scavenging system and oxidative - dependent channels should be clarified between ductal and acinar cells . similarly , the administration of high dose of pde inhibitor ici 153,110 for up to 6 months influenced the epithelial cell proliferation including salivary glands . accordingly , long - term administration of rolipram may result in decreased redox control , as reflected by the enhanced antioxidative role of rolipram . in the current study , although we did not examine the long - term administration of rolipram , the involvement of pde4 was found to modulate acute tlr - mediated [ ca]i signaling and ros production .

acinetobacter baumannii is a glucose non - fermentative gram - negative bacillus classified as an opportunistic pathogen responsible for nosocomial infections , especially in intensive care units ( icu ) and burn therapy units ( btu ) ( 1 , 2 ) . a. baumannii is responsible for nosocomial pneumonia , wound infections , bacteremia , urinary tract infection , meningitis , endocarditis , osteomyelitis and keratitis ( 3 ) . also infections of respiratory tract , central nervous system , urinary tract and peritonitis can be caused by a. baumannii especially in immunocompromised patients ( 3 ) . in the recent years , the emergence of multidrug - resistant a. baumannii has complicated the therapy of a. baumannii infections ( 4 ) . multidrug - resistance ( mdr ) is defined as resistance to three or more representatives of the following classes of antibiotics : fluoroquinolones , third generation cephalosporins , aminoglycosides and carbapenems ( 5 ) . mdr is compounded by increasing resistance to broad - spectrum antibiotics including carbapenems as one of the most effective antibiotics against gram - negative rods ( 6 , 7 ) . although , most of a. baumannii isolates were susceptible to carbapenems previously and imipenem was the most effective treatment for a. baumannii infections , widespread use of carbapenem has caused emergence of resistant strains ( 8 , 9 ) . the emergence of carbapenem resistance in a. baumannii is a significant public health concern ( 3 ) . one of the major mechanisms of carbapenem resistance in this pathogen is production of carbapenem hydrolyzing beta- lactamases mostly oxacillinase ( oxa ) types carbapenemases ( 10 ) . carbapenem resistance in a. baumannii is mediated by acquisition of a class b or class d beta - lactamase genes , such as oxacillinase genes ( 11 ) . oxa - type carbapenamases have been divided into eight subgroups , four of which have been identified in a. baumannii : oxa-23-like , oxa-24-like , oxa-58-like and oxa-51-like ( 12 ) . recently , it has been suggested that enzymes belonging to the oxa-51-like subgroup are intrinsic to a. baumannii , occurring in most or all strains , although they are very variably expressed ( 13 ) . to control and prevent dissemination of resistant isolates , molecular method for typing of mdr a. baumannii the significant contribution of oxacillinase genes to carbapenem resistance has been emphasized , particularly when they are accompanied by isaba1 and isaba3 in the naturally occurring plasmids ( 2 , 15 ) . several studies on these genes have been performed in the world and in some parts of iran ; however , there is no data on the distribution of blaoxa - type genes in a. baumannii isolates in shiraz . this study can help us to know the prevalence of blaoxa - type genes in a. baumannii isolates in our hospital settings for better infection control and treatment in hospitals in shiraz . the aim of this study was to determine antimicrobial susceptibility patterns and distribution of blaoxa - type carbapenemase genes among a. baumannii isolates from hospitalized patients in four teaching hospitals , in shiraz , southwest iran . from december 2013 to may 2013 , 200 a. baumannii were isolated from different clinical specimens , including urine , wound , blood , sputum , endotracheal tube ( ett ) , body fluid , nose , throat and eye from four shiraz teaching hospitals ( faghihi , aliasghar , ghotebedin and nemazee ) in shiraz , southwest iran . some relevant information including sex , sample type and ward name was recorded . the specimens were cultured on macconkey agar ( merck , germany ) and blood agar ( merck , germany ) , then incubated at 37c for 24 - 48 hours . these isolates were identified using standard biochemical tests , including oxidase test , tsi ( triple sugar iron agar ) medium ( merck , germany ) and sim ( sulfide , indole , motility ) medium ( merck , germany ) . negative result for oxidase test , no motility and non - fermentation and growth in temperature of 42 - 44c was considered as the elementary criteria for a. baumannii detection . they were confirmed by microgen kits ( mast , uk ) according to the manufacturer instructions ( www.microgenbioproducts.com ) . antimicrobial susceptibility testing was performed by disk diffusion method according to the clinical and laboratory standards institute ( clsi ) ( 4 , 16 ) . disk diffusion method was performed on muller - hinton agar ( merck , germany ) , using an inoculum of 10 cfu . antibiotic disks ( mast , uk ) containing ampicillin ( 10 g ) , ampicillin - sulbactam ( 10 g ) , piperacillin ( 100 g ) , piperacillin - tazobactam ( 110 g ) , amikacin ( 30 g ) , gentamicin ( 120 g ) , imipenem ( 10 g ) , meropenem ( 10 g ) , ciprofloxacin ( 5 g ) , levofloxacin ( 5 g ) , ceftazidime ( 30 g ) , cefoxitine ( 30 g ) , aztreonam ( 30 g ) , colistin ( 25 g ) , polymyxin b ( 300 unit ) and tigecycline ( 15 g ) . these antibiotic disks were then placed on agar plates and incubated at 37c for 24 hours . escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality controls strain in each susceptibility determination ( 6 ) . 2 - 3 grown bacterial colonies on muller - hinton agar plates were suspended in 180 l pbs . then dna of the bacteria was extracted by phenol - chloroform ( cinnagen , iran ) method with some modifications as described before ( 14 ) . they were used as template in polymerase chain reaction ( pcr ) assay to amplify blaoxa carbapenemase genes ( 17 ) . identification of a. baumannii was confirmed using blaoxa-51-like pcr assay by specific primers ( gene fanavaran , iran ) ( 18 ) . to amplify the blaoxa-51-like gene , each reaction final volume of 25 l was considered , containing 1x pcr buffer ( cinnagen , iran ) , 1 u taq polymerase ( cinnagen , iran ) , 1.5 mm mgcl2 ( cinnagen , iran ) , 200 m of dntp ( cinnagen , iran ) , 10 pmol of each primer and 1 l of extracted dna . pcr conditions were programed in an eppendorf mastercycler as follows : initial denaturation at 94c for 3 min , 35 cycles of 94c for 45 s , 57c for 45 s , 72c for 1 min and 5 minute final extension of 72c . pcr products were electrophoresed ( bio rad , usa ) on 1.2% agarose gel ( cinnagen , iran ) , stained with ethidium bromide solution ( 1 mg / ml ) ( cinnagen , iran ) and placed under uv gel transilluminator . negative control was also included in each pcr reaction , containing all components except the dna template which was replaced by distilled water ( 6 , 14 ) . a multiplex pcr targeting blaoxa-23-like , blaoxa-24-like and blaoxa-58-like genes was used to screen the isolates as previously described ( 17 ) . all the isolates were subjected to multiplex pcr performed to detect blaoxa-23-like , blaoxa-24-like and blaoxa-58-like using specific primers ( gene fanavaran , iran ) , as listed in table 1 ( 19 ) . final volume of 25 l included 1x pcr buffer , 1 u taq polymerase , 2 mm mgcl2 , 200 m of dntp , 0.2 m of each primer and 1 l of template dna . amplification protocol was initial denaturation at 94c for 5 min , 94c for 30 s , 53c for 40 s and 72c for 50 s and final extension at 72c for 6 min . the pcr products were run in 2.5% agarose gel electrophoresis , stained with ethidium bromide ( cinnagene , iran ) and visualized under uv gel transilluminator . a. baumannii reference strains nctc 13304 , nctc 13302 and nctc 13305 were used as positive control for blaoxa-23-like , blaoxa-24-like and blaoxa-58-like , respectively ( 6 , 14 ) . to determine the association between variables , data was analyzed by chi - square and fisher s exact test using spss , version 21 ( spss inc . from december 2013 to may 2013 , 200 a. baumannii were isolated from different clinical specimens , including urine , wound , blood , sputum , endotracheal tube ( ett ) , body fluid , nose , throat and eye from four shiraz teaching hospitals ( faghihi , aliasghar , ghotebedin and nemazee ) in shiraz , southwest iran . some relevant information including sex , sample type and ward name was recorded . the specimens were cultured on macconkey agar ( merck , germany ) and blood agar ( merck , germany ) , then incubated at 37c for 24 - 48 hours . these isolates were identified using standard biochemical tests , including oxidase test , tsi ( triple sugar iron agar ) medium ( merck , germany ) and sim ( sulfide , indole , motility ) medium ( merck , germany ) . negative result for oxidase test , no motility and non - fermentation and growth in temperature of 42 - 44c was considered as the elementary criteria for a. baumannii detection . they were confirmed by microgen kits ( mast , uk ) according to the manufacturer instructions ( www.microgenbioproducts.com ) . antimicrobial susceptibility testing was performed by disk diffusion method according to the clinical and laboratory standards institute ( clsi ) ( 4 , 16 ) . disk diffusion method was performed on muller - hinton agar ( merck , germany ) , using an inoculum of 10 cfu . antibiotic disks ( mast , uk ) containing ampicillin ( 10 g ) , ampicillin - sulbactam ( 10 g ) , piperacillin ( 100 g ) , piperacillin - tazobactam ( 110 g ) , amikacin ( 30 g ) , gentamicin ( 120 g ) , imipenem ( 10 g ) , meropenem ( 10 g ) , ciprofloxacin ( 5 g ) , levofloxacin ( 5 g ) , ceftazidime ( 30 g ) , cefoxitine ( 30 g ) , aztreonam ( 30 g ) , colistin ( 25 g ) , polymyxin b ( 300 unit ) and tigecycline ( 15 g ) . these antibiotic disks were then placed on agar plates and incubated at 37c for 24 hours . escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality controls strain in each susceptibility determination ( 6 ) . 2 - 3 grown bacterial colonies on muller - hinton agar plates were suspended in 180 l pbs . then dna of the bacteria was extracted by phenol - chloroform ( cinnagen , iran ) method with some modifications as described before ( 14 ) . they were used as template in polymerase chain reaction ( pcr ) assay to amplify blaoxa carbapenemase genes ( 17 ) . identification of a. baumannii was confirmed using blaoxa-51-like pcr assay by specific primers ( gene fanavaran , iran ) ( 18 ) . to amplify the blaoxa-51-like gene , each reaction final volume of 25 l was considered , containing 1x pcr buffer ( cinnagen , iran ) , 1 u taq polymerase ( cinnagen , iran ) , 1.5 mm mgcl2 ( cinnagen , iran ) , 200 m of dntp ( cinnagen , iran ) , 10 pmol of each primer and 1 l of extracted dna . pcr conditions were programed in an eppendorf mastercycler as follows : initial denaturation at 94c for 3 min , 35 cycles of 94c for 45 s , 57c for 45 s , 72c for 1 min and 5 minute final extension of 72c . pcr products were electrophoresed ( bio rad , usa ) on 1.2% agarose gel ( cinnagen , iran ) , stained with ethidium bromide solution ( 1 mg / ml ) ( cinnagen , iran ) and placed under uv gel transilluminator . negative control was also included in each pcr reaction , containing all components except the dna template which was replaced by distilled water ( 6 , 14 ) . a multiplex pcr targeting blaoxa-23-like , blaoxa-24-like and blaoxa-58-like genes was used to screen the isolates as previously described ( 17 ) . all the isolates were subjected to multiplex pcr performed to detect blaoxa-23-like , blaoxa-24-like and blaoxa-58-like using specific primers ( gene fanavaran , iran ) , as listed in table 1 ( 19 ) . final volume of 25 l included 1x pcr buffer , 1 u taq polymerase , 2 mm mgcl2 , 200 m of dntp , 0.2 m of each primer and 1 l of template dna . amplification protocol was initial denaturation at 94c for 5 min , 94c for 30 s , 53c for 40 s and 72c for 50 s and final extension at 72c for 6 min . the pcr products were run in 2.5% agarose gel electrophoresis , stained with ethidium bromide ( cinnagene , iran ) and visualized under uv gel transilluminator . a. baumannii reference strains nctc 13304 , nctc 13302 and nctc 13305 were used as positive control for blaoxa-23-like , blaoxa-24-like and blaoxa-58-like , respectively ( 6 , 14 ) . to determine the association between variables , data was analyzed by chi - square and fisher s exact test using spss , version 21 ( spss inc . of two hundred isolates , 109 ( 54.5% ) were from male and 91 ( 45.5% ) from female patients . isolates were collected from four hospitals ; 82 ( 41% ) from nemazee hospital , 53 ( 26.5% ) from aliasghar hospital , 48 ( 24% ) from faghihi hospital and 17 ( 8.5% ) from ghotbedin hospital . the rates of isolation from different wards were as follows ; icu 143 ( 71.5% ) , surgeries ward 16 ( 8% ) , neurosurgical icu 14 ( 7% ) , neonates 8 ( 4% ) , female internal ward 7 ( 3.5% ) , male internal ward 7 ( 3.5% ) and organ transplantation ward 5 ( 2.5% ) . of two hundred isolates , 81 ( 40.5% ) were from sputum , 43 ( 21.5% ) from endotracheal tube , 22 ( 11% ) from wound , 16 ( 8% ) from urine , 12 ( 6% ) from blood , 12 ( 6% ) from body fluids , 4 ( 2% ) from nose , 3 ( 1.5% ) from throat , 2 ( 1% ) from csf , 1 ( 0.5% ) from eye specimens and 4 ( 2% ) other samples . all a. baumannii isolates ( n = 200 ) in this study had positive results for blaoxa-51-like by pcr ( figure 1 ) . 1 , 2 , 3 , 4 , 5 , clinical isolates of a. baumannii with blaoxa-51-like gene ; c- , negative control ; c+ , positive control ; m , 100 bp dna ladder . all the isolates were susceptible to colistin and polymyxin b and all were mdr as defined before , they were totally resistant to piperacillin , piperacillin - tazobactam , ampicillin , ceftazidime , cefoxitin and aztreonam ( table 2 ) . the association between source of isolates and antibiotic resistance pattern was not statistically significant ( p > 0.05 ) . all isolates of a. baumannii were tested by multiplex pcr for the presence of oxacillinase genes . eighty ( 40% ) of 200 isolates had positive results for blaoxa-23-like , 14 ( 7% ) for blaoxa-24-like and 1 ( 0.5% ) isolate for blaoxa-58-like . the co - existence of two different blaoxa genes in the samples was detected for blaoxa-23-like plus blaoxa-24-like in nine ( 4.5% ) isolates ( figure 2 ) . 1 , clinical isolate of a. baumannii containing blaoxa-23-like and blaoxa-24-like genes ; 2 , clinical isolate of a. baumannii having blaoxa-58-like gene ; 3,4 , clinical isolates of a. baumannii having blaoxa-23-like genes ; 5 , clinical isolate of a. baumannii with blaoxa-24-like gene ; c- , negative control ; c+ , positive control ( a. baumannii nctc 13304 , nctc 13302 , nctc 13305 for blaoxa-23-like , blaoxa-24-like , blaoxa-58-like ) ; m , 100 bp dna ladder . of two hundred isolates , 109 ( 54.5% ) were from male and 91 ( 45.5% ) from female patients . isolates were collected from four hospitals ; 82 ( 41% ) from nemazee hospital , 53 ( 26.5% ) from aliasghar hospital , 48 ( 24% ) from faghihi hospital and 17 ( 8.5% ) from ghotbedin hospital . the rates of isolation from different wards were as follows ; icu 143 ( 71.5% ) , surgeries ward 16 ( 8% ) , neurosurgical icu 14 ( 7% ) , neonates 8 ( 4% ) , female internal ward 7 ( 3.5% ) , male internal ward 7 ( 3.5% ) and organ transplantation ward 5 ( 2.5% ) . of two hundred isolates , 81 ( 40.5% ) were from sputum , 43 ( 21.5% ) from endotracheal tube , 22 ( 11% ) from wound , 16 ( 8% ) from urine , 12 ( 6% ) from blood , 12 ( 6% ) from body fluids , 4 ( 2% ) from nose , 3 ( 1.5% ) from throat , 2 ( 1% ) from csf , 1 ( 0.5% ) from eye specimens and 4 ( 2% ) other samples . all a. baumannii isolates ( n = 200 ) in this study had positive results for blaoxa-51-like by pcr ( figure 1 ) . 1 , 2 , 3 , 4 , 5 , clinical isolates of a. baumannii with blaoxa-51-like gene ; c- , negative control ; c+ , positive control ; m , 100 bp dna ladder . all the isolates were susceptible to colistin and polymyxin b and all were mdr as defined before , they were totally resistant to piperacillin , piperacillin - tazobactam , ampicillin , ceftazidime , cefoxitin and aztreonam ( table 2 ) . the association between source of isolates and antibiotic resistance pattern was not statistically significant ( p > 0.05 ) . all isolates of a. baumannii were tested by multiplex pcr for the presence of oxacillinase genes . eighty ( 40% ) of 200 isolates had positive results for blaoxa-23-like , 14 ( 7% ) for blaoxa-24-like and 1 ( 0.5% ) isolate for blaoxa-58-like . the co - existence of two different blaoxa genes in the samples was detected for blaoxa-23-like plus blaoxa-24-like in nine ( 4.5% ) isolates ( figure 2 ) . 1 , clinical isolate of a. baumannii containing blaoxa-23-like and blaoxa-24-like genes ; 2 , clinical isolate of a. baumannii having blaoxa-58-like gene ; 3,4 , clinical isolates of a. baumannii having blaoxa-23-like genes ; 5 , clinical isolate of a. baumannii with blaoxa-24-like gene ; c- , negative control ; c+ , positive control ( a. baumannii nctc 13304 , nctc 13302 , nctc 13305 for blaoxa-23-like , blaoxa-24-like , blaoxa-58-like ) ; m , 100 bp dna ladder . acinetobacter baumannii is an emerging nosocomial pathogen which is in part due to its capacity of acquiring resistance to multiple antimicrobial agents ( 20 ) . occurrence of multidrug - resistance and pandrug - resistant ( pdr ) in a. baumannii is a growing concern ( 7 ) . all of the isolates in this study were multidrug - resistant and resistant to most of the antibiotics ( table 2 ) . this pathogen is an important gram - negative bacterium involved in nosocomial infections , especially in icu wards ( 18 ) . as shown in our study , 71.5% of the isolates were obtained from hospitalized patients in icu . this result confirms the fact that a. baumannii is often an important cause of infection in hospitalized patients in icu . to detect antibiotic resistant patterns , our results revealed that 98.5% and 99.5% of the isolates were resistant to imipenem and meropenem , respectively . in some studies in tehran , 50.9% , 52.5% , 62% and 67.5% of the isolates were resistant to imipenem and 51.8% , 52.5% , 62% and 84.5% to meropenem in 2008 , 2009 , 2011 and 2013 , respectively ( 6 , 12 , 18 , 20 ) . in another study in tehran in 2013 , resistance rates of 99% and 91.5% were reported to imipenem and meropenem , respectively ( 22 ) . mirnejad and vafaei , reported 76% resistance to imipenem and 69% resistance to meropenem among tested isolates in tehran in 2013 ( 23 ) . in two separate studies , one in ahvaz in 2013 , 96.1% resistance to imipenem and meropenem ( 14 ) , and another in kermanshah in 2013 , 79.8% resistance to imipenem and 75% resistance to meropenem were reported ( 15 ) . therefore , our results showed that resistance to carbapenems has an increasing trend which is probably because of dissemination of highly resistant lineages of a. baumannii in our area . increased resistance to carbapenem causes a real concern over an imminent threat of untreatable a. baumannii infections ( 3 ) . the current study showed low susceptibility rates to most of available antimicrobial agents for the treatment of infections caused by a. baumannii , except for polymyxin b and colistin . in our study , all of the isolates were sensitive to colistin and polymyxin b , while other studies conducted in tehran demonstrated 12% resistance to colistin and 3% resistance to polymyxin b in 2011 ( 17 ) . moreover , in two studies in tehran and ahvaz in 2013 , all of the isolates were sensitive to these antibiotics ( 12 , 14 ) . in a study in kermanshah in 2013 , all isolates were sensitive to polymyxin b and colistin ( 15 ) . sensitivity of all a. baumannii isolates to colistin was also reported in tehran in 2013 ( 22 ) . also in a study in tehran in 2013 , among all antibiotic tested , the lowest resistance rate to polymyxin b ( 3% ) was observed ( 23 ) . the current study also described the important role of class d carbapenem hydrolyzing beta - lactamases . production of class d oxacillinase by a. baumannii distributed worldwide is the main mechanism of resistance to carbapenems in this organism . the major carbapenemase genes involved in carbapenem resistance in a. baumannii are blaoxa-23-like , blaoxa-24-like and blaoxa-58-like ( 8 , 14 ) . alleles encoding oxa-23-like , oxa-24-like and oxa-58-like enzymes were consistently associated with resistance or at least reduced susceptibility to carbapenemases ( 24 ) . the blaoxa-51-like genes are located intrinsically in chromosome of all a. baumannii strains ( 8) . this result provides evidence that detection of blaoxa-51-like can be used as a simple and reliable way of identifying a. baumannii ( 13 ) . genbank submissions describing variants from isolates of a. baumannii from many different countries distributed over four continents clearly suggest that blaoxa-51-like is ubiquitous in a. baumannii ( 25 ) . oxa-23-like was the first oxa - type beta - lactamase identified in a. baumannii ( 26 ) . the results obtained in this study indicated that most of our isolates ( 40% ) carried the blaoxa-23-like gene . in other studies investigating outbreaks of oxa-23-producing a. baumannii strains , the rate of blaoxa-23-like ranged 31% to 94% in different parts of the world ( 14 ) . so our results for blaoxa-23-like gene are in the reported ranges . in both separate studies in tehran , in 2008 and 2009 , 25% of the isolates had positive results for blaoxa-23-like gene ( 6 , 20 ) . moreover , in other two studies by karmostaji et al . ( 22 ) , in tehran in 2013 , 81.3% and 55.7% had positive findings for this gene , respectively . 85% and 55.7% positivity rates were reported for blaoxa-23-like gene in studied a. baumannii isolates in ahvaz and kermanshah , respectively ( 14 , 15 ) . in our study , the reported frequency rate of this gene has been previously reported as 0 - 85.4% in different parts of the world ( 14 ) . in a study in tehran in 2008 , 17.9% of isolates contained blaoxa-24-like gene ( 6 ) ; in addition , in another study in tehran in 2009 , 15% had positive results for this gene ( 20 ) . three studies in 2013 in tehran , ahvaz and kermanshah showed frequencies of 8.13% , 8.7% and 19.2% for this gene in the studied isolates , respectively ( 12 , 14 , 15 ) . some authors reported blaoxa-58-like frequency as 2 - 84.9% in a. baumannii isolates in different parts of the world ( 14 ) . in several studies in tehran in 2008 , 2009 and 2013 , 9% , 21.2% and 0.8% of the isolates had positive results for blaoxa-58-like , respectively ( 6 , 12 , 20 ) . moreover , a study conducted in ahvaz and kermanshah , in 2013 , revealed that blaoxa-58-like gene was not detected ( 14 , 15 ) . in our study , we identified blaoxa-58-like gene only in one a. baumannii isolate ( 0.5% ) . we identified nine isolates ( 4.5% ) with co - existence of two different blaoxa-23-like plus blaoxa-24-like . in two studies in 2013 , in tehran and kermanshah , 5.7% and 16.4% of a. baumannii isolates had such a co - existence ( 12 , 15 ) . in another study in 2013 in ahvaz , no co - existence between these genes was reported ( 14 ) . of note , 105 a. baumannii isolates resistant to imipenem and meropenem in our study possessed only the intrinsic blaoxa-51-like , but they had negative result for other investigated genes . resistance to carbapenems in those isolates may be due to other mechanisms other than oxacillinase production ( 14 , 27 ) . in conclusion , such a. baumannii with different blaoxa - carbapenemase genes were isolated from hospitalized patients at shiraz teaching hospitals ( nemazee , faghihi , aliasghar , ghotbedin ) in different wards . the blaoxa-51-like genes were the most prevalent subgroup , as they are intrinsic to a. baumannii . moreover , blaoxa-23-like gene is another most prevalent resistance gene among mdr a. baumannii isolates . controlling infections of mdr a. baumannii in hospitals needs a common strategy issued by decision makers and health - care authorities to make hospitals a safer place for patients .

background : the emergence of multidrug - resistant acinetobacter baumannii complicates the therapy of the related infections . hospital isolates of a. baumannii are usually multidrug - resistant . the problem is compounded by increasing resistance to broad - spectrum antibiotics including carbapenems.objectives:the aim of this study was to determine antimicrobial susceptibility patterns and distribution of blaoxa - type carbapenemases genes among a. baumannii isolates from hospitalized patients in shiraz , southwest iran.materials and methods : two hundred a. baumannii isolates were recovered from different clinical specimens in four shiraz teaching hospitals . isolates were detected as a. baumannii by microgen kit and pcr with specific primers of blaoxa-51-like gene . antimicrobial susceptibility testing was determined by disk diffusion method for all the isolates . multiplex pcr assays were performed for detection of blaoxa-23-like , blaoxa-24-like and blaoxa-58-like genes.results:all the isolates were susceptible to colistin and polymyxin b. moreover , all of them were resistant to piperacillin , piperacillin - tazobactam , ampicillin , ceftazidime , cefoxitin and aztreonam . eighty ( 40% ) isolates had positive results for blaoxa-23-like , 14 ( 7% ) for blaoxa-24-like and 1 ( 0.5% ) isolate for blaoxa-58-like . the co - existence of studied genes was detected for blaoxa-23-like plus blaoxa-24-like in nine ( 4.5% ) isolates.conclusions:the prevalence of carbapenem resistant a. baumannii isolates in shiraz hospitals is high . the blaoxa-23-like gene was the most frequent carbapenemase identified among resistant a. baumannii isolated in shiraz hospitals . the increasing incidence of a. baumannii is a serious concern , therefore control of this pathogen and taking preventive measures are emphasized .

1. Background 2. Objectives 3. Materials and Methods 3.1. Bacterial Strains 3.2. Antimicrobial Susceptibility Testing 3.3. DNA Extraction 3.4. Detection of blaOXA-51-like Gene 3.5. Multiplex PCR Assay for Detection of Oxacillinase Genes 3.6. Statistical Analyses 4. Results 4.1. Bacterial Isolates 4.2. Antimicrobial Susceptibility Testing 4.3. Detection of Oxacillinase Genes 5. Discussion

acinetobacter baumannii is a glucose non - fermentative gram - negative bacillus classified as an opportunistic pathogen responsible for nosocomial infections , especially in intensive care units ( icu ) and burn therapy units ( btu ) ( 1 , 2 ) . in the recent years , the emergence of multidrug - resistant a. baumannii has complicated the therapy of a. baumannii infections ( 4 ) . multidrug - resistance ( mdr ) is defined as resistance to three or more representatives of the following classes of antibiotics : fluoroquinolones , third generation cephalosporins , aminoglycosides and carbapenems ( 5 ) . mdr is compounded by increasing resistance to broad - spectrum antibiotics including carbapenems as one of the most effective antibiotics against gram - negative rods ( 6 , 7 ) . although , most of a. baumannii isolates were susceptible to carbapenems previously and imipenem was the most effective treatment for a. baumannii infections , widespread use of carbapenem has caused emergence of resistant strains ( 8 , 9 ) . the emergence of carbapenem resistance in a. baumannii is a significant public health concern ( 3 ) . one of the major mechanisms of carbapenem resistance in this pathogen is production of carbapenem hydrolyzing beta- lactamases mostly oxacillinase ( oxa ) types carbapenemases ( 10 ) . carbapenem resistance in a. baumannii is mediated by acquisition of a class b or class d beta - lactamase genes , such as oxacillinase genes ( 11 ) . several studies on these genes have been performed in the world and in some parts of iran ; however , there is no data on the distribution of blaoxa - type genes in a. baumannii isolates in shiraz . this study can help us to know the prevalence of blaoxa - type genes in a. baumannii isolates in our hospital settings for better infection control and treatment in hospitals in shiraz . the aim of this study was to determine antimicrobial susceptibility patterns and distribution of blaoxa - type carbapenemase genes among a. baumannii isolates from hospitalized patients in four teaching hospitals , in shiraz , southwest iran . from december 2013 to may 2013 , 200 a. baumannii were isolated from different clinical specimens , including urine , wound , blood , sputum , endotracheal tube ( ett ) , body fluid , nose , throat and eye from four shiraz teaching hospitals ( faghihi , aliasghar , ghotebedin and nemazee ) in shiraz , southwest iran . antimicrobial susceptibility testing was performed by disk diffusion method according to the clinical and laboratory standards institute ( clsi ) ( 4 , 16 ) . antibiotic disks ( mast , uk ) containing ampicillin ( 10 g ) , ampicillin - sulbactam ( 10 g ) , piperacillin ( 100 g ) , piperacillin - tazobactam ( 110 g ) , amikacin ( 30 g ) , gentamicin ( 120 g ) , imipenem ( 10 g ) , meropenem ( 10 g ) , ciprofloxacin ( 5 g ) , levofloxacin ( 5 g ) , ceftazidime ( 30 g ) , cefoxitine ( 30 g ) , aztreonam ( 30 g ) , colistin ( 25 g ) , polymyxin b ( 300 unit ) and tigecycline ( 15 g ) . identification of a. baumannii was confirmed using blaoxa-51-like pcr assay by specific primers ( gene fanavaran , iran ) ( 18 ) . a multiplex pcr targeting blaoxa-23-like , blaoxa-24-like and blaoxa-58-like genes was used to screen the isolates as previously described ( 17 ) . all the isolates were subjected to multiplex pcr performed to detect blaoxa-23-like , blaoxa-24-like and blaoxa-58-like using specific primers ( gene fanavaran , iran ) , as listed in table 1 ( 19 ) . a. baumannii reference strains nctc 13304 , nctc 13302 and nctc 13305 were used as positive control for blaoxa-23-like , blaoxa-24-like and blaoxa-58-like , respectively ( 6 , 14 ) . from december 2013 to may 2013 , 200 a. baumannii were isolated from different clinical specimens , including urine , wound , blood , sputum , endotracheal tube ( ett ) , body fluid , nose , throat and eye from four shiraz teaching hospitals ( faghihi , aliasghar , ghotebedin and nemazee ) in shiraz , southwest iran . antimicrobial susceptibility testing was performed by disk diffusion method according to the clinical and laboratory standards institute ( clsi ) ( 4 , 16 ) . antibiotic disks ( mast , uk ) containing ampicillin ( 10 g ) , ampicillin - sulbactam ( 10 g ) , piperacillin ( 100 g ) , piperacillin - tazobactam ( 110 g ) , amikacin ( 30 g ) , gentamicin ( 120 g ) , imipenem ( 10 g ) , meropenem ( 10 g ) , ciprofloxacin ( 5 g ) , levofloxacin ( 5 g ) , ceftazidime ( 30 g ) , cefoxitine ( 30 g ) , aztreonam ( 30 g ) , colistin ( 25 g ) , polymyxin b ( 300 unit ) and tigecycline ( 15 g ) . identification of a. baumannii was confirmed using blaoxa-51-like pcr assay by specific primers ( gene fanavaran , iran ) ( 18 ) . a multiplex pcr targeting blaoxa-23-like , blaoxa-24-like and blaoxa-58-like genes was used to screen the isolates as previously described ( 17 ) . all the isolates were subjected to multiplex pcr performed to detect blaoxa-23-like , blaoxa-24-like and blaoxa-58-like using specific primers ( gene fanavaran , iran ) , as listed in table 1 ( 19 ) . a. baumannii reference strains nctc 13304 , nctc 13302 and nctc 13305 were used as positive control for blaoxa-23-like , blaoxa-24-like and blaoxa-58-like , respectively ( 6 , 14 ) . the rates of isolation from different wards were as follows ; icu 143 ( 71.5% ) , surgeries ward 16 ( 8% ) , neurosurgical icu 14 ( 7% ) , neonates 8 ( 4% ) , female internal ward 7 ( 3.5% ) , male internal ward 7 ( 3.5% ) and organ transplantation ward 5 ( 2.5% ) . of two hundred isolates , 81 ( 40.5% ) were from sputum , 43 ( 21.5% ) from endotracheal tube , 22 ( 11% ) from wound , 16 ( 8% ) from urine , 12 ( 6% ) from blood , 12 ( 6% ) from body fluids , 4 ( 2% ) from nose , 3 ( 1.5% ) from throat , 2 ( 1% ) from csf , 1 ( 0.5% ) from eye specimens and 4 ( 2% ) other samples . all a. baumannii isolates ( n = 200 ) in this study had positive results for blaoxa-51-like by pcr ( figure 1 ) . 1 , 2 , 3 , 4 , 5 , clinical isolates of a. baumannii with blaoxa-51-like gene ; c- , negative control ; c+ , positive control ; m , 100 bp dna ladder . all the isolates were susceptible to colistin and polymyxin b and all were mdr as defined before , they were totally resistant to piperacillin , piperacillin - tazobactam , ampicillin , ceftazidime , cefoxitin and aztreonam ( table 2 ) . all isolates of a. baumannii were tested by multiplex pcr for the presence of oxacillinase genes . eighty ( 40% ) of 200 isolates had positive results for blaoxa-23-like , 14 ( 7% ) for blaoxa-24-like and 1 ( 0.5% ) isolate for blaoxa-58-like . the co - existence of two different blaoxa genes in the samples was detected for blaoxa-23-like plus blaoxa-24-like in nine ( 4.5% ) isolates ( figure 2 ) . 1 , clinical isolate of a. baumannii containing blaoxa-23-like and blaoxa-24-like genes ; 2 , clinical isolate of a. baumannii having blaoxa-58-like gene ; 3,4 , clinical isolates of a. baumannii having blaoxa-23-like genes ; 5 , clinical isolate of a. baumannii with blaoxa-24-like gene ; c- , negative control ; c+ , positive control ( a. baumannii nctc 13304 , nctc 13302 , nctc 13305 for blaoxa-23-like , blaoxa-24-like , blaoxa-58-like ) ; m , 100 bp dna ladder . the rates of isolation from different wards were as follows ; icu 143 ( 71.5% ) , surgeries ward 16 ( 8% ) , neurosurgical icu 14 ( 7% ) , neonates 8 ( 4% ) , female internal ward 7 ( 3.5% ) , male internal ward 7 ( 3.5% ) and organ transplantation ward 5 ( 2.5% ) . of two hundred isolates , 81 ( 40.5% ) were from sputum , 43 ( 21.5% ) from endotracheal tube , 22 ( 11% ) from wound , 16 ( 8% ) from urine , 12 ( 6% ) from blood , 12 ( 6% ) from body fluids , 4 ( 2% ) from nose , 3 ( 1.5% ) from throat , 2 ( 1% ) from csf , 1 ( 0.5% ) from eye specimens and 4 ( 2% ) other samples . all a. baumannii isolates ( n = 200 ) in this study had positive results for blaoxa-51-like by pcr ( figure 1 ) . 1 , 2 , 3 , 4 , 5 , clinical isolates of a. baumannii with blaoxa-51-like gene ; c- , negative control ; c+ , positive control ; m , 100 bp dna ladder . all the isolates were susceptible to colistin and polymyxin b and all were mdr as defined before , they were totally resistant to piperacillin , piperacillin - tazobactam , ampicillin , ceftazidime , cefoxitin and aztreonam ( table 2 ) . all isolates of a. baumannii were tested by multiplex pcr for the presence of oxacillinase genes . eighty ( 40% ) of 200 isolates had positive results for blaoxa-23-like , 14 ( 7% ) for blaoxa-24-like and 1 ( 0.5% ) isolate for blaoxa-58-like . the co - existence of two different blaoxa genes in the samples was detected for blaoxa-23-like plus blaoxa-24-like in nine ( 4.5% ) isolates ( figure 2 ) . 1 , clinical isolate of a. baumannii containing blaoxa-23-like and blaoxa-24-like genes ; 2 , clinical isolate of a. baumannii having blaoxa-58-like gene ; 3,4 , clinical isolates of a. baumannii having blaoxa-23-like genes ; 5 , clinical isolate of a. baumannii with blaoxa-24-like gene ; c- , negative control ; c+ , positive control ( a. baumannii nctc 13304 , nctc 13302 , nctc 13305 for blaoxa-23-like , blaoxa-24-like , blaoxa-58-like ) ; m , 100 bp dna ladder . acinetobacter baumannii is an emerging nosocomial pathogen which is in part due to its capacity of acquiring resistance to multiple antimicrobial agents ( 20 ) . occurrence of multidrug - resistance and pandrug - resistant ( pdr ) in a. baumannii is a growing concern ( 7 ) . all of the isolates in this study were multidrug - resistant and resistant to most of the antibiotics ( table 2 ) . as shown in our study , 71.5% of the isolates were obtained from hospitalized patients in icu . this result confirms the fact that a. baumannii is often an important cause of infection in hospitalized patients in icu . to detect antibiotic resistant patterns , our results revealed that 98.5% and 99.5% of the isolates were resistant to imipenem and meropenem , respectively . in some studies in tehran , 50.9% , 52.5% , 62% and 67.5% of the isolates were resistant to imipenem and 51.8% , 52.5% , 62% and 84.5% to meropenem in 2008 , 2009 , 2011 and 2013 , respectively ( 6 , 12 , 18 , 20 ) . therefore , our results showed that resistance to carbapenems has an increasing trend which is probably because of dissemination of highly resistant lineages of a. baumannii in our area . in our study , all of the isolates were sensitive to colistin and polymyxin b , while other studies conducted in tehran demonstrated 12% resistance to colistin and 3% resistance to polymyxin b in 2011 ( 17 ) . moreover , in two studies in tehran and ahvaz in 2013 , all of the isolates were sensitive to these antibiotics ( 12 , 14 ) . in a study in kermanshah in 2013 , all isolates were sensitive to polymyxin b and colistin ( 15 ) . sensitivity of all a. baumannii isolates to colistin was also reported in tehran in 2013 ( 22 ) . the major carbapenemase genes involved in carbapenem resistance in a. baumannii are blaoxa-23-like , blaoxa-24-like and blaoxa-58-like ( 8 , 14 ) . this result provides evidence that detection of blaoxa-51-like can be used as a simple and reliable way of identifying a. baumannii ( 13 ) . genbank submissions describing variants from isolates of a. baumannii from many different countries distributed over four continents clearly suggest that blaoxa-51-like is ubiquitous in a. baumannii ( 25 ) . oxa-23-like was the first oxa - type beta - lactamase identified in a. baumannii ( 26 ) . the results obtained in this study indicated that most of our isolates ( 40% ) carried the blaoxa-23-like gene . in other studies investigating outbreaks of oxa-23-producing a. baumannii strains , the rate of blaoxa-23-like ranged 31% to 94% in different parts of the world ( 14 ) . so our results for blaoxa-23-like gene are in the reported ranges . in both separate studies in tehran , in 2008 and 2009 , 25% of the isolates had positive results for blaoxa-23-like gene ( 6 , 20 ) . 85% and 55.7% positivity rates were reported for blaoxa-23-like gene in studied a. baumannii isolates in ahvaz and kermanshah , respectively ( 14 , 15 ) . in a study in tehran in 2008 , 17.9% of isolates contained blaoxa-24-like gene ( 6 ) ; in addition , in another study in tehran in 2009 , 15% had positive results for this gene ( 20 ) . some authors reported blaoxa-58-like frequency as 2 - 84.9% in a. baumannii isolates in different parts of the world ( 14 ) . in several studies in tehran in 2008 , 2009 and 2013 , 9% , 21.2% and 0.8% of the isolates had positive results for blaoxa-58-like , respectively ( 6 , 12 , 20 ) . moreover , a study conducted in ahvaz and kermanshah , in 2013 , revealed that blaoxa-58-like gene was not detected ( 14 , 15 ) . in our study , we identified blaoxa-58-like gene only in one a. baumannii isolate ( 0.5% ) . we identified nine isolates ( 4.5% ) with co - existence of two different blaoxa-23-like plus blaoxa-24-like . in two studies in 2013 , in tehran and kermanshah , 5.7% and 16.4% of a. baumannii isolates had such a co - existence ( 12 , 15 ) . in another study in 2013 in ahvaz , no co - existence between these genes was reported ( 14 ) . of note , 105 a. baumannii isolates resistant to imipenem and meropenem in our study possessed only the intrinsic blaoxa-51-like , but they had negative result for other investigated genes . in conclusion , such a. baumannii with different blaoxa - carbapenemase genes were isolated from hospitalized patients at shiraz teaching hospitals ( nemazee , faghihi , aliasghar , ghotbedin ) in different wards . the blaoxa-51-like genes were the most prevalent subgroup , as they are intrinsic to a. baumannii . moreover , blaoxa-23-like gene is another most prevalent resistance gene among mdr a. baumannii isolates .

recently , there has been a growing demand for medical care due to social trends such as diversity of population structure , raising awareness of rights , and changes in the social system . nurses are the biggest professional workforce at the hospital and have become a key factor in improving productivity and competitiveness of hospitals . the nursing profession because of its inherent nature , organizational strategies , extensive communication with other members of the health team and patients and their families exposure to more potential problems and can cause a variety of effects of stress and discomfort , leading to exhaustion and burnout . physical environment problems , heavy workload , conflict with patients and management of their companions violence , dealing with health and safety risks , lack of support from managers , absenteeism of physicians in emergency situations , and lack of facilities as the stressor sources for iranian nurses . turnover intention occurs when an employee encounters in his current job a bad work environment with high stress , which , in turn , can give them the intention to leave . in consequence , most nurses ( 71.42% ) every day think to leave the nursing profession . according to a study conducted on iranian nurses , the average burnout intention ( 3.030.75 ) is high . regardless of the strategies to retain the staff , critical shortage of nurses not only continues but will worsen over time . health authorities that spend much time and human and financial resources to hire nurses should be aware that it is equally important and affordable to attract and retain their employees utilize similar efforts . coping style is defined as persistent psychological and behavioral strategies to overcome or endure the internal and external challenges or stressors . how individuals use coping strategies may vary according to cultural , individual and psychological factors and is determined by their internal and external resources , including beliefs , health , support , responsibilities , material resources , and social skills . ineffective coping strategies of nurses have resulted in a worsening of relationship between the nurse and patient , and experience of more failures causing a gradual sense of loss of personal development . on the contrary , with successful coping style , the nurses are able to achieve their objectives and enhance their professional effectiveness . issues related to intention to leave and coping among the nurses are of global concerns of all administrators and managers in the healthcare field . as to the prevention of burnout , the mainstream thought is that the more money earned by employees in the organization , the more the chances of staying . researchers believe that staff retention will not be successful only with payments in the long run . they emphasize the plan to create and promote an environment in which employees choose to stay by themselves , and believe it is achievable by group efforts of employees , training and professional improvement of performance . clinical management , encouragement and support , monitoring programs , culture of support of teamwork and experienced work force are presented as the ways to stay in the nursing profession . also , the use of management development programs to stimulate nurses to remain in the nursing profession is emphasized . in a quantitative study in iran , nurses used self - control methods and positive reappraisal to deal with stress and the least used strategy in them was responsibility . in this study , more nurses had used the coping- focused strategies , while problem - focused approaches were used less . according to several studies , it seems that the methods used for retention of nurses in clinical practice are different , and as we have seen , each of the studies have pointed to various aspects and since these methods are usually influenced by cultural , social and religious fields ; therefore , it reveals the need to do qualitative research to understand , identify and describe the coping strategies with a tendency to leave the bedside among the nurses to be constructive for further research to strengthen , modify , adapt and change coping mechanisms . the specific aim of this study was to gain a deeper understanding of coping strategies among iranian nurses to deal with intention to leave . so the acquired knowledge will be useful in the formulation of recommendations for improving the health of the nurses , thereby improving the quality of care provided in hospitals . nurses who participated in this study were selected from several teaching hospitals affiliated with tabriz and uremia universities of medical sciences in iran . the sample selection process was based on the following criteria : 1 ) having a baccalaureate degree or higher , 2 ) having at least one year of work experience in clinical nursing practice , 3 ) being willing to participate in the study , and 4 ) having the ability to express their experiences . a total of 13 nurses met the inclusion criteria and agreed to enroll in the study . from may to february , 2014,(during ten months ) the participants attended semi - structured interviews by asking open - ended questions to investigate the coping strategies used by iranian nurses for overcoming the intention to leave the clinical practice . researchers interviewed each participant individually for 40 to 60 minutes at work ( n=8 ) , and outside of work environment ( n=5 ) . the interview began with a general question about coping strategies used by iranian nurses for overcoming intention to leave and moved to more specific , detailed questions as the interview advanced , such as how do you try to provide the scenarios / situations more for yourself ? , what things help you along the way ? . verbatim raw data were computer coded using maxqda10 ( version 10 r 160410 by udo kuckartz , berlin , germany ) before analysis . among the 13 nurses who participated in this study , there were 11 women and 2 men aged 24 to 47 years ; 9 of them had a baccalaureate degree , four had a master s degree . they had 2 - 15 years of clinical nursing experience in internal medicine , surgery , infectious disease , poison control , intensive adult care and emergency nursing care . eight participants were married and five were single . content analysis method as described by hsieh and shannon was applied for its appropriate fit to meet the objectives of this study . through inductive process , data were coded and categorized . classified codes were categorized , compared and interpreted within the context of general transcripts . during the study , for reporting of qualitative study finding , trustworthiness of methods instead of validity and reliability are widely considered and for this study four supporting processes of trustworthiness such as conformability , dependability , credibility and transferability were applied . moreover , member check was used in addition to prolonged involvement of the researcher to increase the credibility of the data . also , after encoding , the interview transcripts were returned to the participants to ensure the accuracy of the codes and the relevant interpretations . dependability was established by detailed and descriptive data analysis and direct references to individual professional experiences . raw data were translated by a professional translator from farsi ( persian ) into english and back translated to preserve maximum accuracy of the participant expressions within the context . conformability and consistency of analysis were maintained through research team meetings to discuss and analyze the preliminary findings . thematic analysis and coding process occurred through consensus ; and transferability of the findings was increased by descriptive statistics and the participants demographic features to represent the nursing views within the professional context . the present study was approved by the ethics committee of tabriz university of medical sciences ( grant no . , researchers obtained an oral and written informed consent to ensure anonymity , privacy and confidentiality and emphasized their voluntary enrollment . information on the study objectives and goals were detailed and contact information for the principal investigator was offered to answer participants questions . among the 13 nurses who participated in this study , there were 11 women and 2 men aged 24 to 47 years ; 9 of them had a baccalaureate degree , four had a master s degree . they had 2 - 15 years of clinical nursing experience in internal medicine , surgery , infectious disease , poison control , intensive adult care and emergency nursing care . content analysis method as described by hsieh and shannon was applied for its appropriate fit to meet the objectives of this study . through inductive process , data were coded and categorized . during the study , specific methods were used to ensure rigor and trustworthiness of data . for reporting of qualitative study finding , trustworthiness of methods instead of validity and reliability are widely considered and for this study four supporting processes of trustworthiness such as conformability , dependability , credibility and transferability were applied . moreover , member check was used in addition to prolonged involvement of the researcher to increase the credibility of the data . also , after encoding , the interview transcripts were returned to the participants to ensure the accuracy of the codes and the relevant interpretations . dependability was established by detailed and descriptive data analysis and direct references to individual professional experiences . raw data were translated by a professional translator from farsi ( persian ) into english and back translated to preserve maximum accuracy of the participant expressions within the context . conformability and consistency of analysis were maintained through research team meetings to discuss and analyze the preliminary findings . thematic analysis and coding process occurred through consensus ; and transferability of the findings was increased by descriptive statistics and the participants demographic features to represent the nursing views within the professional context . the present study was approved by the ethics committee of tabriz university of medical sciences ( grant no . , researchers obtained an oral and written informed consent to ensure anonymity , privacy and confidentiality and emphasized their voluntary enrollment . information on the study objectives and goals were detailed and contact information for the principal investigator was offered to answer participants questions . content and thematic analysis revealed three major categories : i ) self - empowerment , ii ) self - control , and iii ) pursuing opportunities for advancement and promotion . participants reflections for each category and subcategory were further expanded and later compared with other published studies . categories and subcategories coping strategies among iranian nurses to deal with intention to leave nurses had used five strategies to manage problems and stressors perceived in different aspects that lead to creation of a desire to leave the bedside in them , and gaining new energy and vitality in the organization . from this category , five subcategories emerged as i.1 ) practical knowledge increase , i.2 ) responsibility , i.3 ) finding the nurse identity , i.4 ) balancing work and life , i.5 ) searching for support , and i.6 ) humanitarian tendencies . practical knowledge increase : it was one of the subcategories derived from interviews and led to promotion in self - reliance and empowerment of nurses . for example , when in the ward i m dealing with some cases , i refer to my books . i participate in the courses that are held to teach how to work with the devices . ( participant # 2 , age 29 , 5 years of work experience ) i.2 . responsibility : there is another subcategory that leads to empowerment of the participants . in this i care for the patient wholeheartedly and from now on i go and check point by point my tasks . i got this decision myself ; i say because i am in the hospital environment . but , the patient is helpless and it is difficult for me and i get my colleagues annoyed . if his vein is ruined , i perform vein puncture for him ; i keep telling myself that this is easy ; vein puncture is not so hard ; i should do vein puncture for him . if the tubing was spoiled or had expired , i changed it . ( participant # 3 , age 26 , 3 years of work experience ) i.3 . finding the nurse identity : after i entered the research team of hospital , i began with the doctor residing in icu . we just ran processes clinically ( such as the gavages , how to suction , drug interactions , how to properly care for the patients , etc . ( participant # 9 , age 36 , 3 years of work experience ) i.4 . balancing work and life : establishing a balance between family and career roles is a big challenge for nurses . on the one hand , they are obliged to carry out their tasks within the rules and regulations of the work and on the other hand they must perform family duties , and in this context conflict between family and work is inevitable . one day i work in the morning shift- one day in the evening shift- and another day in the evening shift . but i know the program i plan- for example , i am planning to do it the morning of the tomorrow if i work in the evening , or conversely , if i work in the tomorrow morning , i have to do something . because of the shift , i am not too annoyed because i know the program . ( participant # 11 , age 37 , 3 years of work experience ) i.5 . search of support : another nurses strategy to overcome the tendency to leave service was support search . our head nurse says that if you want to go , i will not let you . because of what i do for the patient , i feel that i have satisfied others . that is , i have satisfied the head nurse that does not let me go . ( participant # 13 , age 25 , 2 years of work experience ) i.6 . humanitarian tendencies : altruistic motives are another reason for the participants to tolerate the clinical situation . despite all the problems that exist in nursing , considering that in this profession the opposite side of nurse is patient that needs him and nursing care is effective on relieving the patient s pain and suffering and precipitates the recovery . this inner satisfaction in the nurse , as result improvements of patient s condition , can be achieved , especially in our culture it has a significant impact on tolerance clinical condition . when i go to the patient s bedside and i just do my duties in a right way when i feel satisfied and the patient is pleased , it is the most enjoyable stage for me . and even i frankly say that i m just happy that i can help somebody . ( participant # 6 , age41 , 8 years of work experience ) the control of personal moods , behavior and reactions in situations of work and family is one of the abstracted individual coping strategies in relation to the desire to leave the bedside , which contains the following three categories : 1 ) tolerance , 2 ) avoidance , and 3 ) routine -based performance to overcome the pressure of work . 1 . tolerance : the results indicate that one of the strategies of self - control is tolerance . in this regard , ( participant # 7 , age24 , 3 years of work experience ) ii . 2 . avoidance : some colleagues do nt answer their phone- they do nt answer the hospital calls , because nurses know that they are called to go for a shift or have not done something for the patient ; that is , they stress me even in the house . participants have found from experience that in many cases nurses have no choice but to deal with and do things routinely . according to the participants , doing things routinely in the conditions governing the treatment system in the country , particularly in terms of the shortage of nurses , has the highest rate . you have to work routinely if you want to do something ; it is clear that the number of patients and workload are very high in the ward with only two nurses . ( participant # 8 , age 25 , 2 years of work experience ) to reduce stress and workload , you have to learn to act professionally , gain acceptance of others and also learn better skills to carry out their routine . this partly causes the nurses to acquire a degree of confidence and not to be humiliated and rejected by colleagues . my colleague worked as a routine and had more time opportunities and finished his work sooner , but i , instead of a minute to give drug to the patient- get the symptoms , maybe i spent 5 - 10 minutes for patients ; i had learned it , but my colleagues had said i did nt work correctly . they said my tasks were not done and the request of drug was not provided . the shift ended but i still had not finished . as a result , it caused dissatisfaction of colleagues maybe he does nt want always to work with me or work with me at the same shift . because he thought my duties ( participant # 1 , age 34 , 4 years of work experience ) iii . communication development : development of relationships with managers and colleagues was the other subcategory ; these relationships benefit the nurses and provide opportunities in the bedside to overcome the desire to leave the bedside . i saw only expertise is not enough to go to the special ward ; i should be able to show myself to head nurse and doctor ; my interactions with them must be strong ( participant # 10 , age 33 , 1.5 years of work experience ) iii . 2 . planning for higher education : following studies in nursing was the other aspect of the development opportunities . participants mentioned that motivation to follow studies in nursing is an effort to promote their position in the bedside and interest in nursing management tenure . because i was interested in learning , i always planned to follow my studies so that i can become the educational supervisor or perhaps i can go to the other ward where there is not so much labor . the medical ward is a general ward ; i thought maybe if i go to the special ward , my problems reduce . ( participant # 12 , age 34 , 5 years of work experience ) the final analysis of the findings showed that nurses individually think the use of coping strategies is working as much as possible to manage clinical problems and tensions in the bedside in order to provide job satisfaction and desire to stay in bedside and also be able to provide relief to the patients . nurses had used five strategies to manage problems and stressors perceived in different aspects that lead to creation of a desire to leave the bedside in them , and gaining new energy and vitality in the organization . from this category , five subcategories emerged as i.1 ) practical knowledge increase , i.2 ) responsibility , i.3 ) finding the nurse identity , i.4 ) balancing work and life , i.5 ) searching for support , and i.6 ) humanitarian tendencies . practical knowledge increase : it was one of the subcategories derived from interviews and led to promotion in self - reliance and empowerment of nurses . for example , when in the ward i m dealing with some cases , i refer to my books . i participate in the courses that are held to teach how to work with the devices . ( participant # 2 , age 29 , 5 years of work experience ) i.2 . responsibility : there is another subcategory that leads to empowerment of the participants . in this i care for the patient wholeheartedly and from now on i go and check point by point my tasks . i got this decision myself ; i say because i am in the hospital environment . but , the patient is helpless and it is difficult for me and i get my colleagues annoyed . if his vein is ruined , i perform vein puncture for him ; i keep telling myself that this is easy ; vein puncture is not so hard ; i should do vein puncture for him . ( participant # 3 , age 26 , 3 years of work experience ) i.3 . finding the nurse identity : one of the participants said : . after i entered the research team of hospital , i began with the doctor residing in icu . we just ran processes clinically ( such as the gavages , how to suction , drug interactions , how to properly care for the patients , etc . ) . ( participant # 9 , age 36 , 3 years of work experience ) i.4 . balancing work and life : establishing a balance between family and career roles is a big challenge for nurses . on the one hand , they are obliged to carry out their tasks within the rules and regulations of the work and on the other hand they must perform family duties , and in this context conflict between family and work is inevitable . one day i work in the morning shift- one day in the evening shift- and another day in the evening shift . but i know the program i plan- for example , i am planning to do it the morning of the tomorrow if i work in the evening , or conversely , if i work in the tomorrow morning , i have to do something . because of the shift , i am not too annoyed because i know the program . ( participant # 11 , age 37 , 3 years of work experience ) i.5 . search of support : another nurses strategy to overcome the tendency to leave service was support search . our head nurse says that if you want to go , i will not let you . because of what i do for the patient , i feel that i have satisfied others . that is , i have satisfied the head nurse that does not let me go . ( participant # 13 , age 25 , 2 years of work experience ) i.6 . humanitarian tendencies : altruistic motives are another reason for the participants to tolerate the clinical situation . despite all the problems that exist in nursing , considering that in this profession the opposite side of nurse is patient that needs him and nursing care is effective on relieving the patient s pain and suffering and precipitates the recovery . this inner satisfaction in the nurse , as result improvements of patient s condition , can be achieved , especially in our culture it has a significant impact on tolerance clinical condition . when i go to the patient s bedside and i just do my duties in a right way when i feel satisfied and the patient is pleased , it is the most enjoyable stage for me . and even i frankly say that i m just happy that i can help somebody . the control of personal moods , behavior and reactions in situations of work and family is one of the abstracted individual coping strategies in relation to the desire to leave the bedside , which contains the following three categories : 1 ) tolerance , 2 ) avoidance , and 3 ) routine -based performance to overcome the pressure of work . 1 . tolerance : the results indicate that one of the strategies of self - control is tolerance . in this regard , one of the participants says : i try not to touch them . ( participant # 7 , age24 , 3 years of work experience ) ii . 2 . avoidance : some colleagues do nt answer their phone- they do nt answer the hospital calls , because nurses know that they are called to go for a shift or have not done something for the patient ; that is , they stress me even in the house . participants have found from experience that in many cases nurses have no choice but to deal with and do things routinely . according to the participants , doing things routinely in the conditions governing the treatment system in the country , particularly in terms of the shortage of nurses , has the highest rate . you have to work routinely if you want to do something ; it is clear that the number of patients and workload are very high in the ward with only two nurses . if you want to function appropriately , you can not . ( participant # 8 , age 25 , 2 years of work experience ) to reduce stress and workload , you have to learn to act professionally , gain acceptance of others and also learn better skills to carry out their routine . this partly causes the nurses to acquire a degree of confidence and not to be humiliated and rejected by colleagues . my colleague worked as a routine and had more time opportunities and finished his work sooner , but i , instead of a minute to give drug to the patient- get the symptoms , maybe i spent 5 - 10 minutes for patients ; i had learned it , but my colleagues had said i did nt work correctly . they said my tasks were not done and the request of drug was not provided . the shift ended but i still had not finished . as a result , it caused dissatisfaction of colleagues maybe he does nt want always to work with me or work with me at the same shift . because he thought my duties 1 . communication development : development of relationships with managers and colleagues was the other subcategory ; these relationships benefit the nurses and provide opportunities in the bedside to overcome the desire to leave the bedside . i saw only expertise is not enough to go to the special ward ; i should be able to show myself to head nurse and doctor ; my interactions with them must be strong ( participant # 10 , age 33 , 1.5 years of work experience ) iii . 2 . planning for higher education : following studies in nursing was the other aspect of the development opportunities . participants mentioned that motivation to follow studies in nursing is an effort to promote their position in the bedside and interest in nursing management tenure . because i was interested in learning , i always planned to follow my studies so that i can become the educational supervisor or perhaps i can go to the other ward where there is not so much labor . the medical ward is a general ward ; i thought maybe if i go to the special ward , my problems reduce . ( participant # 12 , age 34 , 5 years of work experience ) the final analysis of the findings showed that nurses individually think the use of coping strategies is working as much as possible to manage clinical problems and tensions in the bedside in order to provide job satisfaction and desire to stay in bedside and also be able to provide relief to the patients . according to the results of this study , to address the perceived problems in the workplace that led to the desire to leave the bedside , the nurses used the strategy of empowerment and self - controlling and seek out opportunities for advancement and promotion . the results of this study have some similarities and differences with the national and international studies . the interesting finding of this research which is in contrast with those of similar studies was that coping strategies to overcome the tendency to leave the bedside among the nurses were mostly individualized and managers and the role of organizations in this context was not that important . another difference between this study and similar studies was that unlike other studies nurses were not tired and had some degree of willingness to leave the bedside . in the present study , it was found that the most common strategy used by nurses was self - empowerment . the methods that nurses used in this study for self - empowerment included increasing practical knowledge and responsibility , finding identification of the nurse , balancing work and life , and seeking support and humanitarian interests . self - empowerment was very valuable for nurses and reduced their stress , boosted their morale and satisfaction and increased their willingness to stay in the bedside . the results of the investigations also showed that empowering is a predictor of job satisfaction and retention factor of employees in the workplace to avoid leaving the profession . active coping was positively associated with professional efficacy , and negatively with fatigue and emotional exhaustion and pessimism . empowered and independent behavior , decentralized decision - making , relationships , open communication and collaboration with doctors , and cooperation with other clinical nurses caused job satisfaction and finally led to quality of care , retention of nurses . in this study , one of the strategies for self - empowerment of nurses was searching support . therefore , increase in social support for nurses is related to high power to deal with stress , in a very critical and stressful profession like nursing , increased attention to social support through improved management of nursing and administrative communication ; also , better social security may be useful for nurses . nursing administrators are advised to pay more attention to nurses who work in vulnerable conditions and ensure that they have enough support in the workplace . personal feelings about the working group is as important as seeking social support and enjoyment of the job . a study on newly graduated nurses during 5 years of graduation demonstrated that support from colleagues and nursing team was of secondary importance which was one of the reasons for leaving their first position . moreover , it is a positive and flexible professional identity to perform multiple roles of nurses in patient quality care , reduce the stress of workplace , and overcome the tendency to leave the bedside is important . one of the very important concepts and categories that emerged in interviews with nurses was humanitarian approach with the characteristics of loving patient as a human and enjoying helping fellow . the feeling was clean , strong and valuable so that despite the problems and stresses of work , nurses are encouraged to continue working and serving ; having a humanitarian approach , the participants in this study ignored their work problems and preferred health and well - being of patients and actually showed devotion . the findings of a study on nurses in special wards are consistent with the subcategory of humanitarian approach in our study because , despite certain mental conditions governing the patients in the special wards , it has strengthened helping the fellows , sense of being useful in care , motivation to continue work in these wards . the second most common strategy used by the iranian nurses , self - controlling , was recognized when nurses spoke of tolerance , avoiding conflicts and the routine - based performance to overcome the pressure of work . previous research has shown that self - controlling is the main coping strategy of nurses in japan , thailand and china . also , the three most common coping mechanisms were positive reappraisal , full problem solving , and self - control . the major source of stress for nurses was work conditions and applied positive assessment , self - controlling skills and social support to cope with job stress . in another study , 25% of female preoperative nurses employed avoidance coping strategies , while 83% of the males used problem - solving strategies . and the third theme , pursuing opportunities for advancement and promotion , was recognized by community development and planning for higher education . findings of this inquiry support the work of others ; for example , nurses intentions to stay primarily depends on relationships with patients , colleagues and administrators ; conditions in the workplace ; reward of work ; organizational support ; and psychological response to work . these results look logical because when employees are satisfied with their job , they will stay with the organization . most respondents in another study felt that nursing profession was unable to provide professional development opportunities and mental challenges for nurses . so these people have acquired these strategies by applying for graduate studies and by starting a new career . as was observed in this study , turnover can be useful for the individual nurse . changing workplace and career could provide an opportunity for nurses to move into better positions which is appropriate with motivations , ambitions , skills and career goals . according to this study , we think that it would be beneficial for the health care system to keep this group of nurses in the profession by opening new opportunities and challenges in nursing . more in - depth research is needed to better understand why nurses leave , and even more significantly and how we can motivate young generation of nurses to stay in nursing . as in other qualitative studies , one of the limitations of the present study is generalizability of the results . accordingly restricted the field of study to teaching hospitals was another limitation ; so , it is recommended that , in future studies , experiences of people from non - teaching hospitals should be considered . this study only focused on coping strategies of nurses in the background and culture of iran , so it is necessary that more studies be carried out in different fields and cultures in order to document and track the results of a recent study ; also , increasing our knowledge as to various aspects of coping strategies of nurses is recommended . iranian nurses use mainly self - empowerment , self - controlling , and pursuing opportunities for advancement and promotion it is crucial for health organizations to address these issues promptly in order to avoid high attrition and turnover rates which affect the effective functioning of iran health - care delivery system . thus , it is recommended that nursing managers recognize the need to provide appropriate strategies for nurses and maintain them in the profession . creating magnet workplace , staff appreciation , taking the nurses suggestions into consideration , providing communication and interactions based on mutual respect , and finally taking the nurses mental and emotional needs into account can all increase job satisfaction , sense of commitment and responsibility among nurses , and ultimately promote the quality of care . nursing educators and teachers can also foster efficient , competent and resistant manpower to clinical challenges by teaching coping strategies .

background : due to the high clinical challenges , differences in coping strategies , and high workload in nurses , there is a need to develop strategies to keep them in the profession . the aim of the present study was to explore the iranian nurses coping strategies to deal with intention to leave.methods:a qualitative content analysis was used to obtain rich data . we performed 13 in - depth face - to - face semi - structured interviews with nurses working in hospitals affiliated to tabriz and urmia universities of medical sciences in iran , selected through purposive sampling . constant comparative method was used for data analysis.results:three categories and eleven subcategories emerged during data analysis . the extracted categories and sub - categories consisted of ( i ) self - empowerment ( practical knowledge increase , responsibility , finding identification of the nurse , balancing work and life , seek support and humanitarian interests ) , ( ii ) self - controlling ( tolerance , avoidance , the routine - based performance ) , and ( iii ) pursuing opportunities for advancement and promotion ( community development , planning for higher education).conclusion : nurses make attempts to individually manage problems and stressors perceived from bedside that have led them to leave the bedside ; these efforts have been effective in some cases but sometimes they are ineffective due to discontinuous training and relative competence in terms of how to manage and deal with problems . it is suggested that nurses should learn strategies scientifically to meet the challenges of bedside . through enabling and supporting behaviors and creating opportunities for growth and professional development , nursery managers can help nurses to stay and achieve improvement of the quality of cares .

I M Sample and Setting Data Analysis Trustworthiness of the Study Ethical Considerations R I: Self-Empowerment II: Self-Controlling III. Pursuing Opportunities for Advancement and Promotion D C

recently , there has been a growing demand for medical care due to social trends such as diversity of population structure , raising awareness of rights , and changes in the social system . the nursing profession because of its inherent nature , organizational strategies , extensive communication with other members of the health team and patients and their families exposure to more potential problems and can cause a variety of effects of stress and discomfort , leading to exhaustion and burnout . physical environment problems , heavy workload , conflict with patients and management of their companions violence , dealing with health and safety risks , lack of support from managers , absenteeism of physicians in emergency situations , and lack of facilities as the stressor sources for iranian nurses . turnover intention occurs when an employee encounters in his current job a bad work environment with high stress , which , in turn , can give them the intention to leave . in consequence , most nurses ( 71.42% ) every day think to leave the nursing profession . according to a study conducted on iranian nurses , the average burnout intention ( 3.030.75 ) is high . regardless of the strategies to retain the staff , critical shortage of nurses not only continues but will worsen over time . health authorities that spend much time and human and financial resources to hire nurses should be aware that it is equally important and affordable to attract and retain their employees utilize similar efforts . how individuals use coping strategies may vary according to cultural , individual and psychological factors and is determined by their internal and external resources , including beliefs , health , support , responsibilities , material resources , and social skills . ineffective coping strategies of nurses have resulted in a worsening of relationship between the nurse and patient , and experience of more failures causing a gradual sense of loss of personal development . issues related to intention to leave and coping among the nurses are of global concerns of all administrators and managers in the healthcare field . as to the prevention of burnout , the mainstream thought is that the more money earned by employees in the organization , the more the chances of staying . they emphasize the plan to create and promote an environment in which employees choose to stay by themselves , and believe it is achievable by group efforts of employees , training and professional improvement of performance . clinical management , encouragement and support , monitoring programs , culture of support of teamwork and experienced work force are presented as the ways to stay in the nursing profession . also , the use of management development programs to stimulate nurses to remain in the nursing profession is emphasized . in a quantitative study in iran , nurses used self - control methods and positive reappraisal to deal with stress and the least used strategy in them was responsibility . according to several studies , it seems that the methods used for retention of nurses in clinical practice are different , and as we have seen , each of the studies have pointed to various aspects and since these methods are usually influenced by cultural , social and religious fields ; therefore , it reveals the need to do qualitative research to understand , identify and describe the coping strategies with a tendency to leave the bedside among the nurses to be constructive for further research to strengthen , modify , adapt and change coping mechanisms . the specific aim of this study was to gain a deeper understanding of coping strategies among iranian nurses to deal with intention to leave . so the acquired knowledge will be useful in the formulation of recommendations for improving the health of the nurses , thereby improving the quality of care provided in hospitals . nurses who participated in this study were selected from several teaching hospitals affiliated with tabriz and uremia universities of medical sciences in iran . the sample selection process was based on the following criteria : 1 ) having a baccalaureate degree or higher , 2 ) having at least one year of work experience in clinical nursing practice , 3 ) being willing to participate in the study , and 4 ) having the ability to express their experiences . from may to february , 2014,(during ten months ) the participants attended semi - structured interviews by asking open - ended questions to investigate the coping strategies used by iranian nurses for overcoming the intention to leave the clinical practice . researchers interviewed each participant individually for 40 to 60 minutes at work ( n=8 ) , and outside of work environment ( n=5 ) . the interview began with a general question about coping strategies used by iranian nurses for overcoming intention to leave and moved to more specific , detailed questions as the interview advanced , such as how do you try to provide the scenarios / situations more for yourself ? content analysis method as described by hsieh and shannon was applied for its appropriate fit to meet the objectives of this study . moreover , member check was used in addition to prolonged involvement of the researcher to increase the credibility of the data . also , after encoding , the interview transcripts were returned to the participants to ensure the accuracy of the codes and the relevant interpretations . the present study was approved by the ethics committee of tabriz university of medical sciences ( grant no . among the 13 nurses who participated in this study , there were 11 women and 2 men aged 24 to 47 years ; 9 of them had a baccalaureate degree , four had a master s degree . content analysis method as described by hsieh and shannon was applied for its appropriate fit to meet the objectives of this study . moreover , member check was used in addition to prolonged involvement of the researcher to increase the credibility of the data . also , after encoding , the interview transcripts were returned to the participants to ensure the accuracy of the codes and the relevant interpretations . the present study was approved by the ethics committee of tabriz university of medical sciences ( grant no . content and thematic analysis revealed three major categories : i ) self - empowerment , ii ) self - control , and iii ) pursuing opportunities for advancement and promotion . categories and subcategories coping strategies among iranian nurses to deal with intention to leave nurses had used five strategies to manage problems and stressors perceived in different aspects that lead to creation of a desire to leave the bedside in them , and gaining new energy and vitality in the organization . from this category , five subcategories emerged as i.1 ) practical knowledge increase , i.2 ) responsibility , i.3 ) finding the nurse identity , i.4 ) balancing work and life , i.5 ) searching for support , and i.6 ) humanitarian tendencies . practical knowledge increase : it was one of the subcategories derived from interviews and led to promotion in self - reliance and empowerment of nurses . for example , when in the ward i m dealing with some cases , i refer to my books . i participate in the courses that are held to teach how to work with the devices . responsibility : there is another subcategory that leads to empowerment of the participants . but , the patient is helpless and it is difficult for me and i get my colleagues annoyed . balancing work and life : establishing a balance between family and career roles is a big challenge for nurses . on the one hand , they are obliged to carry out their tasks within the rules and regulations of the work and on the other hand they must perform family duties , and in this context conflict between family and work is inevitable . but i know the program i plan- for example , i am planning to do it the morning of the tomorrow if i work in the evening , or conversely , if i work in the tomorrow morning , i have to do something . despite all the problems that exist in nursing , considering that in this profession the opposite side of nurse is patient that needs him and nursing care is effective on relieving the patient s pain and suffering and precipitates the recovery . this inner satisfaction in the nurse , as result improvements of patient s condition , can be achieved , especially in our culture it has a significant impact on tolerance clinical condition . when i go to the patient s bedside and i just do my duties in a right way when i feel satisfied and the patient is pleased , it is the most enjoyable stage for me . ( participant # 6 , age41 , 8 years of work experience ) the control of personal moods , behavior and reactions in situations of work and family is one of the abstracted individual coping strategies in relation to the desire to leave the bedside , which contains the following three categories : 1 ) tolerance , 2 ) avoidance , and 3 ) routine -based performance to overcome the pressure of work . tolerance : the results indicate that one of the strategies of self - control is tolerance . avoidance : some colleagues do nt answer their phone- they do nt answer the hospital calls , because nurses know that they are called to go for a shift or have not done something for the patient ; that is , they stress me even in the house . participants have found from experience that in many cases nurses have no choice but to deal with and do things routinely . according to the participants , doing things routinely in the conditions governing the treatment system in the country , particularly in terms of the shortage of nurses , has the highest rate . you have to work routinely if you want to do something ; it is clear that the number of patients and workload are very high in the ward with only two nurses . communication development : development of relationships with managers and colleagues was the other subcategory ; these relationships benefit the nurses and provide opportunities in the bedside to overcome the desire to leave the bedside . planning for higher education : following studies in nursing was the other aspect of the development opportunities . participants mentioned that motivation to follow studies in nursing is an effort to promote their position in the bedside and interest in nursing management tenure . because i was interested in learning , i always planned to follow my studies so that i can become the educational supervisor or perhaps i can go to the other ward where there is not so much labor . the medical ward is a general ward ; i thought maybe if i go to the special ward , my problems reduce . ( participant # 12 , age 34 , 5 years of work experience ) the final analysis of the findings showed that nurses individually think the use of coping strategies is working as much as possible to manage clinical problems and tensions in the bedside in order to provide job satisfaction and desire to stay in bedside and also be able to provide relief to the patients . nurses had used five strategies to manage problems and stressors perceived in different aspects that lead to creation of a desire to leave the bedside in them , and gaining new energy and vitality in the organization . from this category , five subcategories emerged as i.1 ) practical knowledge increase , i.2 ) responsibility , i.3 ) finding the nurse identity , i.4 ) balancing work and life , i.5 ) searching for support , and i.6 ) humanitarian tendencies . practical knowledge increase : it was one of the subcategories derived from interviews and led to promotion in self - reliance and empowerment of nurses . for example , when in the ward i m dealing with some cases , i refer to my books . i participate in the courses that are held to teach how to work with the devices . responsibility : there is another subcategory that leads to empowerment of the participants . i got this decision myself ; i say because i am in the hospital environment . but , the patient is helpless and it is difficult for me and i get my colleagues annoyed . finding the nurse identity : one of the participants said : . balancing work and life : establishing a balance between family and career roles is a big challenge for nurses . on the one hand , they are obliged to carry out their tasks within the rules and regulations of the work and on the other hand they must perform family duties , and in this context conflict between family and work is inevitable . but i know the program i plan- for example , i am planning to do it the morning of the tomorrow if i work in the evening , or conversely , if i work in the tomorrow morning , i have to do something . this inner satisfaction in the nurse , as result improvements of patient s condition , can be achieved , especially in our culture it has a significant impact on tolerance clinical condition . when i go to the patient s bedside and i just do my duties in a right way when i feel satisfied and the patient is pleased , it is the most enjoyable stage for me . the control of personal moods , behavior and reactions in situations of work and family is one of the abstracted individual coping strategies in relation to the desire to leave the bedside , which contains the following three categories : 1 ) tolerance , 2 ) avoidance , and 3 ) routine -based performance to overcome the pressure of work . tolerance : the results indicate that one of the strategies of self - control is tolerance . in this regard , one of the participants says : i try not to touch them . avoidance : some colleagues do nt answer their phone- they do nt answer the hospital calls , because nurses know that they are called to go for a shift or have not done something for the patient ; that is , they stress me even in the house . participants have found from experience that in many cases nurses have no choice but to deal with and do things routinely . according to the participants , doing things routinely in the conditions governing the treatment system in the country , particularly in terms of the shortage of nurses , has the highest rate . you have to work routinely if you want to do something ; it is clear that the number of patients and workload are very high in the ward with only two nurses . my colleague worked as a routine and had more time opportunities and finished his work sooner , but i , instead of a minute to give drug to the patient- get the symptoms , maybe i spent 5 - 10 minutes for patients ; i had learned it , but my colleagues had said i did nt work correctly . communication development : development of relationships with managers and colleagues was the other subcategory ; these relationships benefit the nurses and provide opportunities in the bedside to overcome the desire to leave the bedside . planning for higher education : following studies in nursing was the other aspect of the development opportunities . participants mentioned that motivation to follow studies in nursing is an effort to promote their position in the bedside and interest in nursing management tenure . because i was interested in learning , i always planned to follow my studies so that i can become the educational supervisor or perhaps i can go to the other ward where there is not so much labor . the medical ward is a general ward ; i thought maybe if i go to the special ward , my problems reduce . ( participant # 12 , age 34 , 5 years of work experience ) the final analysis of the findings showed that nurses individually think the use of coping strategies is working as much as possible to manage clinical problems and tensions in the bedside in order to provide job satisfaction and desire to stay in bedside and also be able to provide relief to the patients . according to the results of this study , to address the perceived problems in the workplace that led to the desire to leave the bedside , the nurses used the strategy of empowerment and self - controlling and seek out opportunities for advancement and promotion . the interesting finding of this research which is in contrast with those of similar studies was that coping strategies to overcome the tendency to leave the bedside among the nurses were mostly individualized and managers and the role of organizations in this context was not that important . another difference between this study and similar studies was that unlike other studies nurses were not tired and had some degree of willingness to leave the bedside . in the present study , it was found that the most common strategy used by nurses was self - empowerment . the methods that nurses used in this study for self - empowerment included increasing practical knowledge and responsibility , finding identification of the nurse , balancing work and life , and seeking support and humanitarian interests . self - empowerment was very valuable for nurses and reduced their stress , boosted their morale and satisfaction and increased their willingness to stay in the bedside . the results of the investigations also showed that empowering is a predictor of job satisfaction and retention factor of employees in the workplace to avoid leaving the profession . empowered and independent behavior , decentralized decision - making , relationships , open communication and collaboration with doctors , and cooperation with other clinical nurses caused job satisfaction and finally led to quality of care , retention of nurses . in this study , one of the strategies for self - empowerment of nurses was searching support . therefore , increase in social support for nurses is related to high power to deal with stress , in a very critical and stressful profession like nursing , increased attention to social support through improved management of nursing and administrative communication ; also , better social security may be useful for nurses . personal feelings about the working group is as important as seeking social support and enjoyment of the job . moreover , it is a positive and flexible professional identity to perform multiple roles of nurses in patient quality care , reduce the stress of workplace , and overcome the tendency to leave the bedside is important . one of the very important concepts and categories that emerged in interviews with nurses was humanitarian approach with the characteristics of loving patient as a human and enjoying helping fellow . the feeling was clean , strong and valuable so that despite the problems and stresses of work , nurses are encouraged to continue working and serving ; having a humanitarian approach , the participants in this study ignored their work problems and preferred health and well - being of patients and actually showed devotion . the second most common strategy used by the iranian nurses , self - controlling , was recognized when nurses spoke of tolerance , avoiding conflicts and the routine - based performance to overcome the pressure of work . previous research has shown that self - controlling is the main coping strategy of nurses in japan , thailand and china . also , the three most common coping mechanisms were positive reappraisal , full problem solving , and self - control . the major source of stress for nurses was work conditions and applied positive assessment , self - controlling skills and social support to cope with job stress . in another study , 25% of female preoperative nurses employed avoidance coping strategies , while 83% of the males used problem - solving strategies . and the third theme , pursuing opportunities for advancement and promotion , was recognized by community development and planning for higher education . findings of this inquiry support the work of others ; for example , nurses intentions to stay primarily depends on relationships with patients , colleagues and administrators ; conditions in the workplace ; reward of work ; organizational support ; and psychological response to work . changing workplace and career could provide an opportunity for nurses to move into better positions which is appropriate with motivations , ambitions , skills and career goals . according to this study , we think that it would be beneficial for the health care system to keep this group of nurses in the profession by opening new opportunities and challenges in nursing . more in - depth research is needed to better understand why nurses leave , and even more significantly and how we can motivate young generation of nurses to stay in nursing . as in other qualitative studies , one of the limitations of the present study is generalizability of the results . accordingly restricted the field of study to teaching hospitals was another limitation ; so , it is recommended that , in future studies , experiences of people from non - teaching hospitals should be considered . this study only focused on coping strategies of nurses in the background and culture of iran , so it is necessary that more studies be carried out in different fields and cultures in order to document and track the results of a recent study ; also , increasing our knowledge as to various aspects of coping strategies of nurses is recommended . iranian nurses use mainly self - empowerment , self - controlling , and pursuing opportunities for advancement and promotion it is crucial for health organizations to address these issues promptly in order to avoid high attrition and turnover rates which affect the effective functioning of iran health - care delivery system . thus , it is recommended that nursing managers recognize the need to provide appropriate strategies for nurses and maintain them in the profession . creating magnet workplace , staff appreciation , taking the nurses suggestions into consideration , providing communication and interactions based on mutual respect , and finally taking the nurses mental and emotional needs into account can all increase job satisfaction , sense of commitment and responsibility among nurses , and ultimately promote the quality of care . nursing educators and teachers can also foster efficient , competent and resistant manpower to clinical challenges by teaching coping strategies .

it is estimated that annually there are 180,000 people encountering osteoporosis - related fractures in england and wales . postmenopausal women with bone loss were considered at high risk of bone fractures , which greatly impaired their life quality and led to mortality . an appropriate and timely management for preventing osteoporotic fracture is extremely important . at present , antiresorptive agents are still the major treatments . besides the novel denosumab , which is a human monoclonal antibody of receptor activator of nf-b ligand ( rankl ) and potently suppresses osteoclastic bone resorption , alendronate ( aln ) , the most widely prescribed bisphosphonates , and raloxifene ( rlx ) , the only food and drug administration approved selective estrogen receptor modulators ( serms ) , are the most evident antiresorptive agents for prevention and treatment of postmenopausal osteoporosis [ 2 , 3 ] . for deciding the therapeutic strategy , it is highly imperative to know an estimate of the difference in fracture risk reduction between aln and rlx [ 4 , 5 ] . although both therapies have established efficacy from randomized controlled trials ( rcts ) , rlx was suggested to be less effective compared to aln , mainly in preventing nonvertebral fractures [ 3 , 68 ] and was therefore not recommended as a first - line treatment option for this population or considered as an alternative for young women with lower nonvertebral risk [ 3 , 9 ] . however , so far the efficacy inferiority of rlx under aln for postmenopausal women , especially the most relevant outcome fractures prevention , was still inconclusive . ( a ) the evidence was mainly derived from the indirect comparison with placebo , of which the inherent defects should be respected [ 10 , 11 ] . with the statistical methods of indirect comparison such as the bayesian method and the network meta - analysis [ 6 , 12 , 13 ] , in particular , the population of the previous systematic reviews and meta - analyses were consisted of elderly osteoporotic men and patients with glucocorticosteroid - induced osteoporosis apart from postmenopausal women [ 6 , 8 , 12 ] . in addition , the adverse effects ( aes ) of two agents , which would highly provide reference during clinical decision making , were not thoroughly compared in previous meta - analyses . ( b ) recently , a large - scaled retrospective study conducted by foster et al . emphasized that after adherent treatment there was a similar risk reduction for the both drugs in both vertebral and nonvertebral fractures of women up to 8 years , which was inconsistent with the previous prospective evidence [ 6 , 12 , 13 ] . ( c ) there were emerging head - to - head rcts to evaluate the comparative effectiveness of the two agents , the results of which were mainly limited by the small sample size but those available comparative data should be well summarized and taken into consideration [ 1521 ] . taken together , this meta - analysis with all the available 7 head - to - head rcts involving 4054 participants was conducted to summarize the comparative efficacy of bone mass increment and fracture prevention between aln and rlx for postmenopausal women [ 1521 ] . their safety profiles were also reviewed in a head - to - head comparative manner . besides , we aimed to evaluate clinically - related and design - related factors which might contribute to the difference in efficacy and aes . electronic databases ( pubmed , medline , embase , clinical trial registry and the cochrane data base of systematic reviews , and the cochrane central register of controlled trials ) were searched without limit by two independent investigators ( lin and ying ) , which were last updated on october , 2013 . ( alendronate or bisphosphonate ) and ( raloxifene or selective estrogen receptor modulators ) and postmenopausal women and ( osteoporosis or fracture ) . reference lists of all the selected articles were hand - searched for any additional trials . the trials were reviewed in which ( a ) the target population were consisted of postmenopausal women with low bone mass , ( b ) the interventions at least included both aln and rlx therapies , ( c ) the outcomes at least comprised one of the following assessments : fracture incidence , bmd , or safety profile , and ( d ) the trials were randomized controlled trials ( rcts ) . the trials were excluded if ( a ) patients had a prior history of metastatic bone disease , ( b ) phase - i or observational studies , case reports , and reviews , and ( c ) the same rcts were reanalyzed . two reviewers ( lin and ying ) independently assessed the study validity with cochrane collaboration 's tool for assessing the risk of bias , which addresses six specific domains such as sequence generation , allocation concealment , blinding , incomplete outcome data , and selective outcome reporting . whether the included trials were similar in baseline , adopting similar cointerventions , and applying intention - to - treat ( itt ) analysis was also evaluated . disagreement was evaluated by means of kappa ( ) test and resolved by discussion . for each eligible trial , two of us ( lin and ying ) independently extracted the relevant data and checked the accuracy . in particular , we abstracted study design , sample size , demographic data ( age , body mass index , and baseline bmd ) , intervention protocol , duration of the trial , loss to followup , trial outcomes ( fracture incidence , bmd , and incidence of adverse events ) , and industrial funding . we contacted the first or the corresponding author of each eligible trial to verify the accuracy of the data abstraction as well as our methodological assessment . the overall incidences of vertebral or nonvertebral fractures ( hip , upper leg , lower leg , pelvis , hummers , wrist / forearm , clavicle / rib , and other ) in the two groups were our primary outcome . we also evaluated the bmd percentage changes from the baseline at lumbar spine ( ls ) , femoral neck ( fn ) , and total hip ( th ) in both groups . the safety profile comprised the reported discontinuations due to aes , aes probably related to aln ( upper gastrointestinal disorders ( gi ) and diarrhea ) , and aes probably related to rlx ( vasomotor events and venous thrombosis ) . we took fracture risk reduction , ls bmd , and risk of upper gastrointestinal ( gi ) disorders at the end of follow - up as our main meta - analysis on basis of their sufficient trials for subgroup analysis . if the data were not reported in the original article , we extrapolated them from the accompanying graphs . to maximize data availability , we applied percentage change data for bmd and serum lipid outcome . if percentage change data were unavailable in bmd outcome , we imputed the percentage change data using ( endpoint data , baseline data ) divided by baseline data then multiplying 100 times . for the missing standard errors ( ses ) of bmd data , the maximum ses extracted from muscoso et al . the sensitivity analysis was performed through omitting trials with imputed ses to assess the variation in overall effect . the fracture incidence and the safety profile outcomes were presented as risk ratio ( rr ) with 95% confidence intervals ( ci ) and combined using the mantel - haenszel method . bmd were pooled with the inverse variance method and presented as weighted mean differences ( wmd ) and 95% ci . we calculated the statistical heterogeneity using a chi - squared ( ) test with the significance at 0.1 . we also assessed the inconsistency i to describe the percentage of the variability in effect estimates due to the heterogeneity . fixed effects model would be applied if there were no statistical heterogeneity among the studies ; otherwise , we used the random effects model . if substantial heterogeneities across studies ( i > 50% ) were detected in the index five main meta - analysis , we performed post hoc sensitivity analysis by omitting the outlier studies to determine the sources of cochran 's heterogeneity . the outliers were detected as the studies with confidence interval of the estimated effect size were not well overlapping with the pooled overall effect size . the subgroup analyses in the main meta - analysis were performed by baseline characteristics of the studies : patterns of treatments in aln groups ( daily or weekly ) , mean age of participants ( > 65 or 65 ) , methodological quality , sample size ( 400 or < 400 ) , and industrial funding . bmi of participants and dose of agents could not be analyzed in subgroup analysis due to the difficulties in determining cut - off values . to determine the influence of outlier studies , after omitting the two detected outliers , the pooled - analysis and the subgroup analyses were repeated in the main analysis with statistical heterogeneities . results of subgroup analysis were presented only if each subgroup comprised at least two trials . to comprehensively identify the clinical - related modifiers , metaregression with covariates ( age , bmi of participants , patterns of aln administration ) were carried out in the fracture ( vertebral fracture analysis was not performed as only 3 trials included ) and gi disorder analysis . to evaluate the publication bias , we used begg 's test and egger 's test with trials from fracture outcomes analysis , including 6 trials in total fractures , 3 trials in vertebral fractures , and 4 trials in nonvertebral fractures . metaregression analysis , begg 's test , and egger 's test were performed through stata 11.0 ( stata corp , college station , tx , usa ) . the criteria of the grading of recommendations assessment , development , and evaluation ( grade ) were used to evaluate the quality of evidence by each outcome . literature search initially yielded 731 relevant articles ; of which 224 overlapped publications were excluded . from the remaining 507 articles , 497 were excluded since they did not fulfill the selection criteria based on their titles and abstracts . after full - text checking of the rest of 10 rcts [ 1521 , 2527 ] , 3 rcts were excluded as their outcomes did not meet the inclusion criteria [ 2527 ] . finally , 7 studies with usable information were included in our meta - analysis [ 1521 ] ( figure 1 ) . the weighted kappa for the agreement on eligibility between reviewers was 0.81 ( 95% ci : 0.720.90 ) . the characteristics of the included 7 trials were shown in table 1 [ 1521 ] . four of the trials were double blinded and placebo - used , multicenter rcts [ 16 , 17 , 19 , 20 ] . there were two studies designed with fractures as endpoint in a two - year followup [ 18 , 19 ] . other five studies [ 1517 , 20 , 21 ] with one - year followup used bmd as surrogates for antifracture assessment , and the fractures was reported as aes [ 16 , 17 , 20 ] or secondary outcomes [ 15 , 18 ] . four studies only comprised aln and rlx treatments [ 15 , 17 , 19 , 20 ] . the other three studies contained combined treatment [ 16 , 21 ] or other therapies as well . only one trial was considered to have substantial loss to follow - up ( more than 20% ) , while the rates were acceptable among the other studies ( range , 1.7% to 19.7% ) . two studies were funded by aln company ( merck & co. ) [ 17 , 20 ] and two were funded by rlx company ( eli lilly & co. ) [ 16 , 19 ] , while the left three did not involve any industrial funding [ 15 , 18 , 21 ] . in terms of the patterns of administrations in aln groups , four studies treated women once daily [ 15 , 16 , 18 , 19 ] , while the other three adopted once weekly strategy [ 17 , 20 , 21 ] . patients in the both groups took calcium and vitamin d as supplementations equally in all eligible studies . the methodological quality was evaluated independently by two reviewers ( lin and ying ) with cochrane collaboration 's tool for assessing the risk of bias and showed in table 2 . four trials [ 6 , 7 , 9 , 10 ] described explicit adequately randomization , concealment of allocation assignment , proper blinding , and applying intention to treat analysis , which were low risk of bias [ 16 , 17 , 19 , 20 ] , while the other three trials with inexplicit randomization and inadequate blinding were considered moderate risk of bias [ 15 , 18 , 21 ] . the weighted kappa for the agreement on the trial quality between reviewers was 0.84 95% ci : ( 0.750.93 ) . no differences in total , vertebral or nonvertebral fracture incidences were demonstrated between the aln groups and rlx groups . ( total : 6 studies , fixed - effects rr ( 95% ci ) : 1.19 ( 0.75 , 1.68 ) , p = 0.58 , i = 0% ; vertebral : 3 studies , fixed - effects rr ( 95% ci ) : 1.30 ( 0.66 to 2.54 ) , p = 0.45 , and i = 0% ; nonvertebral : 4 studies , fixed - effects rr ( 95% ci ) : 0.95 ( 0.54 , 1.68 ) , p = 0.87 , and i = 0% , figure 2 ) . our meta - analysis indicated moderate quality evidence of equivalent efficacies between the two medications in fractures prevention ( table 3 ) . all of the included studies reported bmd data measured by dxa at least at one skeleton site . both aln and rlx increased bmd significantly at ls , fn , and th after 6 , 12 , and 24 months related to the baseline . aln obtained bone mass increment to a greater extent than rlx ( table 4 ) , and the differences were widening as the treatment continued . both aln and rlx were well tolerated , no fatal aes related to treatment were reported . it was similar in drop out due to aes , upper gi disorders , venous thrombosis , and vasodilatation in the both groups : ( aln versus rlx : drop out due to aes : 5 studies , rr : 1.03 ( 0.77 to 1.36 ) , p = 0.85 , and i = 0% ; upper gi disorders : 6 studies , rr : 1.10 ( 0.77 to 1.58 ) , p = 0.60 , and i = 52% ; venous thrombosis : 3 studies , rr : 0.52 ( 0.10 to 2.86 ) , p = 0.45 , and i = 0% ; vasodilatation : 3 studies , rr : 0.74 ( 0.54 to 1.01 ) , p = 0.06 , and i = 0% , figure 3 ) . and the evidence quality for the differences among those aes risks was moderate to high with the exception for aln increase greater risks of upper gi disorders than rlx , which was supported by low quality evidence ( the quality of evidence turned out to be high if excluding the outlier study ) ( table 3 ) . moderate to high quality evidence showed that aln would increase 133% risks of diarrhea while avoid 57% risks of vasomotor events compared to rlx ( aln versus rlx : diarrhea : 3 studies , rr : 2.33 ( 1.21 to 4.49 ) , p = 0.01 , and i = 0% ; vasomotor : 2 studies , rr : 0.47 ( 0.27 to 0.81 ) , p = 0.006 , and i = 0% , figure 3 ) . of the main meta - analysis , substantial heterogeneities were detected in outcomes of ls bmd ( at 12 months : p < 0.01 , i = 95% ) and upper gi disorders ( p = 0.60 , i = 52% ) . in ls bmd comparison ( at 12 months ) , iwamoto ' study was found as an outlier . after omitting this study , the results showed insignificant heterogeneities across studies ( p = 0.19 , i = 35% ) and the estimate effect size ( wmd ) in ls bmd was only reduced from 2.92 ( 95% ci : 2.23 to 3.62 ) to 2.37 ( 2.17 to 2.58 ) . the heterogeneities in gi disorders were simultaneously reduced to be minimal ( p = 0.83 , i = 0% ) after excluding this study while the differences between aln and rlx in risks of gi disorders turned out to be statistical ( aln versus rlx : rr : 1.30 ( 1.04 , 1.63 ) , p = 0.02 ) . since the two outlier studies were identified , the subgroup analysis were repeated after excludion of them in ls bmd at 12 months and gi disorders respectively . the overall results of main meta - analysis were not significantly altered by omitting trials with imputed ses . our subgroup analysis suggested that patterns of administrations in aln groups , participants ' age , methodological quality , sample size , or industrial funding of included studies were not associated with the overall effect size of the differences in fracture reduction . the outlier studies did not alter the results of the subgroup analysis in incidences of gi disorders [ 15 , 20 ] . the higher risk of aln in upper gi disorders compared to rlx was detected in the subgroups containing studies with daily administrated aln ( aln versus rlx : rr : 1.34 ( 1.04 , 1.72 ) , p = 0.02 ) and with participants over 65 years old ( aln versus rlx : rr : 1.32 ( 1.01 , 1.73 ) , p = 0.04 ) , which remained unchanged either including or excluding sambrook et al . . notwithstanding , after excluding iwamoto 's study , the studies involving weekly treated aln groups contributed to a greater difference in ls bone gain between aln and rlx groups compared to those which adopted daily strategies in aln groups ( weekly versus daily : wmd difference : 0.36 , p = 0.01 ) , while the difference was not statistical ( p = 0.26 ) under the presence of iwamoto 's study ( table 5 ) . women 's age , bmi , and pattern of aln administration have no obvious impacts on the results of fracture ( total and nonvertebral fractures ) analysis in our metaregression analysis . though it was insignificant , a widening difference was observed that aln had more upper gi disorders over rlx when women 's propensity to adopt daily aln administration or participants ' age increased ( supplementary file 1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/796510 ) . we found no evidence for publication bias both in vertebral fractures ( 3 trials ) and nonvertebral fractures ( 4 trials ) , according to both begg 's test and egger 's test . although the begg 's test funnel plot indicated a potential absence of small size studies which favored rlx groups in total fractures ( 6 trials ) , a trim and fill analysis suggested there were probably 2 missed small trials and the effect size ( rr ) would be more close to 1 by including them ( supplementary file 2 ) . our meta - analysis suggested no superiority of aln over rlx in reducing the risk of both vertebral fractures and nonvertebral fractures within a followup of 1224 months . aln reduced the risk of vasomotor by 57% but increased the risk of diarrhea by 133% compared to rlx . our subgroup analysis further indicated that the difference between aln and rlx in fracture reduction was not materially altered by administration pattern , age , methodological quality , sample size , or industrial funding . the weekly strategy of aln would further reduce the upper gi disorders and might gain more bone mass increment compared to its daily treatment . our meta - analysis was the first to exclusively comprise head - to - head rcts , target postmenopausal women and comprehensively evaluate the fracture risk , bmd , and the adverse effects . the previous systematic reviews and the network meta - analyses had indirectly compared the two agents within their multiple agents [ 2 , 6 , 8 ] . based on the data of the individual agent compared with the placebo , however , their results had poor consistency and great bias due to the variation in the baseline characteristics of participants and the administration pattern of drugs among the trials [ 10 , 11 ] . the validity of our findings was further strengthened by strictly following cochrane handbook for systematic reviews of interventions 5.0.2 . in particular , we developed the clear criteria of inclusion and exclusion , thoroughly assessed the methodological quality of the included studies , and embarked on the quantitative analysis . identification of the outlier studies and the sensitivity analysis was to sort out the source of heterogeneity in the present analysis , with the purpose of verifying the results . we also performed the subgroup analysis to comprehensively evaluate the multiple factors potentially influencing the comparative effect . finally , we used the grade system to rigidly assess the quality of evidence , which we aimed to recommend for both agents . generally , our grade analysis showed the evidence of moderate to high quality in most endpoints , which was higher than the previous pooled analyses [ 6 , 12 ] ( table 3 ) . ( a ) the combined sample size in current meta - analysis was still limited . however , a large - scale comparative rct trying to achieve significant fracture prevention difference of the two agents was destined to be infeasible and unnecessary , as the sample size would be unfortunately and unbelievably huge ( given the risk fracture of aln and rlx in the present analysis was 2.71% and 2.96% , it would need over 100,000 patients to confirm the theoretical difference of 0.25% ) . in fact , a well - conducted meta - analysis would always economically and adequately reflect the results of the large - scaled rct . the present analysis involving all available comparative rcts with moderate to high quality evidence on the two therapies for postmenopausal women may provide important information for health care providers to supplement the clinical trial evidence . ( b ) the outlier was identified as iwamoto et al . in ls bmd and sambrook et al . in gi disorders . the age of each study ( iwamoto et al . : 69.4 , sambrook et al . : 61.6 ) which differed from that of the other trials ( 62.1~67.5 ) might be the contributor ( table 1 ) . ( c ) three studies , lacking adequate randomization , blinding , and concealment of allocation , were considered as the moderate quality [ 15 , 18 , 21 ] . however , our subgroup analysis suggested the conclusions were not overall influenced by the trial quality . ( d ) four studies [ 16 , 17 , 19 , 20 ] were sponsored by the pharmaceutical companies related to the either agent . although the bias of the selective reporting should be considered , the industrial funding was found not to alter the overall results . aln could tightly bound to trabecular surfaces where osteoclasts attached and then disrupted their function after its ingestion . as for rlx , it bound to the raloxifene - estrogen receptor and activated a specific sequence of dna known as the raloxifene responding element . the subsequent increasing expression of specific cell proteins , which acted as estrogen agonist , resulted in osteoclast suppression . briefly , the more significant efficacy of aln over rlx in bmd increment is probably due to their different pathways of antiremodeling effect [ 28 , 34 , 35 ] . however , a discrepancy between the statistical difference in bone mass increment and their similar efficacy in vertebral and nonvertebral fracture prevention in the current analysis ought to be cautiously considered . one point should be borne in mind that the bmd decline only partially accounted for the osteoporotic bone fracture . literatures indicated that the contribution of the increase in bmd accounted for only 4% of the reduction of vertebral fracture with rlx compared with 17% with aln [ 3639 ] . even though rlx obtained lower bone mass increment , its adequate risk prevention of vertebral fracture has been well established in more studies . in addition , aln allowed fairly accumulation of microdamage in the vertebra , which would be offset by its increase in bone volume though , while the positive effect of rlx on biomechanical properties might adequately cover the inferior bone mass increment , which ultimately bridge the gap in vertebral fracture prevention between both agents . currently , rlx was infrequently prescribed for women with high risk of nonvertebral fractures [ 24 , 6 , 8 ] . in the more study , rlx 60 mg / day did not significant decrease nonvertebral fracture ( rr : 0.91 ( 0.77 , 1.07 ) ) compared with placebo . a recent network meta - analysis performed by murad et al . also demonstrated aln other than rlx achieved a significant reduction in nonvertebral fracture compared to placebo ( aln : odds ratio ( or ) : 0.78 ( 0.66 , 0.92 ) ; rlx : 0.90 ( 0.76 , 1.03 ) ) . but the inferiority of rlx under aln in nonvertebral fractures is still highly inconclusive as the definitive difference was not found in rcts or systematic reviews . a latest database study of over 100,000 postmenopausal women using inverse probability of treatment weights ( iptws ) method for adjustment highlighted that patients treated with either rlx or aln had similar rates in nonvertebral fracture after 8 years of adherent treatment . our pooled data of head - to - head rcts also questioned the difference of risk reduction in nonvertebral fractures between both agents . patients ' adherence to drugs , highly influenced by their tolerance , would substantially affect the benefits of drugs [ 4143 ] . therefore the potential risk of side effects should be thoroughly considered during a decision making . generally , our review suggested that both drugs were well tolerated with no fatal aes reported . in particular , aln increased the incidence of diarrhea while decreased vasomotor events compared to rlx , which did not require extra medication and seldom caused discontinuation . however , in our meta - analysis , only 4 venous thrombosis were found ( 1/990 in aln , 3/975 in rlx ) , which was really rare . nevetheless , we agreed that rlx should be contradicted for postmenopausal women who are at high risk of deep vein thrombosis . it was previously demonstrated that postmenopausal women had a greater propensity to adhere to rlx and higher satisfaction on drug administration compared with aln mainly due to more gi disorders associated with aln [ 47 , 48 ] . in our current analysis , however , the difference of upper gi events and the discontinuation due to aes between the two agents were balanced . nevertheless , the greater risk of upper gi disorder of aln over rlx was observed when we restricted the analysis to subgroups with daily administration of aln or subgroups with the age over 65 . it implied that the daily aln other than the weekly aln increased the frequency of gi irritation . besides , the aged women had more difficulty in taking aln properly , which contributed to the more gi symptoms [ 49 , 50 ] . although there is not any case reported in the included studies due to the short - term followup , the long term risk of atypical fractures and jaw necrosis with aln treatment should be under careful surveillance . although the moderate - to - high - quality evidence supported that aln was more effective in increasing the bone mass than rlx , the moderate - quality evidence suggested no difference in risk prevention of either vertebral or nonvertebral fractures within a followup of 1224 months . for aln the weekly strategy would further reduce the upper gi disorders and might gain more bone mass increment compared to the daily treatment . in addition , more diarrhea episodes but less vasomotor events with aln should also be considered for enhancing the patient compliance during decision making . which agent , aln or rlx , should be preferred for postmenopausal women remained a patient - oriented matter .

purpose . the aim of this study was to directly compare the efficacy and the safety of the two agents for postmenopausal women . methods / principal findings . electronic databases were searched for relevant articles that met our predefined inclusion criteria . seven randomized controlled trials ( rcts ) involving 4054 women were identified and included . although aln was more effective than rlx in increasing bone mineral density ( bmd ) , no statistical differences were observed in reducing the risk of neither vertebral fractures ( p = 0.45 ) nor nonvertebral fractures ( p = 0.87 ) up to two - year followup . aln reduced the risk of vasomotor ( p = 0.006 ) but increased the risk of diarrhea compared to rlx ( p = 0.01 ) . our subgroup analysis further indicated the difference between aln and rlx in fracture risk and was not materially altered by the administration pattern , the age . the weekly strategy of aln would further reduce the upper gastrointestinal ( gi ) disorders and might gain more bone mass increment at lumbar spine compared to its daily treatment . conclusion . there was no evidence of difference of fracture risk reduction between aln and rlx . in addition , age did not obviously influence their relative antifracture efficacy . for aln the weekly strategy would further reduce the upper gi disorders and gain more bone mass increment compared to the daily treatment . during clinical decision making , the patients ' adherence and the related side - effects associated with both drugs should also be taken into account .

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

postmenopausal women with bone loss were considered at high risk of bone fractures , which greatly impaired their life quality and led to mortality . besides the novel denosumab , which is a human monoclonal antibody of receptor activator of nf-b ligand ( rankl ) and potently suppresses osteoclastic bone resorption , alendronate ( aln ) , the most widely prescribed bisphosphonates , and raloxifene ( rlx ) , the only food and drug administration approved selective estrogen receptor modulators ( serms ) , are the most evident antiresorptive agents for prevention and treatment of postmenopausal osteoporosis [ 2 , 3 ] . for deciding the therapeutic strategy , it is highly imperative to know an estimate of the difference in fracture risk reduction between aln and rlx [ 4 , 5 ] . although both therapies have established efficacy from randomized controlled trials ( rcts ) , rlx was suggested to be less effective compared to aln , mainly in preventing nonvertebral fractures [ 3 , 68 ] and was therefore not recommended as a first - line treatment option for this population or considered as an alternative for young women with lower nonvertebral risk [ 3 , 9 ] . however , so far the efficacy inferiority of rlx under aln for postmenopausal women , especially the most relevant outcome fractures prevention , was still inconclusive . with the statistical methods of indirect comparison such as the bayesian method and the network meta - analysis [ 6 , 12 , 13 ] , in particular , the population of the previous systematic reviews and meta - analyses were consisted of elderly osteoporotic men and patients with glucocorticosteroid - induced osteoporosis apart from postmenopausal women [ 6 , 8 , 12 ] . in addition , the adverse effects ( aes ) of two agents , which would highly provide reference during clinical decision making , were not thoroughly compared in previous meta - analyses . emphasized that after adherent treatment there was a similar risk reduction for the both drugs in both vertebral and nonvertebral fractures of women up to 8 years , which was inconsistent with the previous prospective evidence [ 6 , 12 , 13 ] . ( c ) there were emerging head - to - head rcts to evaluate the comparative effectiveness of the two agents , the results of which were mainly limited by the small sample size but those available comparative data should be well summarized and taken into consideration [ 1521 ] . taken together , this meta - analysis with all the available 7 head - to - head rcts involving 4054 participants was conducted to summarize the comparative efficacy of bone mass increment and fracture prevention between aln and rlx for postmenopausal women [ 1521 ] . besides , we aimed to evaluate clinically - related and design - related factors which might contribute to the difference in efficacy and aes . electronic databases ( pubmed , medline , embase , clinical trial registry and the cochrane data base of systematic reviews , and the cochrane central register of controlled trials ) were searched without limit by two independent investigators ( lin and ying ) , which were last updated on october , 2013 . the trials were reviewed in which ( a ) the target population were consisted of postmenopausal women with low bone mass , ( b ) the interventions at least included both aln and rlx therapies , ( c ) the outcomes at least comprised one of the following assessments : fracture incidence , bmd , or safety profile , and ( d ) the trials were randomized controlled trials ( rcts ) . two reviewers ( lin and ying ) independently assessed the study validity with cochrane collaboration 's tool for assessing the risk of bias , which addresses six specific domains such as sequence generation , allocation concealment , blinding , incomplete outcome data , and selective outcome reporting . in particular , we abstracted study design , sample size , demographic data ( age , body mass index , and baseline bmd ) , intervention protocol , duration of the trial , loss to followup , trial outcomes ( fracture incidence , bmd , and incidence of adverse events ) , and industrial funding . the overall incidences of vertebral or nonvertebral fractures ( hip , upper leg , lower leg , pelvis , hummers , wrist / forearm , clavicle / rib , and other ) in the two groups were our primary outcome . we also evaluated the bmd percentage changes from the baseline at lumbar spine ( ls ) , femoral neck ( fn ) , and total hip ( th ) in both groups . the safety profile comprised the reported discontinuations due to aes , aes probably related to aln ( upper gastrointestinal disorders ( gi ) and diarrhea ) , and aes probably related to rlx ( vasomotor events and venous thrombosis ) . we took fracture risk reduction , ls bmd , and risk of upper gastrointestinal ( gi ) disorders at the end of follow - up as our main meta - analysis on basis of their sufficient trials for subgroup analysis . the fracture incidence and the safety profile outcomes were presented as risk ratio ( rr ) with 95% confidence intervals ( ci ) and combined using the mantel - haenszel method . we also assessed the inconsistency i to describe the percentage of the variability in effect estimates due to the heterogeneity . the subgroup analyses in the main meta - analysis were performed by baseline characteristics of the studies : patterns of treatments in aln groups ( daily or weekly ) , mean age of participants ( > 65 or 65 ) , methodological quality , sample size ( 400 or < 400 ) , and industrial funding . bmi of participants and dose of agents could not be analyzed in subgroup analysis due to the difficulties in determining cut - off values . to determine the influence of outlier studies , after omitting the two detected outliers , the pooled - analysis and the subgroup analyses were repeated in the main analysis with statistical heterogeneities . to comprehensively identify the clinical - related modifiers , metaregression with covariates ( age , bmi of participants , patterns of aln administration ) were carried out in the fracture ( vertebral fracture analysis was not performed as only 3 trials included ) and gi disorder analysis . to evaluate the publication bias , we used begg 's test and egger 's test with trials from fracture outcomes analysis , including 6 trials in total fractures , 3 trials in vertebral fractures , and 4 trials in nonvertebral fractures . after full - text checking of the rest of 10 rcts [ 1521 , 2527 ] , 3 rcts were excluded as their outcomes did not meet the inclusion criteria [ 2527 ] . there were two studies designed with fractures as endpoint in a two - year followup [ 18 , 19 ] . other five studies [ 1517 , 20 , 21 ] with one - year followup used bmd as surrogates for antifracture assessment , and the fractures was reported as aes [ 16 , 17 , 20 ] or secondary outcomes [ 15 , 18 ] . four studies only comprised aln and rlx treatments [ 15 , 17 , 19 , 20 ] . in terms of the patterns of administrations in aln groups , four studies treated women once daily [ 15 , 16 , 18 , 19 ] , while the other three adopted once weekly strategy [ 17 , 20 , 21 ] . the methodological quality was evaluated independently by two reviewers ( lin and ying ) with cochrane collaboration 's tool for assessing the risk of bias and showed in table 2 . ( total : 6 studies , fixed - effects rr ( 95% ci ) : 1.19 ( 0.75 , 1.68 ) , p = 0.58 , i = 0% ; vertebral : 3 studies , fixed - effects rr ( 95% ci ) : 1.30 ( 0.66 to 2.54 ) , p = 0.45 , and i = 0% ; nonvertebral : 4 studies , fixed - effects rr ( 95% ci ) : 0.95 ( 0.54 , 1.68 ) , p = 0.87 , and i = 0% , figure 2 ) . our meta - analysis indicated moderate quality evidence of equivalent efficacies between the two medications in fractures prevention ( table 3 ) . both aln and rlx increased bmd significantly at ls , fn , and th after 6 , 12 , and 24 months related to the baseline . aln obtained bone mass increment to a greater extent than rlx ( table 4 ) , and the differences were widening as the treatment continued . both aln and rlx were well tolerated , no fatal aes related to treatment were reported . it was similar in drop out due to aes , upper gi disorders , venous thrombosis , and vasodilatation in the both groups : ( aln versus rlx : drop out due to aes : 5 studies , rr : 1.03 ( 0.77 to 1.36 ) , p = 0.85 , and i = 0% ; upper gi disorders : 6 studies , rr : 1.10 ( 0.77 to 1.58 ) , p = 0.60 , and i = 52% ; venous thrombosis : 3 studies , rr : 0.52 ( 0.10 to 2.86 ) , p = 0.45 , and i = 0% ; vasodilatation : 3 studies , rr : 0.74 ( 0.54 to 1.01 ) , p = 0.06 , and i = 0% , figure 3 ) . and the evidence quality for the differences among those aes risks was moderate to high with the exception for aln increase greater risks of upper gi disorders than rlx , which was supported by low quality evidence ( the quality of evidence turned out to be high if excluding the outlier study ) ( table 3 ) . moderate to high quality evidence showed that aln would increase 133% risks of diarrhea while avoid 57% risks of vasomotor events compared to rlx ( aln versus rlx : diarrhea : 3 studies , rr : 2.33 ( 1.21 to 4.49 ) , p = 0.01 , and i = 0% ; vasomotor : 2 studies , rr : 0.47 ( 0.27 to 0.81 ) , p = 0.006 , and i = 0% , figure 3 ) . of the main meta - analysis , substantial heterogeneities were detected in outcomes of ls bmd ( at 12 months : p < 0.01 , i = 95% ) and upper gi disorders ( p = 0.60 , i = 52% ) . in ls bmd comparison ( at 12 months ) , iwamoto ' study was found as an outlier . after omitting this study , the results showed insignificant heterogeneities across studies ( p = 0.19 , i = 35% ) and the estimate effect size ( wmd ) in ls bmd was only reduced from 2.92 ( 95% ci : 2.23 to 3.62 ) to 2.37 ( 2.17 to 2.58 ) . the heterogeneities in gi disorders were simultaneously reduced to be minimal ( p = 0.83 , i = 0% ) after excluding this study while the differences between aln and rlx in risks of gi disorders turned out to be statistical ( aln versus rlx : rr : 1.30 ( 1.04 , 1.63 ) , p = 0.02 ) . since the two outlier studies were identified , the subgroup analysis were repeated after excludion of them in ls bmd at 12 months and gi disorders respectively . our subgroup analysis suggested that patterns of administrations in aln groups , participants ' age , methodological quality , sample size , or industrial funding of included studies were not associated with the overall effect size of the differences in fracture reduction . the outlier studies did not alter the results of the subgroup analysis in incidences of gi disorders [ 15 , 20 ] . the higher risk of aln in upper gi disorders compared to rlx was detected in the subgroups containing studies with daily administrated aln ( aln versus rlx : rr : 1.34 ( 1.04 , 1.72 ) , p = 0.02 ) and with participants over 65 years old ( aln versus rlx : rr : 1.32 ( 1.01 , 1.73 ) , p = 0.04 ) , which remained unchanged either including or excluding sambrook et al . notwithstanding , after excluding iwamoto 's study , the studies involving weekly treated aln groups contributed to a greater difference in ls bone gain between aln and rlx groups compared to those which adopted daily strategies in aln groups ( weekly versus daily : wmd difference : 0.36 , p = 0.01 ) , while the difference was not statistical ( p = 0.26 ) under the presence of iwamoto 's study ( table 5 ) . women 's age , bmi , and pattern of aln administration have no obvious impacts on the results of fracture ( total and nonvertebral fractures ) analysis in our metaregression analysis . though it was insignificant , a widening difference was observed that aln had more upper gi disorders over rlx when women 's propensity to adopt daily aln administration or participants ' age increased ( supplementary file 1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/796510 ) . we found no evidence for publication bias both in vertebral fractures ( 3 trials ) and nonvertebral fractures ( 4 trials ) , according to both begg 's test and egger 's test . although the begg 's test funnel plot indicated a potential absence of small size studies which favored rlx groups in total fractures ( 6 trials ) , a trim and fill analysis suggested there were probably 2 missed small trials and the effect size ( rr ) would be more close to 1 by including them ( supplementary file 2 ) . our meta - analysis suggested no superiority of aln over rlx in reducing the risk of both vertebral fractures and nonvertebral fractures within a followup of 1224 months . aln reduced the risk of vasomotor by 57% but increased the risk of diarrhea by 133% compared to rlx . our subgroup analysis further indicated that the difference between aln and rlx in fracture reduction was not materially altered by administration pattern , age , methodological quality , sample size , or industrial funding . the weekly strategy of aln would further reduce the upper gi disorders and might gain more bone mass increment compared to its daily treatment . our meta - analysis was the first to exclusively comprise head - to - head rcts , target postmenopausal women and comprehensively evaluate the fracture risk , bmd , and the adverse effects . the previous systematic reviews and the network meta - analyses had indirectly compared the two agents within their multiple agents [ 2 , 6 , 8 ] . based on the data of the individual agent compared with the placebo , however , their results had poor consistency and great bias due to the variation in the baseline characteristics of participants and the administration pattern of drugs among the trials [ 10 , 11 ] . identification of the outlier studies and the sensitivity analysis was to sort out the source of heterogeneity in the present analysis , with the purpose of verifying the results . however , a large - scale comparative rct trying to achieve significant fracture prevention difference of the two agents was destined to be infeasible and unnecessary , as the sample size would be unfortunately and unbelievably huge ( given the risk fracture of aln and rlx in the present analysis was 2.71% and 2.96% , it would need over 100,000 patients to confirm the theoretical difference of 0.25% ) . the present analysis involving all available comparative rcts with moderate to high quality evidence on the two therapies for postmenopausal women may provide important information for health care providers to supplement the clinical trial evidence . : 61.6 ) which differed from that of the other trials ( 62.1~67.5 ) might be the contributor ( table 1 ) . however , our subgroup analysis suggested the conclusions were not overall influenced by the trial quality . although the bias of the selective reporting should be considered , the industrial funding was found not to alter the overall results . briefly , the more significant efficacy of aln over rlx in bmd increment is probably due to their different pathways of antiremodeling effect [ 28 , 34 , 35 ] . however , a discrepancy between the statistical difference in bone mass increment and their similar efficacy in vertebral and nonvertebral fracture prevention in the current analysis ought to be cautiously considered . in addition , aln allowed fairly accumulation of microdamage in the vertebra , which would be offset by its increase in bone volume though , while the positive effect of rlx on biomechanical properties might adequately cover the inferior bone mass increment , which ultimately bridge the gap in vertebral fracture prevention between both agents . currently , rlx was infrequently prescribed for women with high risk of nonvertebral fractures [ 24 , 6 , 8 ] . also demonstrated aln other than rlx achieved a significant reduction in nonvertebral fracture compared to placebo ( aln : odds ratio ( or ) : 0.78 ( 0.66 , 0.92 ) ; rlx : 0.90 ( 0.76 , 1.03 ) ) . but the inferiority of rlx under aln in nonvertebral fractures is still highly inconclusive as the definitive difference was not found in rcts or systematic reviews . our pooled data of head - to - head rcts also questioned the difference of risk reduction in nonvertebral fractures between both agents . therefore the potential risk of side effects should be thoroughly considered during a decision making . in particular , aln increased the incidence of diarrhea while decreased vasomotor events compared to rlx , which did not require extra medication and seldom caused discontinuation . nevetheless , we agreed that rlx should be contradicted for postmenopausal women who are at high risk of deep vein thrombosis . it was previously demonstrated that postmenopausal women had a greater propensity to adhere to rlx and higher satisfaction on drug administration compared with aln mainly due to more gi disorders associated with aln [ 47 , 48 ] . in our current analysis , however , the difference of upper gi events and the discontinuation due to aes between the two agents were balanced . nevertheless , the greater risk of upper gi disorder of aln over rlx was observed when we restricted the analysis to subgroups with daily administration of aln or subgroups with the age over 65 . it implied that the daily aln other than the weekly aln increased the frequency of gi irritation . besides , the aged women had more difficulty in taking aln properly , which contributed to the more gi symptoms [ 49 , 50 ] . although there is not any case reported in the included studies due to the short - term followup , the long term risk of atypical fractures and jaw necrosis with aln treatment should be under careful surveillance . although the moderate - to - high - quality evidence supported that aln was more effective in increasing the bone mass than rlx , the moderate - quality evidence suggested no difference in risk prevention of either vertebral or nonvertebral fractures within a followup of 1224 months . for aln the weekly strategy would further reduce the upper gi disorders and might gain more bone mass increment compared to the daily treatment . in addition , more diarrhea episodes but less vasomotor events with aln should also be considered for enhancing the patient compliance during decision making .

recombinant interferon ( ifn ) is one of the most widely used first line therapies in multiple sclerosis ( ms ) , but up to 50% of patients under treatment continue to experience relapses and progression of the disease and are considered suboptimal responders.1 it is , therefore , strongly desirable to count with biomarkers that allow prediction of the therapeutic response to ifn. the tumour necrosis factor - related apoptosis inducing ligand ( trail)/trail receptor system has been shown to be implicated in ms pathogenesis24 as well as in the mechanisms of action of ifn.5 6 expression of trail and its four surface receptors in different cell types during ifn treatment has been studied,7 8 and trail mrna expression in leucocytes has been proposed as a biomarker for the therapeutic response to ifn in patients with ms.5 nevertheless , none of these studies has taken into account the different splicing variants of each gene , which could lead to the expression of non - functional proteins . three different splice variants have been discovered for trail : trail- , the functional isoform ; trail- , which lacks exon 3 ; and trail- , which lacks exon 2 and 3 . the loss of exon 3 leads to the lack of the extracellular domain and , therefore , to the absence of apoptotic potential.9 trailr-2 has two different apoptotic variants : trailr-2 isoform 1 ( trick2b ) , which has an 87 nucleotides insertion and trailr-2 isoform 2 ( trick2a ) , which is homologous to trailr-1.10 finally , there are two different splice variants for trailr-4 : trailr-4 , the long isoform , and trailr-4 , which lacks exon 3.11 the loss of this exon leads to the truncation of the first cysteine rich domain 1 , involved in the ligand receptor complex , which may alter its ability to bind trail . in this study , we monitored the expression kinetics of trail and its receptors after induction with ifn. although kinetics of trail expression have previously been reported,12 it remains unknown whether trail receptors follow the same pattern . the aim of the present work was to assess the effects of ifn treatment on the expression of the splice variants of trail and its receptors in different cell subpopulations from patients with ms , and to determine whether these expressions discriminated responders from non - responders to ifn therapy . forty - two spanish patients with ms prone to start ifn treatment were recruited from the multiple sclerosis unit at malaga regional university hospital ( malaga , spain ) and followed for a minimum of 2 years of treatment . study inclusion criteria included definite ms according to the mcdonald criteria,13 and treatment - naive at baseline for at least 6 months . once they started therapy with ifn , patients had to be on a stable dose for at least 24 months . blood samples were obtained during remissions before the first ifn administration ( baseline ) and again after 1214 months ( serum and peripheral blood mononuclear cells ( pbmc ) ) and 24 months ( serum ) on that therapy . as controls ( hc ) , samples were processed following standard procedures and frozen immediately after they were received by the mlaga regional hospital biobank , as part of andalusian public health system biobank . all individuals participating in the study gave their informed consent and protocols were approved by an institutional ethical committee ( comisin de tica y de investigacin del hospital regional universitario carlos haya ) . two patients initially taking part in the study were excluded due to the presence of high and permanent titres of neutralising antibodies ( nabs ) against ifn. the demographic and clinical variables of the remaining 40 patients with ms and hc are listed in table 1 . demographic and clinical characteristics of the participants at baseline quantitative data are presented as median ( iqr ) . * p values : refers to p values obtained following comparisons between healthy controls , responders and non - responders by means of a kruskal - wallis test ( age ) and test ( gender ) , as well as p values obtained following comparisons between responders and non - responders by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . edss , expanded disability status scale ; ifn , interferon ; rr , relapsing remitting ; sp , secondary progressive ; trail , tumour necrosis factor - related apoptosis inducing ligand . patients were classified as optimal responders if they had neither relapses within the previous year nor progression of the disease over the same follow - up period , and as non - responders if they experienced one or more relapses or an increase of at least one point in the expanded disability status scale ( edss ) that persisted for a minimum of two consecutive visits separated by a 6-month interval.14 the presence of nabs in serum was checked after 12 and 24 months of treatment by the ifn - induced inhibition of virus cytopathic effect on human cells in culture , following the who recommendations as previously described.15 the neutralisation titre of serum samples was calculated according to kawade et al.16 titres 20 tru / ml were considered as positive . owing to the limited yield of pbmc obtained from each patient , gene expression kinetics were carried out in an immortalised human t lymphoblastoid cell line . jurkat cells ( clone e.6 , american type culture collection ) were cultured in 12-well plates , at 1.510 cells / ml , in rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) , 1.3% hepes , 50 g / ml gentamicin and 2 mm l - glutamine . after incubation at 37c and 5% co2 for 24 h , cells were washed and incubated for 2 h with the former media without fbs . cells for pre - stimulation condition ( 3 replicates ) were collected in 1 ml of tripure isolation reagent ( roche diagnostic gmbh ) and the remaining cells were stimulated with 200 ui / ml of ifn-1a ( avonex , biogen idec ) and collected in the same way at 4 , 8 , 12 and 24 h after ifn induction . blood samples were taken before the first ifn administration and after 1 year of treatment . monocytes ( cd14 + ) , cd4 + t cells and cd8 + t cells were isolated using immunomagnetic microbeads and ls macs columns ( miltenyi biotec gmbh ) , following the manufacturer 's protocol . after isolation , the cellular subsets were cultured in 96 well plates with medium without fbs . cells were induced with 200 ui / ml of ifn-1a and collected in tripure at 4 and 24 h after induction . total rna was isolated from jurkat cells and pbmc using a modification of the phenol chloroform method.17 total rna yield and quality of product was assessed with a nanodrop 2000 spectrophotometer . for complementary dna ( cdna ) synthesis , 1 g of total rna was reverse transcribed with the m - mlv reverse transcriptase as described elsewhere.18 the cdna was stored at 80c until use . primers for trailr-1 and trailr-3 were designed using primer 3 software.19 primers for trail , trailr-2 and trailr-4 were custom designed to make them complementary to the specific exon boundaries of each splice variant . primers used for amplification of the splice variants of trail and trail receptors trail , tumour necrosis factor - related apoptosis inducing ligand . conventional pcr with temperatures ranging from 57c to 63c were performed to ensure specificity of primer pairs and to determine the optimal annealing temperature . annealing at 57c was optimal for trailr-1 and trail variants , at 60c for gapdh and at 58c for the remaining genes . quantitative pcr was performed in duplicate in a rotor gene q thermocycler ( qiagen gmbh ) in a 20 l reaction mix containing depc - treated water , 20 mm primers ( forward and reverse ) , quantitec sybr green pcr master mix ( qiagen ) and cdna . the programme consisted of a step of 15 min at 95c , followed by 40 cycles of 95c for 30 s , 57c , 58c or 60c for 30 s and 72c for 30 s. a melting step from 65c to 95c was run , increasing 0.5c every 5 s. the relative trail and trail receptor variants messenger rna ( mrna ) expression levels were calculated according to the 2 method , first by normalising to gapdh and then to a calibrator sample . comparisons of demographic characteristics at baseline between hc , responder and non - responder patients with ms were performed by kruskal - wallis test ( age ) and test ( gender ) . comparisons of clinical characteristics at baseline between responders and non - responders were performed by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . a wilcoxon test was used to compare relative expression between pretreatment samples and samples from patients after 1 year of treatment with ifn. a mann - whitney u test was used to compare relative expression of trail and its receptors between responders and suboptimal responders to ifn therapy . receiver operating characteristic ( roc ) analyses were performed to evaluate the predictive value of gene expression before treatment onset on therapeutic response to ifn. forty - two spanish patients with ms prone to start ifn treatment were recruited from the multiple sclerosis unit at malaga regional university hospital ( malaga , spain ) and followed for a minimum of 2 years of treatment . study inclusion criteria included definite ms according to the mcdonald criteria,13 and treatment - naive at baseline for at least 6 months . once they started therapy with ifn , patients had to be on a stable dose for at least 24 months . blood samples were obtained during remissions before the first ifn administration ( baseline ) and again after 1214 months ( serum and peripheral blood mononuclear cells ( pbmc ) ) and 24 months ( serum ) on that therapy . as controls ( hc ) , samples were processed following standard procedures and frozen immediately after they were received by the mlaga regional hospital biobank , as part of andalusian public health system biobank . all individuals participating in the study gave their informed consent and protocols were approved by an institutional ethical committee ( comisin de tica y de investigacin del hospital regional universitario carlos haya ) . two patients initially taking part in the study were excluded due to the presence of high and permanent titres of neutralising antibodies ( nabs ) against ifn. the demographic and clinical variables of the remaining 40 patients with ms and hc are listed in table 1 . demographic and clinical characteristics of the participants at baseline quantitative data are presented as median ( iqr ) . * p values : refers to p values obtained following comparisons between healthy controls , responders and non - responders by means of a kruskal - wallis test ( age ) and test ( gender ) , as well as p values obtained following comparisons between responders and non - responders by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . edss , expanded disability status scale ; ifn , interferon ; rr , relapsing remitting ; sp , secondary progressive ; trail , tumour necrosis factor - related apoptosis inducing ligand . patients were classified as optimal responders if they had neither relapses within the previous year nor progression of the disease over the same follow - up period , and as non - responders if they experienced one or more relapses or an increase of at least one point in the expanded disability status scale ( edss ) that persisted for a minimum of two consecutive visits separated by a 6-month interval.14 the presence of nabs in serum was checked after 12 and 24 months of treatment by the ifn - induced inhibition of virus cytopathic effect on human cells in culture , following the who recommendations as previously described.15 the neutralisation titre of serum samples was calculated according to kawade et al.16 titres 20 tru / ml were considered as positive . owing to the limited yield of pbmc obtained from each patient , gene expression kinetics were carried out in an immortalised human t lymphoblastoid cell line . jurkat cells ( clone e.6 , american type culture collection ) were cultured in 12-well plates , at 1.510 cells / ml , in rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) , 1.3% hepes , 50 g / ml gentamicin and 2 mm l - glutamine . after incubation at 37c and 5% co2 for 24 h , cells were washed and incubated for 2 h with the former media without fbs . cells for pre - stimulation condition ( 3 replicates ) were collected in 1 ml of tripure isolation reagent ( roche diagnostic gmbh ) and the remaining cells were stimulated with 200 ui / ml of ifn-1a ( avonex , biogen idec ) and collected in the same way at 4 , 8 , 12 and 24 h after ifn induction . blood samples were taken before the first ifn administration and after 1 year of treatment . monocytes ( cd14 + ) , cd4 + t cells and cd8 + t cells were isolated using immunomagnetic microbeads and ls macs columns ( miltenyi biotec gmbh ) , following the manufacturer 's protocol . after isolation , the cellular subsets were cultured in 96 well plates with medium without fbs . cells were induced with 200 ui / ml of ifn-1a and collected in tripure at 4 and 24 h after induction . total rna was isolated from jurkat cells and pbmc using a modification of the phenol chloroform method.17 total rna yield and quality of product was assessed with a nanodrop 2000 spectrophotometer . for complementary dna ( cdna ) synthesis , 1 g of total rna was reverse transcribed with the m - mlv reverse transcriptase as described elsewhere.18 the cdna was stored at 80c until use . primers for trailr-1 and trailr-3 were designed using primer 3 software.19 primers for trail , trailr-2 and trailr-4 were custom designed to make them complementary to the specific exon boundaries of each splice variant . primers used for amplification of the splice variants of trail and trail receptors trail , tumour necrosis factor - related apoptosis inducing ligand . conventional pcr with temperatures ranging from 57c to 63c were performed to ensure specificity of primer pairs and to determine the optimal annealing temperature . annealing at 57c was optimal for trailr-1 and trail variants , at 60c for gapdh and at 58c for the remaining genes . quantitative pcr was performed in duplicate in a rotor gene q thermocycler ( qiagen gmbh ) in a 20 l reaction mix containing depc - treated water , 20 mm primers ( forward and reverse ) , quantitec sybr green pcr master mix ( qiagen ) and cdna . the programme consisted of a step of 15 min at 95c , followed by 40 cycles of 95c for 30 s , 57c , 58c or 60c for 30 s and 72c for 30 s. a melting step from 65c to 95c was run , increasing 0.5c every 5 s. the relative trail and trail receptor variants messenger rna ( mrna ) expression levels were calculated according to the 2 method , first by normalising to gapdh and then to a calibrator sample . total rna was isolated from jurkat cells and pbmc using a modification of the phenol chloroform method.17 total rna yield and quality of product was assessed with a nanodrop 2000 spectrophotometer . for complementary dna ( cdna ) synthesis , 1 g of total rna was reverse transcribed with the m - mlv reverse transcriptase as described elsewhere.18 the cdna was stored at 80c until use . primers for trailr-1 and trailr-3 were designed using primer 3 software.19 primers for trail , trailr-2 and trailr-4 were custom designed to make them complementary to the specific exon boundaries of each splice variant . primers used for amplification of the splice variants of trail and trail receptors trail , tumour necrosis factor - related apoptosis inducing ligand . conventional pcr with temperatures ranging from 57c to 63c were performed to ensure specificity of primer pairs and to determine the optimal annealing temperature . annealing at 57c was optimal for trailr-1 and trail variants , at 60c for gapdh and at 58c for the remaining genes . quantitative pcr was performed in duplicate in a rotor gene q thermocycler ( qiagen gmbh ) in a 20 l reaction mix containing depc - treated water , 20 mm primers ( forward and reverse ) , quantitec sybr green pcr master mix ( qiagen ) and cdna . the programme consisted of a step of 15 min at 95c , followed by 40 cycles of 95c for 30 s , 57c , 58c or 60c for 30 s and 72c for 30 s. a melting step from 65c to 95c was run , increasing 0.5c every 5 s. the relative trail and trail receptor variants messenger rna ( mrna ) expression levels were calculated according to the 2 method , first by normalising to gapdh and then to a calibrator sample . comparisons of demographic characteristics at baseline between hc , responder and non - responder patients with ms were performed by kruskal - wallis test ( age ) and test ( gender ) . comparisons of clinical characteristics at baseline between responders and non - responders were performed by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . a wilcoxon test was used to compare relative expression between pretreatment samples and samples from patients after 1 year of treatment with ifn. a mann - whitney u test was used to compare relative expression of trail and its receptors between responders and suboptimal responders to ifn therapy . receiver operating characteristic ( roc ) analyses were performed to evaluate the predictive value of gene expression before treatment onset on therapeutic response to ifn. the expression kinetics of each gene after in vitro induction with ifn was determined in jurkat cells ( figure 1 ) . the three trail variants showed a similar expression pattern : gene expression was upregulated during 14 h after ifn stimulation ( peak at 4 h ) , and declined thereafter , followed by a second increment after 24 h , in agreement with previous data.12 gene expression kinetics of the tumour necrosis factor - related apoptosis inducing ligand ( trail ) and trail receptors splicing variants in jurkat cells on in vitro stimulation with interferon ( ifn ) . ( a ) trail splicing variant genes are upregulated during 1 - 4 h of ifn treatment and decline thereafter . ( b ) death receptor isoforms begin to upregulate at 8 h and reach a maximum at 24 h. ( c ) decoy receptor isoforms begin to be induced at the 12 h time point and beyond . expression levels are represented as relative expression compared with the reference gene gapdh , using the ct method . trailr-1 and trailr-2 shared the same expression pattern , characterised by a decreased expression in the first hours , and a peak 24 h after ifn induction . the decoy receptors also began to be induced at 812 h post - stimulation with ifn , showing a progressive increment with a marked peak at 24 h. thus , in pbmc , the expression of trail and its receptors was assessed at 4 and 24 h after in vitro stimulation with ifn , respectively . on in vitro stimulation with ifn , mrna expression of trail- was significantly increased in hc compared to untreated patients with ms in the three cellular subsets ( p=0.023 in monocytes , p=0.00004 in cd4 + t cells and p= 0.021 in cd8 + t cells ) , as observed in figure 2 . we detected no other significant differences in the expression of trail and receptor variants at the rna level . the tumour necrosis factor - related apoptosis inducing ligand ( trail ) differential expression in healthy controls ( hc ) and untreated patients with multiple sclerosis ( ms ) . expression levels are represented as relative expression compared with the reference gene gapdh , using the ct method . figures show box plots : the horizontal bars are the median and lower and upper edges of the boxes represent the 2575th centiles . the mann - whitney u test was used to determine statistical differences . within the patient group , prior to ifn treatment onset , we found higher mrna expression of trail- in monocytes , cd4 + and cd8 + t cells from patients who will subsequently be classified as non - responders than from responders ( p=0.008 , p=0.00006 and 0.003 , respectively ) , as shown in figure 3 . conversely , the mrna expression of the trail- isoform was significantly higher in cd4 + and cd8 + t cells from patients who will become responders ( p=0.03 and p=0.025 ) , and showed the same trend of expression in monocytes ( p=0.052 ) . additionally , expression of trailr-1 was also significantly higher in monocytes and cd4 + t cells from patients who will become responders . relative expression of tumour necrosis factor - related apoptosis inducing ligand ( trail ) , trail- and trailr-1 , on in vitro stimulation with interferon ( ifn ) , before treatment onset . those patients who will become non - responders ( nr ) to ifn therapy showed higher expression of trail- mrna in the three cellular subsets , while those who will become responders ( r ) showed higher baseline expression of trail- and trailr-1 . expression level is represented as relative expression compared with the reference gene gapdh , using the ct method . figures show box plots : the horizontal bars are the median and lower and upper edges of the boxes represent the 2575th centiles . for biomarkers to predict the therapeutic response to ifn , roc analyses were performed to evaluate the predictive discriminating value of trail- , trail- and trailr-1 expression on in vitro induction with ifn before treatment onset , on different cell subsets from patients with ms . values of areas under the curve ( auc ) , sensitivity , specificity , positive predictive value and negative predictive value are shown in table 3 . expression of trail- in the three cellular subsets better predicted those patients who will subsequently become non - responders to treatment , with auc > 0.7 and sensitivities greater than 80% in the three cases . conversely , expression of trailr-1 on monocytes and cd4 + t cells better predicted those patients who will subsequently become responders to treatment , with auc > 0.8 , sensitivities greater than 90% and specificities greater than 75% . trail- expression did not seem a good biomarker to predict therapeutic response to ifn , as aucs were under 0.7 . sensitivity and specificity of biomarkers to predict the therapeutic response to ifn auc , area under the curve ; ifn , interferon ; npv , negative predictive value ; ppv , positive predictive value ; trail , tumour necrosis factor - related apoptosis inducing ligand . relative expression of trail and trail receptors was significantly modified along the first year of therapy only for two genes . expression of the isoform 2 of the death receptor trailr-2 was significantly reduced in monocytes from first - year responder as well as non - responder patients ( p=0.004 and 0.016 , respectively ) . expression of trail- was significantly increased in both t cell subsets but exclusively in patients who showed a clinical response to systemic ifn treatment ( p=0.004 in cd4 + cells and p=0.002 in cd8 + cells from responders ) . induction of trail- mrna expression in monocytes by ifn therapy was achieved in 13 of 24 responders and , therefore , overall expression in this subset was not significantly increased ( figure 4 ) . effects of long - term treatment with interferon ( ifn ) on tumour necrosis factor - related apoptosis inducing ligand trailr-2 isoform 2 and trail- expression . expression levels are represented as relative expression compared with the reference gene gapdh , using the ct method . expression before and after 1 year of ifn therapy in the same individual patients is plotted . ( a ) long - term therapy with ifn significantly decreased mrna expression of trailr-2 isoform 2 in monocytes from patients who will become responders ( r ) as well as those who will become non - responders ( nr ) to treatment . ( b ) trail- mrna expression in cd4 + and cd8 + t cells significantly increased along with ifn therapy , but only in responders . the expression kinetics of each gene after in vitro induction with ifn was determined in jurkat cells ( figure 1 ) . the three trail variants showed a similar expression pattern : gene expression was upregulated during 14 h after ifn stimulation ( peak at 4 h ) , and declined thereafter , followed by a second increment after 24 h , in agreement with previous data.12 gene expression kinetics of the tumour necrosis factor - related apoptosis inducing ligand ( trail ) and trail receptors splicing variants in jurkat cells on in vitro stimulation with interferon ( ifn ) . ( a ) trail splicing variant genes are upregulated during 1 - 4 h of ifn treatment and decline thereafter . ( b ) death receptor isoforms begin to upregulate at 8 h and reach a maximum at 24 h. ( c ) decoy receptor isoforms begin to be induced at the 12 h time point and beyond . expression levels are represented as relative expression compared with the reference gene gapdh , using the ct method . trailr-1 and trailr-2 shared the same expression pattern , characterised by a decreased expression in the first hours , and a peak 24 h after ifn induction . the decoy receptors also began to be induced at 812 h post - stimulation with ifn , showing a progressive increment with a marked peak at 24 h. thus , in pbmc , the expression of trail and its receptors was assessed at 4 and 24 h after in vitro stimulation with ifn , respectively . on in vitro stimulation with ifn , mrna expression of trail- was significantly increased in hc compared to untreated patients with ms in the three cellular subsets ( p=0.023 in monocytes , p=0.00004 in cd4 + t cells and p= 0.021 in cd8 + t cells ) , as observed in figure 2 . we detected no other significant differences in the expression of trail and receptor variants at the rna level . the tumour necrosis factor - related apoptosis inducing ligand ( trail ) differential expression in healthy controls ( hc ) and untreated patients with multiple sclerosis ( ms ) . expression levels are represented as relative expression compared with the reference gene gapdh , using the ct method . figures show box plots : the horizontal bars are the median and lower and upper edges of the boxes represent the 2575th centiles . within the patient group , prior to ifn treatment onset , we found higher mrna expression of trail- in monocytes , cd4 + and cd8 + t cells from patients who will subsequently be classified as non - responders than from responders ( p=0.008 , p=0.00006 and 0.003 , respectively ) , as shown in figure 3 . conversely , the mrna expression of the trail- isoform was significantly higher in cd4 + and cd8 + t cells from patients who will become responders ( p=0.03 and p=0.025 ) , and showed the same trend of expression in monocytes ( p=0.052 ) . additionally , expression of trailr-1 was also significantly higher in monocytes and cd4 + t cells from patients who will become responders . relative expression of tumour necrosis factor - related apoptosis inducing ligand ( trail ) , trail- and trailr-1 , on in vitro stimulation with interferon ( ifn ) , before treatment onset . those patients who will become non - responders ( nr ) to ifn therapy showed higher expression of trail- mrna in the three cellular subsets , while those who will become responders ( r ) showed higher baseline expression of trail- and trailr-1 . expression level is represented as relative expression compared with the reference gene gapdh , using the ct method . figures show box plots : the horizontal bars are the median and lower and upper edges of the boxes represent the 2575th centiles . for biomarkers to predict the therapeutic response to ifn , roc analyses were performed to evaluate the predictive discriminating value of trail- , trail- and trailr-1 expression on in vitro induction with ifn before treatment onset , on different cell subsets from patients with ms . values of areas under the curve ( auc ) , sensitivity , specificity , positive predictive value and negative predictive value are shown in table 3 . expression of trail- in the three cellular subsets better predicted those patients who will subsequently become non - responders to treatment , with auc > 0.7 and sensitivities greater than 80% in the three cases . conversely , expression of trailr-1 on monocytes and cd4 + t cells better predicted those patients who will subsequently become responders to treatment , with auc > 0.8 , sensitivities greater than 90% and specificities greater than 75% . trail- expression did not seem a good biomarker to predict therapeutic response to ifn , as aucs were under 0.7 . sensitivity and specificity of biomarkers to predict the therapeutic response to ifn auc , area under the curve ; ifn , interferon ; npv , negative predictive value ; ppv , positive predictive value ; trail , tumour necrosis factor - related apoptosis inducing ligand . relative expression of trail and trail receptors was significantly modified along the first year of therapy only for two genes . expression of the isoform 2 of the death receptor trailr-2 was significantly reduced in monocytes from first - year responder as well as non - responder patients ( p=0.004 and 0.016 , respectively ) . expression of trail- was significantly increased in both t cell subsets but exclusively in patients who showed a clinical response to systemic ifn treatment ( p=0.004 in cd4 + cells and p=0.002 in cd8 + cells from responders ) . induction of trail- mrna expression in monocytes by ifn therapy was achieved in 13 of 24 responders and , therefore , overall expression in this subset was not significantly increased ( figure 4 ) . effects of long - term treatment with interferon ( ifn ) on tumour necrosis factor - related apoptosis inducing ligand trailr-2 isoform 2 and trail- expression . expression levels are represented as relative expression compared with the reference gene gapdh , using the ct method . expression before and after 1 year of ifn therapy in the same individual patients is plotted . ( a ) long - term therapy with ifn significantly decreased mrna expression of trailr-2 isoform 2 in monocytes from patients who will become responders ( r ) as well as those who will become non - responders ( nr ) to treatment . ( b ) trail- mrna expression in cd4 + and cd8 + t cells significantly increased along with ifn therapy , but only in responders . trail expression in pbmc has been proposed as a biomarker for therapeutic response to ifn in ms,5 but different splice variants were not taken into account . several diseases have been related to alternative splice variant or aberrant splicing.20 we hypothesise that alternative splice variants could be implicated in response to treatment and , thus , they should be measured independently . in our study , we first determined the expression kinetics of trail and its receptors isoforms after in vitro stimulation with ifn. in agreement with the existing literature , trail isoforms reached a peak 4 h after ifn stimulation.12 conversely , trail receptors reached maximum expression at 24 h after induction . to the best of our knowledge , our group has been the first to assess this issue . it remains unknown whether in vivo gene expression of trail receptors after induction with ifn shares the same pattern as in vitro , or whether they go through a more complex regulation . we are currently performing a longitudinal study , using blood samples drawn at different time points after injection with ifn , to address this question . contrary to previous studies,12 21 22 we found that the expression of trail- was significantly lower in monocytes and t cells from untreated patients with ms than from controls . in agreement , kurne et al found lower soluble trail concentrations in patients with ms than in hc.23 one possible explanation for this finding may be the lower levels of endogenous ifn reported in patients with ms,24 which are unable to induce trail synthesis to the same extent as they can in healthy subjects . cell type - specific induction of trail by ifn in patients with ms has been reported . we have shown upregulation of trail- expression after ifn therapy in monocytes , and in cd4 + and cd8 + t lymphocytes . earlier works found increased levels of trail after ifn-1a injection only on the surfaces of monocytes and/or granulocytes.6 8 25 however , upregulation of trail expression in lymphocytes7 26 27 and t cell lines28 from patients with ms and hc has also been reported . these differences could be attributed to the different culture conditions , kinetics and activation stimulus of the cells . within the patient group , those patients with ms who will be subsequently considered as responders to ifn therapy could be clearly distinguished from non - responders by baseline expression of trail- and trailr-1 , indicating underlying differences in transcriptional regulation of these genes . the in vitro addition of ifn to previously ifn nave cell subsets triggered a higher expression of trail- and a lower expression of trail- in monocytes and t cells from patients who will continue to experience relapses or disease progression while treated with ifn , and a higher expression of trailr-1 in monocytes and cd4 + t cells from those who will have neither relapses nor progression of the disease . wandinger et al5 reported no differences in baseline expression of trail regarding ifn responsiveness , but found higher baseline concentrations of soluble trail in responders , findings that could not be replicated by others.29 conversely , another study found higher levels of trail in monocytes from relapsing remitting quiescent disease patients ( responders).8 comparison among studies regarding biomarkers for therapeutic response to ifn in ms is difficult , due to the unique design and experimental approach of each single study . it is known that trail is upregulated on activation of monocytes24 30 and t cells,28 29 while trailr-1 and trailr-2 , in turn , are downregulated on the rna level when t cells are activated.28 in our roc analysis , baseline trail- expression in the three cellular subsets at the cut - off levels described in table 3 predicts suboptimal response with a sensitivity greater than 80% and a specificity around 65% , while baseline trailr-1 expression over 0.803 in monocytes and over 0.883 in cd4 + t cells predicts a good response to ifn therapy with a sensitivity higher than 90% and a specificity over 75% , promoting the idea of using the expression of trail- and trailr-1 as biomarkers to predict ifn treatment response . probably , the changes in baseline expression of trail- and trailr-1 of such a low magnitude in patients that will become non - responders may not be able to induce the expected changes after a significant increase in trail , such as a strong inhibition of activation of autoreactive t cells , promotion of tregs , or induction of neurons and oligodendrocytes apoptosis after lymphocyte infiltration.31 they may be more probably reflecting a compensatory mechanism to prevent , in part , the activation of autoreactive t cells in those untreated patients with an increased state of activation who will continue to experience relapses or progression . another explanation for this increase in trail- expression at baseline in non - responders may be attributed to a comparatively higher pretreatment serum concentration of endogenous ifn in non - responders than responders , as shown by axtell et al.32 long - term treatment with ifn has been reported to increase trail mrna expression.5 7 8 21 in our study , ifn therapy significantly increases trail- expression in both t cell subsets , but exclusively in responder patients , similar to results reported by others,5 7 8 reaching levels of expression that overcome those found in hc . in this sense , upregulation of trail in responders contributes to the deletion of autoreactive lymphocytes , the inhibition of activation of autoreactive lymphocytes and limitation of the production of proinflammatory cytokines , mechanisms that may contribute to the clinical benefit of ifn therapy . moreover , trail- promotes the induction of endogenous ifn,33 amplifying its effects . although trail- is also known to induce apoptosis of oligodendrocytes and neurons , as ifn enhances the integrity of the blood brain barrier ( bbb ) in responders , the upregulation of trail induced by ifn therapy probably comprises immunoregulatory mechanisms outside the central nervous system ( cns ) more than apoptosis of brain cells . however , although non - responders had higher basal trail- levels than responders , they showed an inability to increase trail- expression in t cells in response to ifn therapy , that is , these patients are already responding to endogenous ifn , probably at maximum level , but still show a disease activity that obtains no benefit from further administration of ifn. this inability to increase trail- expression could , on one hand , lead to a failure in the apoptosis of auto - aggressive t cells invading the cns and , on the other hand , prevent inhibition of the activation of autoreactive t cells contributing to the perpetuation of the inflammatory response and the appearance of relapses and cns damage . moreover , we found that long - term ifn therapy decreased the expression of trailr-2 isoform 2 in monocytes from patients with ms , regardless of the therapeutic responsiveness to this drug . expression of trailr-2 is constitutive in peripheral blood leucocytes and may be involved in the apoptosis of activated lymphocytes22 34 and monocytes . in later stages of tissue pathology , macrophages outnumber t cells in cns infiltrates and are needed to execute myelin phagocytosis in the demyelinating process . in this sense , downregulation of trailr-2 isoform 2 in monocytes could be one of the mechanisms responsible for the clinical benefits of ifn , as apoptosis of peripheral monocytes will be prevented , allowing these cells to differentiate into macrophages , cross the bbb and exert their functions . the present study shows that long - term therapy with ifn increased the expression of trail- in both t cell subsets exclusively from responder patients and decreased the expression of the isoform 2 of the death receptor trailr-2 in monocytes from responders as well as non - responders . roc analysis revealed that expression of trail- in the three cellular subsets , before the onset of ifn therapy , better predicted those patients who will subsequently become non - responders to treatment while expression of trailr-1 on monocytes and cd4 + t cells better predicted those patients who will become responders . further studies are necessary in order to elucidate the pathways implicated in this process , which could reveal the precise mechanism by which ifn is exerting its beneficial effects on patients with ms , but trail- and trailr-1 should be assessed as predictors in future ifn responsiveness studies . however , inconsistent results among different studies regarding therapeutic biomarkers to ifn in ms make us evaluate our results with caution . to prove the effectiveness of these predictive markers , studies with larger sample sizes are warranted .

objectivewe aimed to assess the effects of interferon ( ifn ) treatment on the expression of the splice variants of the tumour necrosis factor - related apoptosis inducing ligand ( trail ) and its receptors in different cell subpopulations ( cd14 + , cd4 + and cd8 + ) from patients with multiple sclerosis ( ms ) , and to determine whether this expression discriminated responders from non - responders to ifn therapy.methodswe examined mrna expression of the trail and trail receptors variants in patients with ms , at baseline and after one year of ifn therapy , according to responsiveness to this drug.resultslong-term therapy with ifn increased the expression of trail- in t cell subsets exclusively from responders and decreased the expression of the isoform 2 of trailr-2 in monocytes from responders as well as non - responders . lower expression of trail- , and higher expression of trail- in monocytes and t cells , was found before the onset of ifn therapy in patients who will subsequently become responders . baseline expression of trailr-1 was also significantly higher in monocytes and cd4 + t cells from responders.conclusionsthe present study shows that long - term ifn treatment has a direct influence on trail- and trailr-2 isoform 2 expression . besides , receiver operating characteristic analysis revealed that the baseline expression of trail- in monocytes and t cells , and that of trailr-1 in monocytes and cd4 + t cells , showed a predictive value of the clinical response to ifn therapy , pointing to a role of trail system in the mechanism of action of ifn in ms that will need further investigation .

Introduction Methods Subjects Definition of response to IFN therapy Detection of Neutralising Antibodies NABs Determination of expression kinetics in the Jurkat T-cell leukaemia cell line Gene expression in patients with MS RNA isolation, primer design and quantitative reverse transcription PCR Statistical analysis Results Gene expression kinetics of TRAIL and TRAIL receptors in Jurkat cells Gene expression of TRAIL and TRAIL receptors in untreated patients with MS and HC Basal gene expression of TRAIL and TRAIL receptors in patients with MS who will subsequently become responders or non-responders to IFN therapy Effects of long-term IFN therapy on TRAIL and TRAIL receptors expression in PBMC from patients with MS Discussion Conclusions

recombinant interferon ( ifn ) is one of the most widely used first line therapies in multiple sclerosis ( ms ) , but up to 50% of patients under treatment continue to experience relapses and progression of the disease and are considered suboptimal responders.1 it is , therefore , strongly desirable to count with biomarkers that allow prediction of the therapeutic response to ifn. the tumour necrosis factor - related apoptosis inducing ligand ( trail)/trail receptor system has been shown to be implicated in ms pathogenesis24 as well as in the mechanisms of action of ifn.5 6 expression of trail and its four surface receptors in different cell types during ifn treatment has been studied,7 8 and trail mrna expression in leucocytes has been proposed as a biomarker for the therapeutic response to ifn in patients with ms.5 nevertheless , none of these studies has taken into account the different splicing variants of each gene , which could lead to the expression of non - functional proteins . the loss of exon 3 leads to the lack of the extracellular domain and , therefore , to the absence of apoptotic potential.9 trailr-2 has two different apoptotic variants : trailr-2 isoform 1 ( trick2b ) , which has an 87 nucleotides insertion and trailr-2 isoform 2 ( trick2a ) , which is homologous to trailr-1.10 finally , there are two different splice variants for trailr-4 : trailr-4 , the long isoform , and trailr-4 , which lacks exon 3.11 the loss of this exon leads to the truncation of the first cysteine rich domain 1 , involved in the ligand receptor complex , which may alter its ability to bind trail . the aim of the present work was to assess the effects of ifn treatment on the expression of the splice variants of trail and its receptors in different cell subpopulations from patients with ms , and to determine whether these expressions discriminated responders from non - responders to ifn therapy . * p values : refers to p values obtained following comparisons between healthy controls , responders and non - responders by means of a kruskal - wallis test ( age ) and test ( gender ) , as well as p values obtained following comparisons between responders and non - responders by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . monocytes ( cd14 + ) , cd4 + t cells and cd8 + t cells were isolated using immunomagnetic microbeads and ls macs columns ( miltenyi biotec gmbh ) , following the manufacturer 's protocol . primers used for amplification of the splice variants of trail and trail receptors trail , tumour necrosis factor - related apoptosis inducing ligand . comparisons of clinical characteristics at baseline between responders and non - responders were performed by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . a mann - whitney u test was used to compare relative expression of trail and its receptors between responders and suboptimal responders to ifn therapy . * p values : refers to p values obtained following comparisons between healthy controls , responders and non - responders by means of a kruskal - wallis test ( age ) and test ( gender ) , as well as p values obtained following comparisons between responders and non - responders by means of a mann - whitney u test ( duration , edss and number of relapses ) and or fisher test ( clinical course and type of ifn ) . edss , expanded disability status scale ; ifn , interferon ; rr , relapsing remitting ; sp , secondary progressive ; trail , tumour necrosis factor - related apoptosis inducing ligand . patients were classified as optimal responders if they had neither relapses within the previous year nor progression of the disease over the same follow - up period , and as non - responders if they experienced one or more relapses or an increase of at least one point in the expanded disability status scale ( edss ) that persisted for a minimum of two consecutive visits separated by a 6-month interval.14 the presence of nabs in serum was checked after 12 and 24 months of treatment by the ifn - induced inhibition of virus cytopathic effect on human cells in culture , following the who recommendations as previously described.15 the neutralisation titre of serum samples was calculated according to kawade et al.16 titres 20 tru / ml were considered as positive . monocytes ( cd14 + ) , cd4 + t cells and cd8 + t cells were isolated using immunomagnetic microbeads and ls macs columns ( miltenyi biotec gmbh ) , following the manufacturer 's protocol . primers used for amplification of the splice variants of trail and trail receptors trail , tumour necrosis factor - related apoptosis inducing ligand . primers used for amplification of the splice variants of trail and trail receptors trail , tumour necrosis factor - related apoptosis inducing ligand . a mann - whitney u test was used to compare relative expression of trail and its receptors between responders and suboptimal responders to ifn therapy . the three trail variants showed a similar expression pattern : gene expression was upregulated during 14 h after ifn stimulation ( peak at 4 h ) , and declined thereafter , followed by a second increment after 24 h , in agreement with previous data.12 gene expression kinetics of the tumour necrosis factor - related apoptosis inducing ligand ( trail ) and trail receptors splicing variants in jurkat cells on in vitro stimulation with interferon ( ifn ) . the decoy receptors also began to be induced at 812 h post - stimulation with ifn , showing a progressive increment with a marked peak at 24 h. thus , in pbmc , the expression of trail and its receptors was assessed at 4 and 24 h after in vitro stimulation with ifn , respectively . on in vitro stimulation with ifn , mrna expression of trail- was significantly increased in hc compared to untreated patients with ms in the three cellular subsets ( p=0.023 in monocytes , p=0.00004 in cd4 + t cells and p= 0.021 in cd8 + t cells ) , as observed in figure 2 . the tumour necrosis factor - related apoptosis inducing ligand ( trail ) differential expression in healthy controls ( hc ) and untreated patients with multiple sclerosis ( ms ) . within the patient group , prior to ifn treatment onset , we found higher mrna expression of trail- in monocytes , cd4 + and cd8 + t cells from patients who will subsequently be classified as non - responders than from responders ( p=0.008 , p=0.00006 and 0.003 , respectively ) , as shown in figure 3 . conversely , the mrna expression of the trail- isoform was significantly higher in cd4 + and cd8 + t cells from patients who will become responders ( p=0.03 and p=0.025 ) , and showed the same trend of expression in monocytes ( p=0.052 ) . additionally , expression of trailr-1 was also significantly higher in monocytes and cd4 + t cells from patients who will become responders . relative expression of tumour necrosis factor - related apoptosis inducing ligand ( trail ) , trail- and trailr-1 , on in vitro stimulation with interferon ( ifn ) , before treatment onset . those patients who will become non - responders ( nr ) to ifn therapy showed higher expression of trail- mrna in the three cellular subsets , while those who will become responders ( r ) showed higher baseline expression of trail- and trailr-1 . for biomarkers to predict the therapeutic response to ifn , roc analyses were performed to evaluate the predictive discriminating value of trail- , trail- and trailr-1 expression on in vitro induction with ifn before treatment onset , on different cell subsets from patients with ms . expression of trail- in the three cellular subsets better predicted those patients who will subsequently become non - responders to treatment , with auc > 0.7 and sensitivities greater than 80% in the three cases . conversely , expression of trailr-1 on monocytes and cd4 + t cells better predicted those patients who will subsequently become responders to treatment , with auc > 0.8 , sensitivities greater than 90% and specificities greater than 75% . sensitivity and specificity of biomarkers to predict the therapeutic response to ifn auc , area under the curve ; ifn , interferon ; npv , negative predictive value ; ppv , positive predictive value ; trail , tumour necrosis factor - related apoptosis inducing ligand . expression of the isoform 2 of the death receptor trailr-2 was significantly reduced in monocytes from first - year responder as well as non - responder patients ( p=0.004 and 0.016 , respectively ) . expression of trail- was significantly increased in both t cell subsets but exclusively in patients who showed a clinical response to systemic ifn treatment ( p=0.004 in cd4 + cells and p=0.002 in cd8 + cells from responders ) . effects of long - term treatment with interferon ( ifn ) on tumour necrosis factor - related apoptosis inducing ligand trailr-2 isoform 2 and trail- expression . ( a ) long - term therapy with ifn significantly decreased mrna expression of trailr-2 isoform 2 in monocytes from patients who will become responders ( r ) as well as those who will become non - responders ( nr ) to treatment . ( b ) trail- mrna expression in cd4 + and cd8 + t cells significantly increased along with ifn therapy , but only in responders . the three trail variants showed a similar expression pattern : gene expression was upregulated during 14 h after ifn stimulation ( peak at 4 h ) , and declined thereafter , followed by a second increment after 24 h , in agreement with previous data.12 gene expression kinetics of the tumour necrosis factor - related apoptosis inducing ligand ( trail ) and trail receptors splicing variants in jurkat cells on in vitro stimulation with interferon ( ifn ) . on in vitro stimulation with ifn , mrna expression of trail- was significantly increased in hc compared to untreated patients with ms in the three cellular subsets ( p=0.023 in monocytes , p=0.00004 in cd4 + t cells and p= 0.021 in cd8 + t cells ) , as observed in figure 2 . the tumour necrosis factor - related apoptosis inducing ligand ( trail ) differential expression in healthy controls ( hc ) and untreated patients with multiple sclerosis ( ms ) . within the patient group , prior to ifn treatment onset , we found higher mrna expression of trail- in monocytes , cd4 + and cd8 + t cells from patients who will subsequently be classified as non - responders than from responders ( p=0.008 , p=0.00006 and 0.003 , respectively ) , as shown in figure 3 . conversely , the mrna expression of the trail- isoform was significantly higher in cd4 + and cd8 + t cells from patients who will become responders ( p=0.03 and p=0.025 ) , and showed the same trend of expression in monocytes ( p=0.052 ) . additionally , expression of trailr-1 was also significantly higher in monocytes and cd4 + t cells from patients who will become responders . relative expression of tumour necrosis factor - related apoptosis inducing ligand ( trail ) , trail- and trailr-1 , on in vitro stimulation with interferon ( ifn ) , before treatment onset . those patients who will become non - responders ( nr ) to ifn therapy showed higher expression of trail- mrna in the three cellular subsets , while those who will become responders ( r ) showed higher baseline expression of trail- and trailr-1 . for biomarkers to predict the therapeutic response to ifn , roc analyses were performed to evaluate the predictive discriminating value of trail- , trail- and trailr-1 expression on in vitro induction with ifn before treatment onset , on different cell subsets from patients with ms . expression of trail- in the three cellular subsets better predicted those patients who will subsequently become non - responders to treatment , with auc > 0.7 and sensitivities greater than 80% in the three cases . conversely , expression of trailr-1 on monocytes and cd4 + t cells better predicted those patients who will subsequently become responders to treatment , with auc > 0.8 , sensitivities greater than 90% and specificities greater than 75% . sensitivity and specificity of biomarkers to predict the therapeutic response to ifn auc , area under the curve ; ifn , interferon ; npv , negative predictive value ; ppv , positive predictive value ; trail , tumour necrosis factor - related apoptosis inducing ligand . expression of the isoform 2 of the death receptor trailr-2 was significantly reduced in monocytes from first - year responder as well as non - responder patients ( p=0.004 and 0.016 , respectively ) . expression of trail- was significantly increased in both t cell subsets but exclusively in patients who showed a clinical response to systemic ifn treatment ( p=0.004 in cd4 + cells and p=0.002 in cd8 + cells from responders ) . effects of long - term treatment with interferon ( ifn ) on tumour necrosis factor - related apoptosis inducing ligand trailr-2 isoform 2 and trail- expression . ( a ) long - term therapy with ifn significantly decreased mrna expression of trailr-2 isoform 2 in monocytes from patients who will become responders ( r ) as well as those who will become non - responders ( nr ) to treatment . ( b ) trail- mrna expression in cd4 + and cd8 + t cells significantly increased along with ifn therapy , but only in responders . contrary to previous studies,12 21 22 we found that the expression of trail- was significantly lower in monocytes and t cells from untreated patients with ms than from controls . we have shown upregulation of trail- expression after ifn therapy in monocytes , and in cd4 + and cd8 + t lymphocytes . earlier works found increased levels of trail after ifn-1a injection only on the surfaces of monocytes and/or granulocytes.6 8 25 however , upregulation of trail expression in lymphocytes7 26 27 and t cell lines28 from patients with ms and hc has also been reported . within the patient group , those patients with ms who will be subsequently considered as responders to ifn therapy could be clearly distinguished from non - responders by baseline expression of trail- and trailr-1 , indicating underlying differences in transcriptional regulation of these genes . the in vitro addition of ifn to previously ifn nave cell subsets triggered a higher expression of trail- and a lower expression of trail- in monocytes and t cells from patients who will continue to experience relapses or disease progression while treated with ifn , and a higher expression of trailr-1 in monocytes and cd4 + t cells from those who will have neither relapses nor progression of the disease . wandinger et al5 reported no differences in baseline expression of trail regarding ifn responsiveness , but found higher baseline concentrations of soluble trail in responders , findings that could not be replicated by others.29 conversely , another study found higher levels of trail in monocytes from relapsing remitting quiescent disease patients ( responders).8 comparison among studies regarding biomarkers for therapeutic response to ifn in ms is difficult , due to the unique design and experimental approach of each single study . it is known that trail is upregulated on activation of monocytes24 30 and t cells,28 29 while trailr-1 and trailr-2 , in turn , are downregulated on the rna level when t cells are activated.28 in our roc analysis , baseline trail- expression in the three cellular subsets at the cut - off levels described in table 3 predicts suboptimal response with a sensitivity greater than 80% and a specificity around 65% , while baseline trailr-1 expression over 0.803 in monocytes and over 0.883 in cd4 + t cells predicts a good response to ifn therapy with a sensitivity higher than 90% and a specificity over 75% , promoting the idea of using the expression of trail- and trailr-1 as biomarkers to predict ifn treatment response . probably , the changes in baseline expression of trail- and trailr-1 of such a low magnitude in patients that will become non - responders may not be able to induce the expected changes after a significant increase in trail , such as a strong inhibition of activation of autoreactive t cells , promotion of tregs , or induction of neurons and oligodendrocytes apoptosis after lymphocyte infiltration.31 they may be more probably reflecting a compensatory mechanism to prevent , in part , the activation of autoreactive t cells in those untreated patients with an increased state of activation who will continue to experience relapses or progression . another explanation for this increase in trail- expression at baseline in non - responders may be attributed to a comparatively higher pretreatment serum concentration of endogenous ifn in non - responders than responders , as shown by axtell et al.32 long - term treatment with ifn has been reported to increase trail mrna expression.5 7 8 21 in our study , ifn therapy significantly increases trail- expression in both t cell subsets , but exclusively in responder patients , similar to results reported by others,5 7 8 reaching levels of expression that overcome those found in hc . however , although non - responders had higher basal trail- levels than responders , they showed an inability to increase trail- expression in t cells in response to ifn therapy , that is , these patients are already responding to endogenous ifn , probably at maximum level , but still show a disease activity that obtains no benefit from further administration of ifn. moreover , we found that long - term ifn therapy decreased the expression of trailr-2 isoform 2 in monocytes from patients with ms , regardless of the therapeutic responsiveness to this drug . in this sense , downregulation of trailr-2 isoform 2 in monocytes could be one of the mechanisms responsible for the clinical benefits of ifn , as apoptosis of peripheral monocytes will be prevented , allowing these cells to differentiate into macrophages , cross the bbb and exert their functions . the present study shows that long - term therapy with ifn increased the expression of trail- in both t cell subsets exclusively from responder patients and decreased the expression of the isoform 2 of the death receptor trailr-2 in monocytes from responders as well as non - responders . roc analysis revealed that expression of trail- in the three cellular subsets , before the onset of ifn therapy , better predicted those patients who will subsequently become non - responders to treatment while expression of trailr-1 on monocytes and cd4 + t cells better predicted those patients who will become responders .

coordinate - based free energy simulation methods accomplish two tasks : sample a reduced - dimension space of generalized coordinates , and estimate the associated free energy landscape . among these , the adaptive biasing force method ( abf ) is an adaptive application of unconstrained thermodynamic integration ( ti ) . in abf , the running estimate of the free energy gradient from ti is used as the biasing force , hence combining the importance sampling and free energy estimation problems . here we investigate a combination of abf with extended - system dynamics as in the car this combination was introduced by lelivre et al . , and is a part of the double - integration orthogonal space tempering method of yang and co - workers , under the name dynamic reference restraining . this extended - system abf algorithm will henceforth be referred to as eabf . instead of applying the abf scheme to generalized variables that depend directly on atomic coordinates , eabf dynamics proceeds in a separate , explicit collective coordinate space with its own equations of motion , and these extended degrees of freedom are harmonically coupled to the collective variables in atomic coordinate space . as eabf does not rely on the free energy surface of interest for importance sampling , a separate estimator of the free energy is needed . one is a gain in flexibility and efficiency due to separating the problems of sampling and free energy estimation , as has been investigated in recent studies . the second benefit is bypassing the stringent technical requirements for collective variables to be usable for abf . the multidimensional ti formalism used by our implementation of abf entails some practical limitations : collective variables are required to be mutually orthogonal , and orthogonal to constraints ; furthermore , a jacobian term , which depends on second derivatives of the coordinates , must be calculated . while defining an effective , low - dimension collective coordinate space for molecular processes is often daunting to begin with , additional restrictions on the coordinates that can be used for abf reduce the applicability of the method in the most challenging cases . eabf relaxes these requirements , making the method applicable to any set of variables whose individual values and gradients can be readily computed . below , we present the principles of eabf dynamics in one and multiple dimensions , as well its combination with standard abf in what we term semi - eabf . we derive some properties of eabf - biased distributions of the coordinates , which are useful for understanding the sampling efficiency of the method . we then describe numerical tests and explain the effects of its parameters on convergence , most notably the strength of coupling between the geometric coordinate and the fictitious variable . we investigate the role of noise reduction through mollification of the measured forces by fluctuations of the coupling spring . we introduce the corrected z - averaged restraint ( czar ) estimator of the true free energy based on eabf trajectories . finally , we test the convergence and accuracy of eabf / czar and compare it to abf as well as an umbrella - integration - based estimator . let us recall the principles and notations of the abf method , the direct parent of eabf . consider a set of particles of coordinates , subject to constraints of the form k(q ) = 0 , and whose physical distribution is the canonical ensemble associated with the potential v(q ) at temperature t. within that ensemble , we wish to sample a vector generalized coordinate ( collective variable ) of physical interest , z = (q ) . abf is an adaptively biased dynamics that produces , in the long - time limit , a uniform distribution of . abf also yields an unbiased estimate of the free energy profile ( or potential of mean force , pmf ) a(z ) , which is defined by1where is the equilibrium probability distribution of . the time - dependent biasing force in abf is the running estimate of the free energy gradient . our implementation of abf relies on multidimensional thermodynamic integration as expressed by ciccotti et al . for each coordinate i , let vi be any vector field on atomic coordinates ( , where n is the number of atoms ) satisfying , for all j and k,23we call vi the inverse gradient of i . a possible choice is vi = jgi , j1 i , provided that the matrix g ( ij)i , j is invertible . this , however , is not often practical : in simpler cases intuition is sufficient to exhibit valid choices of v , whereas in more complex cases , obtaining v in closed form from this expression is difficult , if at all possible . the ith partial derivative of the free energy surface a may then be calculated as the -conditioned ensemble average of the instantaneous collective force fi:4depending on the function , calculation of the divergence vi , which typically implies second derivatives of i , may be onerous or impossible to express in closed form . it then becomes desirable to replace abf dynamics on coordinate with a dynamics that approximates it at a lower cost ( including the cost of developing algorithms and production - quality code ) . eabf consists in performing abf dynamics on a fictitious coordinate that fluctuates around (q ) . we shall now present eabf in detail , first on a scalar variable for simplicity , then in higher dimension . in eabf , we consider the extended system ( q , ) , where is a fictitious , nonphysical degree of freedom with mass m. evolves in time according to langevin dynamics at the same temperature as the rest of the system . to ensure that approximates , we couple these quantities with the harmonic potential ( k/2 ) ( (q ) ) . eabf can therefore be seen as a multiscale simulation method , where t represents a coarse - grained dynamics coupled to the atomic dynamics ( figure 1 ) . we then define eabf on coordinate as standard abf dynamics on the extended coordinate , where ( q , ) . time trajectories of the collective variable (xt ) ( black ) and the fictitious coordinate t ( blue ) for the deca - alanine system ( see computational details for details ) . the obvious choice of inverse gradient v is equal to the gradient , that is , a vector with null components for all q , and 1 for . this satisfies eq 2 and eq 3 because v has no support on coordinates q involved in constraints . the instantaneous force of eq 4 then reduces to the harmonic force from the coupling potential , which can be calculated regardless of the geometry of , and without using the physical forces v(q ) , in contrast to standard abf . the main convergence property of abf applies , that is , the biased dynamics of is less metastable than the unbiased one , and its limiting probability distribution is uniform , yielding a form of optimal sampling . the key intuition behind eabf is that efficient sampling of will result in efficient sampling of , given sufficient coupling between those two variables . we will show numerically that this is verified , and examine the requirement on the coupling strength . in the absence of adaptive bias , the extended potential is5and the equilibrium marginal distribution of depends on k:6the corresponding pmf in is defined by7 in the following , probability distributions are expressed up to a normalization constant , and free energy profiles up to an implicit additive constant , without loss of precision or generality . the integral in ( 6 ) may be recast by inserting ((q ) z ) dz = 1:8that is , the marginal distribution of the extended variable is that of (q ) convolved with a gaussian kernel of variance ( k ) . therefore , in the tight - coupling limit ( large k , small ) , becomes arbitrarily close to , and the extended pmf a , to the physical pmf a. a numerical example comparing these two quantities ( figure 2 ) illustrates a as a smoothed or mollified version of a. comparison of the physical free energy profile a(z ) ( black ) and that of the extended variable , a( ) ( blue ) for the deca - alanine system with loose coupling ( = 0.5 ) . under eabf dynamics , the applied bias on is the running estimate of the average spring force:9where the angle brackets indicate a canonical average conditioned by = *. the system is driven by a time - dependent biased potential t(q , ) that converges toward10which generates the following biased boltzmann distribution:11integrating over q and inserting eq 6 shows that this results in a uniform marginal distribution of , ( ) = constant , which is the abf result for the extended variable . integrating eq 11 over the conditional measure (q)z yields the joint distribution of ( z , ):12integrating over gives the biased marginal in z:13where we have substituted a with its definition ( eq 7 ) . finally , substituting eq 8 into eq 13 , we obtain an explicit relationship between the unbiased and biased z - distributions:14which can be summarized as15where g stands for the gaussian kernel of variance , and * for convolution . in the high - k , low- limit , convolution by the kernel thus , the biased limiting distribution (z ) becomes uniform , and eabf in the high - k limit recovers the behavior of standard abf on coordinate z = (q ) . the approach outlined above may be extended to higher - dimension variables , following the principles of multidimensional abf . given a d - dimensional vector collective variable (q ) = ( i(q))i , we augment the system with a vector of d extended variables = ( i)i , coupled through as many harmonic potentials ( 1/2 ) ki ( (q ) i ) . because the components i may have different physical dimensions , the spring constants ki are not generally commensurable . to recover the multidimensional thermodynamic integration formalism recalled above , we define the inverse gradient vector vi as equal to the gradient (q , ) iext , that is , the vector with component 1 on coordinate i and zero elsewhere . components of the adaptive biasing force are then16where the angle brackets indicate an average with respect to the canonical distribution of ( q , ) conditioned by = *. the case of a two - dimensional coordinate is illustrated in numerical tests below . one may then run a semi - eabf simulation , that is , a simulation in extended space ( q , ) with a harmonic potential coupling and 2(q ) , and an abf bias on the two - dimensional coordinate (q , ) ( 1(q ) , ) . a vector field v1 needs to be defined , verifying v1(q ) 1(q ) = 1 for all q ( eq 2 ) , and orthogonal to constraints ( eq 3 ) . the mutual orthogonality condition of eq 2 is trivially fulfilled because the second coordinate is independent of q. the biasing forces may then be calculated based on1718where the angle brackets indicate averages with respect to the canonical distribution of ( q , ) conditioned by ( 1(q ) = z1 , = * ) . more generally , one may run abf dynamics on a vector variable involving any combination of geometric coordinates and fictitious variables . numerical tests of the semi - eabf approach in two dimensions are presented below , together with an appropriate free - energy estimator . the eabf free energy gradient estimator converges in the long - time limit to a( ) , which is not the true , physical free energy gradient a(z ) . here we discuss how to estimate the physical free energy gradient from an eabf simulation . the simplest estimator uses a as an approximation to the physical pmf , a(z ) . this , however , is not asymptotically unbiased in the long - time limit , because of the convolution in eq 8 , as can be seen in figure 2 . below , we describe and test two other estimators , one of which is asymptotically unbiased regardless of k. another option , since a is related to a by convolution , is to apply a generic deconvolution algorithm ; however , that approach again yields a biased estimator . zheng and yang proposed a free energy estimator in the context of the orthogonal space tempering method , but it is applicable more generally to any eabf - like simulation . considering that each -value is a state wherein z is sampled under a harmonic restraint , the free energy derivative can be written based on umbrella integration . the final estimate of the free energy derivative is a weighted average of the umbrella integration values from different restraining ensembles ( -states):19where ( z| ) is the observed , bias distribution of (q ) conditioned by . umbrella integration further assumes that this distribution is nearly gaussian , hence its log - derivative may be approximated based on its mean z and variance ( z):20this assumption reduces the variance of the estimator , but introduces a bias that formally vanishes only in the high - k regime , when (z| ) becomes peaked and tends toward a gaussian . in the past years , we informally provided the community with an implementation of the zheng / yang estimator as a post - treatment of eabf trajectories , which was inefficient for large amounts of data . this was improved by an on - the - fly implementation that was published recently . we propose here an asymptotically unbiased estimator of a , named corrected z - averaged restraint ( czar ) . below , we introduce this estimator based on physical intuition ( see supporting information for a formal derivation ) . this coordinate only feels the eabf bias through the spring force k( z ) . thus , at a given value of z , the biasing force on coordinate results in an effective average biasing force on z equal to k(z z ) ( where z is the conditional average of at a given value of z ) . the free energy gradient is the log - derivative of the unbiased distribution :21from which we can derive a relationship with the eabf - biased distribution :22the right - hand side can be estimated numerically from the time trajectory of ( z , ) . we note that eq 22 is related to eq 19 by swapping the integration with respect to and differentiation with respect to z. thus , the main difference between the czar and zheng / yang approaches is that the latter relies on a gaussian approximation for the conditional distributions of z. a practical benefit of czar is that it does not require defining discrete -states , which makes implementation simpler and removes a tunable parameter . the czar estimator generalizes to eabf on a vector variable of arbitrary dimension , z = ( zi)i = ( i(q))i . each component of the free energy gradient may be estimated as23the derivation is provided in the supporting information , in dimension 2 for the sake of simplicity . the czar estimator is valid for standard abf as well , if the z - averaged force is replaced with the average force of abf for non - extended coordinates . the log - derivative of the abf - biased distribution vanishes for long times as the distribution becomes uniform . thus , czar may be used transparently in the semi - eabf case , as it provides unbiased estimates of the free energy gradient along both extended and nonextended variables . our eabf implementation in the collective variables module ( colvars ) is essentially a combination of two pre - existing features : extended - system coordinates , and multidimensional abf . the extended lagrangian functionality of the colvars module includes separate velocity - verlet or langevin integrators , independent from that of the underlying md engine , as well as harmonic restraints coupling each extended degree of freedom to its counterpart collective variable . activating extended dynamics introduces two tunable parameters for each extended coordinate : integrating the langevin equation of motion requires that the coordinate be assigned a fictitious mass , and the harmonic spring is defined by an arbitrary force constant . rather than choosing those parameters directly , the colvars implementation relies on an equivalent pair of quantities of the coupling , , which is also the standard deviation of the gaussian kernel in eq 8 , and its time constant ( the period of the oscillator ) . they are determined from the following expressions:2425where m is the fictitious mass and k is the harmonic force constant . broadly speaking , if is to approximate (q ) , then a small value of the tolerance is desirable . conversely , the oscillator period should be much larger than the md time step for stability of the integrator . more refined criteria for choosing these parameters will emerge from the numerical tests described below . one practical benefit of implementing eabf over standard abf , besides doing away with geometric complexity , is estimating the free energy gradient without needing the values of atomic forces . in large - scale parallel applications where namd and lammps are often used , this removes the need to gather these atomic forces on the computing node tasked with executing the abf algorithm . the decrease in interprocess communications should yield scaling improvements when many particles are involved in the definition of the collective variable . it is de facto available for any combination of md engine and platform supported by the colvars , most notably , namd and lammps . the algorithm is lightweight and relies on two accumulators that are updated at every time step : the z - conditioned average forces kzk z , and the biased z marginal distribution , (z ) . these two quantities are only combined at output time , to yield the free energy gradient estimate according to eq 22 . the implementation , as with most colvars functionality , supports an arbitrary number of variables . in one - dimensional cases , the gradient must be integrated in a postprocessing step to yield the free energy surface . to this end , we use our discrete markov chain monte carlo algorithm in this work for practical reasons , but in principle any poisson solver with adequate boundary conditions could be used . the test systems considered here were two small peptides in vacuum that were used in many studies before . the helical peptide deca - alanine was associated with its end - to - end distance ( using the first and last carbon atoms , in the absence of bond constraints ) . for two - dimensional tests , conformation of the dipeptide mimic n - acetyl - n - methyl - l - alanylamide ( nanma ) was described by its central ramachandran angles and . simulations were run with the development version of namd 2.12 , together with the collective variables module version 2016 - 09 - 03 , which will be included in namd 2.12 . in vacuo systems were modeled by the charmm22 force field , and simulated with a time step of 0.5 fs with langevin dynamics at 300 k and a damping coefficient of 5 ps . the zheng and yang umbrella integration estimator was used in its namd / colvars implementation by fu et al . convergence of free energy gradient estimators was assessed as follows . for each set of parameters , 10 simulations were started using different random seeds and stochastic initial velocities , and run for 10 ns each . for assessing the convergence of true free energy estimators , sampling was increased to 20 simulations of 20 ns each to account for their higher variance . the average of the final estimates of the free energy gradient from those replicas was the target for measuring relative convergence toward the limiting value of a given estimator under a given set of parameters . reference abf simulations were run under the same conditions except for the extended variables . the average abf gradient from those reference simulations was the target to test for absolute convergence toward the true free energy gradient . for each individual test simulation , the root - mean - square distance from the estimated gradient to the target was calculated as a function of sampling time . the average and standard deviations of these distances over sets of simulations were used for comparing the accuracy and rate of convergence of estimators , and are plotted in figures in the results section . when estimating the free energy profile a(z ) in an eabf simulation , convergence occurs at two levels . at the level of eabf itself , the free energy gradient estimator converges toward a( ) , and the observed distribution of becomes uniform . this depends on tunable eabf parameters and , and on the delay parameter fullsamples . the second level is convergence of the estimators of a(z ) based on the eabf trajectory ( zt , t ) . the latter depends on the choice of estimator , and in the case of the zheng / yang and czar estimators described above , on good sampling of the joint distribution of z and . because the methods are intended for large - scale molecular simulations where sampling time is necessarily limited , we test the estimators and parameter sets based on their rate of convergence at short and moderate times ( 10 ns for the fast deca - alanine system ) . the fullsamples parameter controls when enough sampling has been collected in one given bin for the adaptive biasing force to be introduced . to ensure continuity of the force as a function of time , the force is scaled by a linear ramp that goes from zero to one as the local sample count evolves between half the value of fullsamples and its full value . the top panel in figure 3 shows convergence rates of test simulations as a function of fullsamples . while the deviation at the end of the 10 ns simulations is equivalent , it can be seen to affect the initial convergence rate . a value of zero leads to transient overcompensation of the underlying free - energy , with strong dispersion of the results in the initial 4 ns for this particular case . this relaxation time could be much longer for more slowly relaxing systems , however , which is why larger values are advisable . even for this toy system , a value of 2000 samples gives the fastest short - time convergence , as well as nearly optimal accuracy in the intermediate regime ( 48 ns ) until the accuracies of all simulation sets converge at 10 ns . the fullsamples parameter is not specific of eabf , yet its role is not necessarily identical in eabf as in standard abf . tests of abf on the same system showed that a value of 0 for fullsamples did not impair convergence . this confirms that short - time scale relaxation properties of abf and eabf are substantially different , as eabf dynamics is dominated by the relatively slow oscillations of the fictitious degree of freedom ( figures 1 and 4 ) . on longer time scales , however , the limiting factor becomes orthogonal relaxation of slow degrees of freedom that are not captured by the chosen transition coordinate . it is not well - captured by a small gas - phase system such as this one , yet slower systems would not lend themselves to precise statistical characterization . convergence rate of eabf simulations as a function of the fullsamples parameter ( upper panel ) , coupling time scale defined in eq 25 ( middle panel ) , and coupling width defined in eq 24 ( lower panel ) . the graphs show the rms deviation of the eabf free energy gradient from an estimate of its limiting value , as a function of sampling time , averaged over ten 10-ns trajectories ( error bars indicate standard deviation over the 10-fold average ) . time trajectories of the spring force in eabf ( red ) and the instantaneous collective force in abf ( black ) for the deca - alanine system with = 0.2 . data points are shown for every md time step , here , every 0.5 fs . like fullsamples , the fluctuation period of the oscillator ( defined in equation 25 ) has no measurable influence on the deviation after 10 ns of simulation ( figure 3 , middle ) . however , it does slightly influence the rate of convergence for shorter times . for this system a shorter time of 50 fs gives similarly fast convergence , yet this is only possible because we use a short md integration time step of 0.5 fs in these gas - phase simulations . for condensed - phase simulations with longer timesteps , larger values of may be required to guarantee the accuracy of the extended - system trajectory : we recommend scaling it with the integration time step of the extended variable . unless otherwise stated , other eabf simulations of the deca - alanine system are performed with fullsamples set to 2000 and a of 100 fs . the key parameter of an eabf simulation is the coupling width , whose effect on convergence is illustrated by the lower panel of figure 3 . in the 0 limit , eabf is equivalent to standard abf . the initial rate of convergence is simply an increasing function of : broader fluctuations of the fictitious degree of freedom appear to accelerate convergence . this order is still valid at the end of the 10 ns interval , although error values have come closer together . this result is surprising in the sense that higher values of make the simulation more approximate or however , this expected loss of accuracy leads to a gain in precision . in abf , fluctuations of the instantaneous force f are dominated by high - frequency terms due to bonded interactions ( figure 4 ) . in eabf , the mean square fluctuation of the spring force f2 is of the order of k/ , which implies:26thus , the choice of is a trade - off between fluctuations of the extended variable ( bias of a as an estimator of a ) and fluctuations of the extended force ( variance of a ) : a high , low k will yield a limiting value of a that is very different from a but is estimated with low variance . however , the bias on a can be corrected by the estimators described below , which displaces the optimal compromise toward a soft ( low k ) rather than a stiff spring ( high k ) . as evidenced numerically in figure 4 , even a moderately soft spring results in considerable noise reduction with respect to abf . the upper panel of figure 5 shows the joint biased distribution (z , ) observed for four different values of . these distributions observed numerically agree well with the prediction of eq 12 based on numerical estimates of a and a ( data not shown ) . the common feature is that the marginal distribution in is nearly uniform , thanks to the eabf bias . setting = 0.1 enforces near equality of the two variables , leading to a very narrow joint distribution and a nearly uniform z - marginal as well ( lower panel of figure 5 ) . for looser coupling , the distribution broadens . at = 0.5 , the high - probability region deviates visibly from the equality line , leading to undersampling of low values of z. at = 1 , sampling along z becomes strongly nonuniform , with variations across 3 orders of magnitude . in this example , eabf sampling is most effective for between 0.2 and 0.5 , that is , where the mollification is significant but not sufficient to impede sampling along z. upper : observed joint distribution of ( z , ) , the physical and extended coordinates , in eabf sampling of the deca - alanine system , for different values of . the scale is logarithmic , with red isovalue lines separated by a factor of 10 in probability density . lower : observed marginal distribution of z = (q ) from eabf sampling at different values of the coupling width for the deca - alanine system , plotted on a logarithmic scale . therefore , the drawback of a softer spring does not directly reside in the greater difference between a and a , but rather in the greater deviation of the limiting biased distribution of z from uniformity , eventually yielding poor sampling as high - free - energy regions in z may be missed despite complete sampling over . since the eabf bias differs from the optimal bias by gaussian convolution ( eq 13 ) , deviations of (z ) from uniformity are related to the difference between and its convolution . considering the convolved of by a gaussian of variance as an approximation to (z ) in the small- limit , it can be shown ( see supporting information ) that the error is bounded by l/2 . that is , deviation from uniform sampling depends on the curvature of (z ) . one may see from figure 5 that sampling of z is reduced in concave regions of the pmf ( hence of ) and increased in convex regions . as a result , the optimal value of for a given application depends on the curvature of . figure 6 shows the convergence toward the true free energy derivative a(z ) of the naive , zheng / yang ( zy ) , and czar estimators compared to standard abf for different values of . the long - time bias of the naive estimator is clearly apparent for larger values of , but this estimator still proves faster - converging than standard abf , and as accurate at t = 20 ns , for equal to 0.1 . convergence of the naive ( top ) , zheng / yang ( middle ) , and czar ( bottom ) free energy estimators , as a function of the coupling parameter . the graphs show the rms deviation from the converged , true free energy gradient of each free energy gradient estimator as a function of sampling time , averaged over 20 20 ns trajectories ( error bars indicate standard deviation over the 20-fold average ) . the dotted line is from a set of standard ( nonextended system ) abf simulations . on this time scale , the asymptotic bias of the zy estimator at finite is only visible for of 0.5 or 1 : on those conditions , the gaussian assumption ( 20 ) for the conditional distribution is not verified . for lower values of , the bias is masked by the error due to variance : the mean error at 20 ns is equivalent to that of czar or standard abf . still , the convergence rate is slightly slower at 0.2 and at 0.1 , with a greater dispersion . since the czar and zy estimators share their mean force term , this could be ascribed to noise in the two - dimensional ( z , ) histogram , which the gaussian approximation may not erase at short times in less - populated regions of the histogram . another contributing factor could be the error due to discretization along . the defining feature of czar is its robustness with respect to , directly deriving from its asymptotically unbiased nature . its rate of convergence , however , depends on efficient sampling along z. for too large values of ( here , 1 ) , enhanced sampling along leaves some metastability or unsampled regions along ( figure 5 ) , leading to slower convergence of the pmf estimator ( blue line ) . in constrast with convergence of the mollified eabf free energy ( figure 3 ) , all estimators seem more accurate at small ( 0.1 ) , but the czar estimator shows much more robustness with respect to . on the two - dimensional coordinate describing the nanma peptide , the pmf estimates from eabf / czar and standard abf are visually indistinguishable after 100 ns of sampling ( figure 7 ) . a more quantitative view of the convergence is given by the upper panel of figure 8 . the optimal value of here is 5. = 1 produces a high initial error , certainly due to the high variance of the 2d ( , ) histogram at short sampling times . at 100 ns , this estimate has reached the error of standard abf . conversely , the = 10 simulation shows a high initial convergence rate , but at later times , its convergence is impeded by poor sampling along ( , ) due to insufficient coupling to ( , ) , similar to the = 1 case for deca - alanine ( figure 5 ) . two - dimensional free energy surface for the ramachandran angles of nanma in vacuum , reconstructed by standard abf ( a ) and eabf with the czar estimator ( b ) . convergence of the eabf / czar estimator of the two - dimensional free - energy gradients of nanma , for a 2d eabf dynamics ( upper panel ) and semi - eabf dynamics ( lower panel ) with an extended coordinate coupled to either angle or . data is provided for three values of . convergence of standard 2d abf is shown for reference ( dotted line ) . the semi - eabf case is a form of 2d - abf dynamics where one of the variables is biased directly , while the other is coupled to a fictitious degree of freedom that is biased . the results are largely similar to the 2d - eabf case , demonstrating the practical effectiveness of the approach . there is one interesting exception : the loose coupling case ( = 10 ) , which shows slower convergence in 2d : eabf converges well when only the angle is treated with an extended variable . this results from the anisotropic shape of the 2d pmf , where the barriers are more pronounced along than along ( figure 7 ) . the eabf approach is a generalization of abf , when applied to fictitious degrees of freedom coupled to generalized coordinates of interest . in contrast to abf , it imposes no requirements as to what combinations of coordinates may be used , and only their gradients , not their second derivatives , are needed . eabf sampling is more effective than abf due to the noise reduction effect of the fluctuations of the extended variable , as well as the nonlocal effect of the biasing force . we introduce czar , a conceptually simple , asymptotically unbiased estimator of the free energy that converges faster than abf for a broad range of coupling strengths . thus , we have shown that the deviation of a from a is not damaging , as long as sampling of the actual geometric coordinate is efficacious . this stems from the essential difference between eabf and abf , that is , separating the force used for sampling from the free energy gradient estimator . in abf , the free energy that is being estimated is used exactly to accelerate the dynamics . eabf decouples the sampling bias and free energy estimate , leaving some flexibility to use a biased estimator with reduced variance and fast convergence for sampling , while still ultimately benefiting from an unbiased free energy estimator . in this sense , eabf is similar in spirit to a recently proposed approach , combining metadynamics for exploration and a separate free energy estimator . indeed , the convergence rate of eabf / czar as a free energy estimator compares favorably to that of standard abf , essentially because it combines efficient exploration on a smoothed free energy surface with an unbiased estimator of the actual free energy . eabf is applicable where abf is not , in particular for elaborate generalized coordinates where calculation of second derivatives is cumbersome , and for sets of coordinates where orthogonality of the inverse gradient ( eq 2 ) is difficult to satisfy . its added algorithmic complexity ( integrator for the extended equations of motion and free energy estimator ) is offset by the reduced geometric calculations . however , we have shown that one of them , the oscillation time , has limited impact on convergence , and the other , the coupling width , has a range of safe values , particularly if a robust , asymptotically unbiased free energy estimator is used . based on these results , it may be argued that eabf / czar is always preferable to abf for its rate of convergence , regardless of practical implementation considerations . this may become clearer when several large - scale , numerically challenging applications are documented .

we report a theoretical description and numerical tests of the extended - system adaptive biasing force method ( eabf ) , together with an unbiased estimator of the free energy surface from eabf dynamics . whereas the original abf approach uses its running estimate of the free energy gradient as the adaptive biasing force , eabf is built on the idea that the exact free energy gradient is not necessary for efficient exploration , and that it is still possible to recover the exact free energy separately with an appropriate estimator . eabf does not directly bias the collective coordinates of interest , but rather fictitious variables that are harmonically coupled to them ; therefore is does not require second derivative estimates , making it easily applicable to a wider range of problems than abf . furthermore , the extended variables present a smoother , coarse - grain - like sampling problem on a mollified free energy surface , leading to faster exploration and convergence . we also introduce czar , a simple , unbiased free energy estimator from eabf trajectories . eabf / czar converges to the physical free energy surface faster than standard abf for a wide range of parameters .

Introduction Theory Results and Discussion Conclusion

among these , the adaptive biasing force method ( abf ) is an adaptive application of unconstrained thermodynamic integration ( ti ) . in abf , the running estimate of the free energy gradient from ti is used as the biasing force , hence combining the importance sampling and free energy estimation problems . instead of applying the abf scheme to generalized variables that depend directly on atomic coordinates , eabf dynamics proceeds in a separate , explicit collective coordinate space with its own equations of motion , and these extended degrees of freedom are harmonically coupled to the collective variables in atomic coordinate space . as eabf does not rely on the free energy surface of interest for importance sampling , a separate estimator of the free energy is needed . the multidimensional ti formalism used by our implementation of abf entails some practical limitations : collective variables are required to be mutually orthogonal , and orthogonal to constraints ; furthermore , a jacobian term , which depends on second derivatives of the coordinates , must be calculated . we introduce the corrected z - averaged restraint ( czar ) estimator of the true free energy based on eabf trajectories . consider a set of particles of coordinates , subject to constraints of the form k(q ) = 0 , and whose physical distribution is the canonical ensemble associated with the potential v(q ) at temperature t. within that ensemble , we wish to sample a vector generalized coordinate ( collective variable ) of physical interest , z = (q ) . abf also yields an unbiased estimate of the free energy profile ( or potential of mean force , pmf ) a(z ) , which is defined by1where is the equilibrium probability distribution of . the time - dependent biasing force in abf is the running estimate of the free energy gradient . the ith partial derivative of the free energy surface a may then be calculated as the -conditioned ensemble average of the instantaneous collective force fi:4depending on the function , calculation of the divergence vi , which typically implies second derivatives of i , may be onerous or impossible to express in closed form . in eabf , we consider the extended system ( q , ) , where is a fictitious , nonphysical degree of freedom with mass m. evolves in time according to langevin dynamics at the same temperature as the rest of the system . eabf can therefore be seen as a multiscale simulation method , where t represents a coarse - grained dynamics coupled to the atomic dynamics ( figure 1 ) . we then define eabf on coordinate as standard abf dynamics on the extended coordinate , where ( q , ) . the obvious choice of inverse gradient v is equal to the gradient , that is , a vector with null components for all q , and 1 for . the instantaneous force of eq 4 then reduces to the harmonic force from the coupling potential , which can be calculated regardless of the geometry of , and without using the physical forces v(q ) , in contrast to standard abf . in the absence of adaptive bias , the extended potential is5and the equilibrium marginal distribution of depends on k:6the corresponding pmf in is defined by7 in the following , probability distributions are expressed up to a normalization constant , and free energy profiles up to an implicit additive constant , without loss of precision or generality . the integral in ( 6 ) may be recast by inserting ((q ) z ) dz = 1:8that is , the marginal distribution of the extended variable is that of (q ) convolved with a gaussian kernel of variance ( k ) . therefore , in the tight - coupling limit ( large k , small ) , becomes arbitrarily close to , and the extended pmf a , to the physical pmf a. a numerical example comparing these two quantities ( figure 2 ) illustrates a as a smoothed or mollified version of a. comparison of the physical free energy profile a(z ) ( black ) and that of the extended variable , a( ) ( blue ) for the deca - alanine system with loose coupling ( = 0.5 ) . under eabf dynamics , the applied bias on is the running estimate of the average spring force:9where the angle brackets indicate a canonical average conditioned by = *. in the high - k , low- limit , convolution by the kernel thus , the biased limiting distribution (z ) becomes uniform , and eabf in the high - k limit recovers the behavior of standard abf on coordinate z = (q ) . to recover the multidimensional thermodynamic integration formalism recalled above , we define the inverse gradient vector vi as equal to the gradient (q , ) iext , that is , the vector with component 1 on coordinate i and zero elsewhere . components of the adaptive biasing force are then16where the angle brackets indicate an average with respect to the canonical distribution of ( q , ) conditioned by = *. one may then run a semi - eabf simulation , that is , a simulation in extended space ( q , ) with a harmonic potential coupling and 2(q ) , and an abf bias on the two - dimensional coordinate (q , ) ( 1(q ) , ) . a vector field v1 needs to be defined , verifying v1(q ) 1(q ) = 1 for all q ( eq 2 ) , and orthogonal to constraints ( eq 3 ) . numerical tests of the semi - eabf approach in two dimensions are presented below , together with an appropriate free - energy estimator . the eabf free energy gradient estimator converges in the long - time limit to a( ) , which is not the true , physical free energy gradient a(z ) . here we discuss how to estimate the physical free energy gradient from an eabf simulation . zheng and yang proposed a free energy estimator in the context of the orthogonal space tempering method , but it is applicable more generally to any eabf - like simulation . considering that each -value is a state wherein z is sampled under a harmonic restraint , the free energy derivative can be written based on umbrella integration . the final estimate of the free energy derivative is a weighted average of the umbrella integration values from different restraining ensembles ( -states):19where ( z| ) is the observed , bias distribution of (q ) conditioned by . in the past years , we informally provided the community with an implementation of the zheng / yang estimator as a post - treatment of eabf trajectories , which was inefficient for large amounts of data . the free energy gradient is the log - derivative of the unbiased distribution :21from which we can derive a relationship with the eabf - biased distribution :22the right - hand side can be estimated numerically from the time trajectory of ( z , ) . we note that eq 22 is related to eq 19 by swapping the integration with respect to and differentiation with respect to z. thus , the main difference between the czar and zheng / yang approaches is that the latter relies on a gaussian approximation for the conditional distributions of z. a practical benefit of czar is that it does not require defining discrete -states , which makes implementation simpler and removes a tunable parameter . each component of the free energy gradient may be estimated as23the derivation is provided in the supporting information , in dimension 2 for the sake of simplicity . thus , czar may be used transparently in the semi - eabf case , as it provides unbiased estimates of the free energy gradient along both extended and nonextended variables . our eabf implementation in the collective variables module ( colvars ) is essentially a combination of two pre - existing features : extended - system coordinates , and multidimensional abf . rather than choosing those parameters directly , the colvars implementation relies on an equivalent pair of quantities of the coupling , , which is also the standard deviation of the gaussian kernel in eq 8 , and its time constant ( the period of the oscillator ) . one practical benefit of implementing eabf over standard abf , besides doing away with geometric complexity , is estimating the free energy gradient without needing the values of atomic forces . these two quantities are only combined at output time , to yield the free energy gradient estimate according to eq 22 . in one - dimensional cases , the gradient must be integrated in a postprocessing step to yield the free energy surface . simulations were run with the development version of namd 2.12 , together with the collective variables module version 2016 - 09 - 03 , which will be included in namd 2.12 . convergence of free energy gradient estimators was assessed as follows . the average of the final estimates of the free energy gradient from those replicas was the target for measuring relative convergence toward the limiting value of a given estimator under a given set of parameters . at the level of eabf itself , the free energy gradient estimator converges toward a( ) , and the observed distribution of becomes uniform . the latter depends on the choice of estimator , and in the case of the zheng / yang and czar estimators described above , on good sampling of the joint distribution of z and . the fullsamples parameter controls when enough sampling has been collected in one given bin for the adaptive biasing force to be introduced . to ensure continuity of the force as a function of time , the force is scaled by a linear ramp that goes from zero to one as the local sample count evolves between half the value of fullsamples and its full value . it is not well - captured by a small gas - phase system such as this one , yet slower systems would not lend themselves to precise statistical characterization . convergence rate of eabf simulations as a function of the fullsamples parameter ( upper panel ) , coupling time scale defined in eq 25 ( middle panel ) , and coupling width defined in eq 24 ( lower panel ) . the graphs show the rms deviation of the eabf free energy gradient from an estimate of its limiting value , as a function of sampling time , averaged over ten 10-ns trajectories ( error bars indicate standard deviation over the 10-fold average ) . like fullsamples , the fluctuation period of the oscillator ( defined in equation 25 ) has no measurable influence on the deviation after 10 ns of simulation ( figure 3 , middle ) . for condensed - phase simulations with longer timesteps , larger values of may be required to guarantee the accuracy of the extended - system trajectory : we recommend scaling it with the integration time step of the extended variable . in the 0 limit , eabf is equivalent to standard abf . in eabf , the mean square fluctuation of the spring force f2 is of the order of k/ , which implies:26thus , the choice of is a trade - off between fluctuations of the extended variable ( bias of a as an estimator of a ) and fluctuations of the extended force ( variance of a ) : a high , low k will yield a limiting value of a that is very different from a but is estimated with low variance . however , the bias on a can be corrected by the estimators described below , which displaces the optimal compromise toward a soft ( low k ) rather than a stiff spring ( high k ) . setting = 0.1 enforces near equality of the two variables , leading to a very narrow joint distribution and a nearly uniform z - marginal as well ( lower panel of figure 5 ) . at = 0.5 , the high - probability region deviates visibly from the equality line , leading to undersampling of low values of z. at = 1 , sampling along z becomes strongly nonuniform , with variations across 3 orders of magnitude . in this example , eabf sampling is most effective for between 0.2 and 0.5 , that is , where the mollification is significant but not sufficient to impede sampling along z. upper : observed joint distribution of ( z , ) , the physical and extended coordinates , in eabf sampling of the deca - alanine system , for different values of . lower : observed marginal distribution of z = (q ) from eabf sampling at different values of the coupling width for the deca - alanine system , plotted on a logarithmic scale . therefore , the drawback of a softer spring does not directly reside in the greater difference between a and a , but rather in the greater deviation of the limiting biased distribution of z from uniformity , eventually yielding poor sampling as high - free - energy regions in z may be missed despite complete sampling over . as a result , the optimal value of for a given application depends on the curvature of . figure 6 shows the convergence toward the true free energy derivative a(z ) of the naive , zheng / yang ( zy ) , and czar estimators compared to standard abf for different values of . the long - time bias of the naive estimator is clearly apparent for larger values of , but this estimator still proves faster - converging than standard abf , and as accurate at t = 20 ns , for equal to 0.1 . convergence of the naive ( top ) , zheng / yang ( middle ) , and czar ( bottom ) free energy estimators , as a function of the coupling parameter . on this time scale , the asymptotic bias of the zy estimator at finite is only visible for of 0.5 or 1 : on those conditions , the gaussian assumption ( 20 ) for the conditional distribution is not verified . its rate of convergence , however , depends on efficient sampling along z. for too large values of ( here , 1 ) , enhanced sampling along leaves some metastability or unsampled regions along ( figure 5 ) , leading to slower convergence of the pmf estimator ( blue line ) . in constrast with convergence of the mollified eabf free energy ( figure 3 ) , all estimators seem more accurate at small ( 0.1 ) , but the czar estimator shows much more robustness with respect to . on the two - dimensional coordinate describing the nanma peptide , the pmf estimates from eabf / czar and standard abf are visually indistinguishable after 100 ns of sampling ( figure 7 ) . conversely , the = 10 simulation shows a high initial convergence rate , but at later times , its convergence is impeded by poor sampling along ( , ) due to insufficient coupling to ( , ) , similar to the = 1 case for deca - alanine ( figure 5 ) . two - dimensional free energy surface for the ramachandran angles of nanma in vacuum , reconstructed by standard abf ( a ) and eabf with the czar estimator ( b ) . convergence of the eabf / czar estimator of the two - dimensional free - energy gradients of nanma , for a 2d eabf dynamics ( upper panel ) and semi - eabf dynamics ( lower panel ) with an extended coordinate coupled to either angle or . the semi - eabf case is a form of 2d - abf dynamics where one of the variables is biased directly , while the other is coupled to a fictitious degree of freedom that is biased . the eabf approach is a generalization of abf , when applied to fictitious degrees of freedom coupled to generalized coordinates of interest . eabf sampling is more effective than abf due to the noise reduction effect of the fluctuations of the extended variable , as well as the nonlocal effect of the biasing force . we introduce czar , a conceptually simple , asymptotically unbiased estimator of the free energy that converges faster than abf for a broad range of coupling strengths . thus , we have shown that the deviation of a from a is not damaging , as long as sampling of the actual geometric coordinate is efficacious . this stems from the essential difference between eabf and abf , that is , separating the force used for sampling from the free energy gradient estimator . in abf , the free energy that is being estimated is used exactly to accelerate the dynamics . eabf decouples the sampling bias and free energy estimate , leaving some flexibility to use a biased estimator with reduced variance and fast convergence for sampling , while still ultimately benefiting from an unbiased free energy estimator . in this sense , eabf is similar in spirit to a recently proposed approach , combining metadynamics for exploration and a separate free energy estimator . indeed , the convergence rate of eabf / czar as a free energy estimator compares favorably to that of standard abf , essentially because it combines efficient exploration on a smoothed free energy surface with an unbiased estimator of the actual free energy . eabf is applicable where abf is not , in particular for elaborate generalized coordinates where calculation of second derivatives is cumbersome , and for sets of coordinates where orthogonality of the inverse gradient ( eq 2 ) is difficult to satisfy . its added algorithmic complexity ( integrator for the extended equations of motion and free energy estimator ) is offset by the reduced geometric calculations . however , we have shown that one of them , the oscillation time , has limited impact on convergence , and the other , the coupling width , has a range of safe values , particularly if a robust , asymptotically unbiased free energy estimator is used . based on these results , it may be argued that eabf / czar is always preferable to abf for its rate of convergence , regardless of practical implementation considerations .

the 5-year survival rate of the treated patients was only about 20% [ 1 , 2 ] . the treatment of osteosarcoma currently involves surgical resection in combination with neoadjuvant chemotherapy . despite advances in the neoadjuvant chemotherapy and in limb - salvage surgery , the disease - free survival rate still remains poor for patients with metastatic , recurrent , or unresectable osteosarcoma . previously , we found that the novel lectin eucheuma serra agglutinin ( esa ) , which was successfully isolated by kawakubo et al . from the marine red alga eucheuma serra , specifically binds to carcinoma cell lines of human adenocarcinoma , human cervical squamous cell carcinoma , and marine adenocarcinoma but not to normal human fibroblasts or lymphocytes . we also revealed , that the specific binding of esa to carcinoma cells is based on specific interactions between esa and the unique sugar chains of high mannose type on the surface of the carcinoma cells . in a more recent study , hori et al . investigated the specific interactions between esa and various unique sugar chains of high mannose type in detail . furthermore , we successfully elaborated the basis for a novel type of drug delivery system ( dds ) for cancer therapy using esa ( i ) as targeting ligand to carcinoma tumors and ( ii ) as inducer of apoptosis due to specific esa binding to carcinoma cells . recently , the general potential of certain types of sugar binding proteins ( lectins ) as promising , alternative antitumor drugs has been emphasized . the antitumor activity of these lectins might be related directly to specific intermolecular interactions between the lectins and the sugar chains on the tumor cell surface . however , whether lectins also have antitumor activities against osteosarcomas has not been clarified yet . generally , carcinomas which originate in epithelial cells and sarcomas which originate in mesenchymal cells ( e.g. , osteosarcoma ) are thought to be quite different in their tumorigenesis as well as in the phenotypes including cytoskeleton , binding molecules , proliferation procedure , and surface glycoproteins [ 9 , 10 ] . therefore , different therapeutic approaches have been employed for the treatment of sarcomas , if compared with the therapies applied for the treatment of carcinomas , except for the surgical treatment . on the other hand , the existence of cell surface - bound sugar chain structures , which are common among carcinomas and sarcomas , but not present in normal cells , has been suggested . moreover , the concept of epithelial - mesenchymal transition in tumors implies common structures and/or mechanism among carcinomas and sarcomas [ 12 , 13 ] . therefore , on the basis of our previous in vitro and in vivo studies with esa bound to span 80 vesicles for targeting carcinoma cells , we found it worthwhile to investigate whether the lectin esa can also be applied in a therapeutic approach against osteosarcomas . span 80 is generally known in the food and cosmetic industries as sorbitan monooleate , although commercial span 80 is a heterogeneous mixture of sorbitan mono- , di- , tri- , and tetra - esters . we have already demonstrated that nonionic vesicles prepared from span 80 have promising physicochemical properties ( high membrane fluidity with temperature dependent fusiogenicity ) which make this type of vesicle an attractive possible alternative to the commonly used liposomes in vitro and in vivo [ 6 , 1422 ] . aim of the work was to clarify the specificity of the binding of esa to either ost cells or lm8 cells , both being osteosarcoma cell lines . furthermore , the potential effectivity of esa as ligand on the surface of span 80 vesicles [ 6 , 14 , 18 , 19 , 21 , 22 ] with targeting function and as possible apoptosis - inducer for the treatment of osteosarcoma was also examined . in the work presented , the interactions between esa and ost cells and between esa and lm8 cells were examined by means of fluorescence microscopy and flow cytometry . as a result of our study , the evidence is presented that esa specifically binds to these two types of osteosarcoma cells , followed by induction of apoptosis due to this specific esa binding to the cells . furthermore , we could demonstrate that esa has a considerable potential as novel type of ligand immobilized onto pegylated span 80 vesicles , an important step towards the potential development of a therapy for the treatment of refractory osteosarcoma , as novel lipidic microcapsule drug - delivery system ( dds ) for transporting and delivering anticancer drugs for the treatment of cancer . eucheuma serra agglutinin ( esa ) was extracted from the red alga eucheuma serra , by means of ethanol precipitation , followed by purification with fast protein liquid chromatography ( fplc ) , using a 10 mmol / l sodium phosphate buffer ( ph = 7.4 ) . propidium iodide ( pi ) , -mannnosidase , -mannnosidase , endoglycosidase h , and rhodamine 6 g were obtained from sigma - aldrich ( st . louis , mo , usa ) . annexin v - pe apoptosis detection kit i which contains annexin v - pe and 7-amino - actinomycin d ( 7-add ) was obtained from becton dickinson biosciences ( franklin lakes , nj , usa ) . the caspase assay system was purchased from promega ( madison , wi , usa ) . fluorescein isothiocyanate , isomer i ( fitc ) , span 80 , cholesterol , and lecithin from soybeans were obtained from wako pure chemical industries ( osaka , japan ) . the phospholipid 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(succinyl ) ( supe ) was obtained from avanti polar lipids ( alabaster , al , usa ) . pbs ( phosphate buffered saline ) was composed of 137 mm nacl , 2.7 mm kcl , 10 mm na2hpo4 and 2 mm kh2po4 , ( ph = 7.4 ) . katsuro tomita ( department of orthopaedic surgery , kanazawa university school of medicine , japan ) , cultured in either erdf medium ( kyokuto pharmaceutical industrial , tokyo , japan ) or dulbecco 's modified eagle medium ( d - mem ) ( wako pure chemical industries , osaka , japan ) supplemented with 10% of fetal bovine serum ( fbs ) at 37c in a humidified atmosphere consisting of 5% co2 . murine osteosarcoma cell line ( lm8 cells ) was obtained from riken ( riken brc cell bank ) . these lm8 cells were grown in d - mem supplemented with 10% of fbs at 37c in a humidified atmosphere consisting of 5% co2 . ost cells and lm8 cells were inoculated in 6-well culture plates at a cell density of 2.0 10 cells / ml suspended in d - mem with 10% fbs . after 16 hours , the medium in each plate was exchanged with 10% fbs d - mem containing various concentration of esa . after incubation during one day , the cell number and the viabilities of both types of cells were evaluated by means of the propidium iodide nucleic acid stain using flow cytometry . the viability assay of ost cells for epv was also performed by the same way as above . in a similar way , time - courses of the viability of both ost cells and lm8 cells were experimentally measured in medium with esa at a concentration of 50 g / ml . apoptosis was analyzed by using the annexin v - pe apoptosis detection kit i according to a previously published protocol [ 2527 ] . ost cells or lm8 cells , at a concentration of 2 10 cells / ml , were suspended in d - mem containing 10% fbs , and then inoculated in 6-well culture plates . after 16 hours inoculation , the medium in each plate was exchanged with 10% fbs , d - mem containing 50 g / ml esa . the cell lines in each plate were incubated for different time periods , followed by twice washing with cold pbs . using a cell counter , the washed cells were diluted to a concentration of 1 10 cells / ml by resuspending the cells in 0.1 m hepes / naoh ( ph 7.4 ) containing 1.4 m nacl and 25 mm cacl2 ( binding buffer ) . volumes of 100 l of the cell suspensions were transferred to 1.5 ml eppendorf tubes . solutions of 5 l of annexinv - pe and 5 l of 7-add were added to the suspensions , followed by vortexing and incubation for 15 min at room temperature in the dark . then , 400 l binding buffer were added to each tube containing the incubated suspension , followed by analysis with a flow cytometer . caspase-3 activity was evaluated spectrophotometrically at = 405 nm with the caspase-3 substrate ac - devd - pna . ost cells were suspended at 2.0 10 cells / ml in d - mem with 10% fbs and then pipetted into 6-well culture plates . after 16 hours of incubation at 37c and 5% co2 , the medium in each plate was exchanged by 10% fbs , d - mem containing either 50 g / ml esa , or 50 g / ml esa + zvad - fmk ( = n - benzyloxycarbonyl - val - ala - asp(o - me ) fluoromethyl ketone ) which is a known caspase-3 inhibitor , or pbs as control . following culturing for 16 hours , the caspase-3 activity in these kinds of cells was measured with the caspase assay system ( promega , madison , wi , usa ) , using a spectrophotometer u-2000 ( hitachi , tokyo , japan ) according to the manufacturer 's instructions . an amount of 1.22 mg / ml esa was fluorescently labeled by addition of 1 mg / ml rhodamine 6 g ( rh6 g ) in 0.15 m sodium carbonate buffer ( ph = 9.0 ) , followed by removal of free fitc by using a pd-10 column ( ge healthcare , ct , usa ) . ost cells and lm8 cells , suspended at a concentration of 2.0 10 cells / ml , were cultured in 10% fbs erdf medium . after 16 hours , the culture medium was exchanged with a culture medium containing 10% fitc - esa solution , both types of cells were separately incubated for 3 , 6 , 9 , 12 , and 24 hours in a co2 incubator at 37c , respectively . after the incubation both cell suspensions were then analyzed by flow cytometry using a facs calibur instrument ( becton dickinson , mansfield , ma , usa ) . in a similar way , the binding activities of esa ( labeled with either rhodamine 6 g ( rh6 g ) or fitc ) to the sugar chains on the surface of ost cells were examined by incubation with -mannnosidase , or -mannnosidase , or endoglycosidase h for 2 hours before adding fluorescenctly labeled esa . after incubation for 1 hour , the esa binding to the ost cells was evaluated by using a fluorescence microscope ( bh2-rfc , olympus corp , tokyo , japan ) and the flow cytometer . esa - supe , a phospholipid - esa conjugate , was prepared as follows : 100 l of a supe solution ( 1.25 mg / ml in chloroform ) were added to a test tube . a thin film of supe formed after evaporation of chloroform under a stream of nitrogen gas . afterwards , 2.5 ml of an esa solution ( 0.675 mg / ml ) were added to the film to react with supe in 0.15 m sodium carbonate buffer ( ph 9.0 ) at room temperature . the reaction mixture was incubated for 2 hours with vortexing for a few seconds every 30 min , followed by letting the suspension stand at 4c overnight . residual supe in the buffer solution was removed by gel filtration with a pd-10 column packed with sephadex g-25 ( ge healthcare ; buckinghamshire , england ) . in the present work , type 1 : span 80 vesicles with immobilized esa and immobilized peg ( epv ) , containing as inner aqueous solution pbs . type 3 : span 80 vesicles with immobilized esa ( ev ) containing encapsulated fitc . type 4 : span 80 vesicles with immobilized esa and immobilized peg ( epv ) containing encapsulated fitc . the vesicles of types 2 , 3 , and 4 contained a 0.15 m sodium carbonate buffer solution ( ph = 9.0 ) containing 1 mg / ml fitc as inner aqueous solution . the vesicles were prepared with the two - step emulsification method in pretty much the same way of as described in the previous papers [ 6 , 19 ] . in this work , some minor modifications were applied for the preparation of epv containing fitc . a volume of 0.6 ml of the inner aqueous solutions ( the sodium carbonate buffer solution containing fitc as mentioned above ) was added to 6 ml of a n - hexane solution containing span 80 ( 264 mg ) , purified lecithin ( 24 mg ) and cholesterol ( 12 mg ) , followed by the first emulsification for 6 min at 17,500 rpm using a micro - homogenizer ns-310e 2 ( microtec co. , ltd . , funabashi , japan ) . afterwards , the solvent was removed in a rotary evaporator at 28c under reduced pressure , yielding a water - lipid emulsion to which 6 ml of the esa - supe solution ( obtained as described above ) containing tween 80 ( 96 mg ) and dspe - peg2000 ( 14.2 mg / ml ) were added , followed by the second emulsification with the homogenizer for 2 min at 3500 rpm to obtain a heterogeneous span 80 vesicle suspension . after stirring with a magnetic stirrer for 3 hours at room temperature , the vesicle suspension the vesicles were then purified by ultracentrifugation ( 50,000 rpm at 4c for 120 min ) in a himac centrifuge cr15b ( hitachi koki co. , ltd . , the lower phase was filtrated through 100-nm nucleopore track - etch polycarbonate membranes ( avanti polar lipids ; alabaster , al , usa ) and purified by gel filtration on a 7 cm ( diameter ) 50 cm ( length ) column containing biogel - a5 m ( bio - rad laboratories , richmond , ca , usa ) . cv containing fitc and ev containing fitc were also prepared in the same manner as above , but without both esa and peg ( for cv ) , and without dspe - peg2000 ( for ev ) , respectively . the diameters of cv , ev , and epv , which contained fitc were 104 7 nm , 100 2 nm , and 103 5 nm , respectively . ost cells were inoculated in 6-well culture plates at a cell density of 2.0 10 cells / ml suspended in d - mem with 10% fbs . , the culture medium was exchanged with 1.8 ml d - mem containing 10% fbs and 0.2 ml pbs , cv containing encapsulated fitc , ev containing encapsulated fitc , or epv containing encapsulated fitc . the cells were then kept for 15 min in a co2 incubator at 37c . after incubation , the ost cells were washed with cold pbs twice , followed by flow cytometric analysis . the viabilities of ost cells and lm8 cells were measured in the concentration range from 10 g / ml to 50 g / ml to evaluate the possible anticancer activity of esa . as shown in figure 1(a ) , the proliferations of both osteosarcoma cell types were inhibited by esa . the inhibitory effect against the cell viability increased with increasing amounts of added esa . addition of 50 g / ml esa , for example , decreased the cell viabilities of ost cells and lm8 cells to 54.7 11.4% and 41.7 12.3% , respectively . furthermore , figure 1(b ) the cell proliferation was inhibited completely by the addition of 50 g / ml esa after incubation for 48 hours . these experiments clearly demonstrate the anticancer activity of esa in the case of these osteosarcoma cells . the findings presented above about the inhibition of sarcoma cell proliferation ( see section 3.1 . ) suggested that esa may also induce apoptosis in sarcoma cells . therefore , apoptosis induction in either ost cells or lm8 cells by esa was examined by means of the double staining test for annexin v - pe and 7-add . the numerical values obtained from this analysis are displayed in figure 2 and summarized in table 1 . as shown in figure 2(a ) and table 1 , the relative amount of cells in the lower right part of the diagram ( indicating early stages of apoptosis ) was 74.8% at an elapsing time of 3 hours after adding esa , while in the case of the control cells ( pbs - treated only , no esa ) , the amount of the cells was 14.2% in the same part . moreover , the amount of cells in the upper right part of the diagram ( indicating dead cells ) increased from 22.5% ( at 3 hours after esa addition ) to 71.0% ( at 24 hours ) . the amount of cells in the lower right part of the diagram increased from 19.8% ( control ) to 68.2% at an elapsing time of 3 hours after adding esa , being similar to the case of ost cells . the amount of cells in the upper right of the diagram also increased from 17.9% ( at 3 hours ) to 23.1% ( at 24 hours ) . thus , esa also induced apoptosis in lm8 cells . from the results in sections 3.1 and 3.2 , it was found that esa specifically binds to ost cells and to lm8 cells , both being osteosarcoma cell lines , followed by induction of apoptosis . in the following investigations we mainly focused on ost cells , the activity of caspase-3 in ost cells was measured by using the caspase-3 assay in combination with the caspase-3 inhibitor zvad - fmk , as outlined in section 2.5 . the values reported on the y - axis of figure 3 are proportional to the amount ( i.e. , the activity ) of expressed caspase-3 , arising from the induced apoptosis in the ost cells . upon addition of esa , a 2.3-fold increase in caspase-3 activity was observed in comparison with the control ( without esa : only pbs ) . on the other hand , the addition of zvad - fmk inhibited the expressed capase-3 to almost the same level as in the case of the control . these data indicate that esa induces apoptotic cell death in ost cells , which confirms the independent results presented in figure 2 . to investigate the binding of esa ( labeled with fitc ) to both ost cells and lm8 cells , flow if esa - fitc binding to cells occurs , a rightward shift of the flow cyotometric curve is expected . this , indeed , was observed in the experiments with ost cells and lm8 cells , as shown in figure 4 . the fluorescence intensity of the cells treated with esa - fitc increased significantly , as compared to the control cells ( treated with pbs only ) . the curve shifts became larger with longer cell - incubation times : with both cell types , the shifts after 12 hours of incubation were larger than the shifts observed after 3 hours . in a previous study it was shown that esa is a lectin that specifically binds to high - mannose type ( hm ) n - glycans . the binding of esa to ost cells that were pretreated with glycosidases was investigated by labeling cell - bound esa with rhodamine 6 g ( rh6 g ) , see section 2.6 . first , the ost cells were pretreated with glycosidases to cleave sugar chains on the cell surface . incubation was for 2 hours using one of the following three glycosidases , -mannnosidase , -mannnosidase , or endoglycosidase h. the method of rh6 g labeling with esa was performed by incubating esa with rh6 g as mentioned in section 2.6 . then , the esa labeled with rh6 g was bound to the cells by incubating the cells for 1 hour , followed by a fluorescence microscopic observation of the labeled cells . as shown in figure 5 , non - treated ost cells ( as control ) displayed rh6 g fluorescence , but other ost cells that were pretreated with a glycosidases showed almost no fluorescence . this means that esa could not recognize the molecular structure of the sugar - chains on the surface of ost cell that were cleaved by glycosidases ; esa only recognized the native structure of the sugar - chains of the ost cells . thus , with these experiments it could be demonstrated that esa specifically binds to ost cells , through recognition of the sugar chains on the surface of the cells . to confirm the specific binding of esa to ost cells , a flow cytometric examination was also performed in a similar way as described in sections 3.4 and 3.5 . the results are shown in figure 6(a ) for cells treated with -mannosidase and -mannosidase , and in figure 6(b ) for cells treated with endoglycosidase h. in both cases , the decreases in fluorescence intensity in those cells that were treated with a glycosidase , if compared to untreated cells , were obvious . the intensity decrease in the case of treatment with -mannosidase seemed to be smaller than in the case of -mannosidase or endoglycosidase h. this is in good agreement with the images shown in figure 5 obtained with an independent analysis . although with rather low intensity only if the treatment was with -mannosidase . in the other two cases , there was no detectable fluorescence ( figure 5 ) . to test whether esa could be used as osteosarcoma - targeting ligand on a vesicular dds , span 80 vesicles with surface bound esa were prepared , and the interaction of these vesicles with ost cells three types of span 80 vesicles were prepared and tested ( see section 2.9 ) : cv ( control vesicles , no esa ) , ev ( vesicles with immobilized esa ) , and epv ( pegylated vesicles with immobilized esa ) . the vesicles were then mixed with ost cells and incubated , as mentioned in section 2.9 . then , flow cytometric measurements were performed . as shown in figure 7 , the fluorescence intensity in both cases was higher than for cells treated with cv containing fitc . this means that both types of vesicles with surface bound esa , epv , and ev bind to ost cells stronger than cv does . furthermore , the fluorescence intensity of the cells treated with epv containing fitc was found to be almost equal to the fluorescence intensity of the cells that were treated with ev containing fitc . therefore , pegylation did not hinder the binding of esa to the sugar chains on the surface of the cells . thus , span 80 vesicles with immobilized esa may be well suited for the development of a dds for targeting osteosarcoma cells . in a final investigation the variation of the ost cells viability as a function of the concentration of added esa ( incubation time was 48 hours ) is shown in figure 8 . epv also clearly showed a strong anticancer activity against ost cells , inhibiting proliferation of ost cells completely in a culture medium that contained 2 g / ml esa . this result is promising as it shows that pegylated span 80 vesicles with immobilized esa are potentially useful as drug carrier system with endogenous antitumor activity against osteosarcoma . in the esa concentration range above about 2 g / ml complete death of the ost cells was observed , as shown in figure 8 . this demonstrates that epv not only can function as targeting unit ( see section 3.7 ) , but also efficiently inhibit ost cell growth . it is known that the carbohydrate structures vary among the different cancer cell lines [ 27 , 28 ] . in this work , we report about our findings that esa has anticancer activity not only against carcinoma but also against sarcoma . this conclusion is based on the observation that both types of osteosarcoma cells , ost cells and lm8 cells , were significantly destroyed if incubated with esa at a concentration of 50 g / ml during a period of 24 hours , as shown in figure 1(a ) , and also destroyed completely if during 48 hours , as shown in figure 1(b ) . the effect of esa on the viabilities of osteosarcroma cells was compared with the effect of esa carcinoma cells studied previously , see s-2 , supplementary material , available online at doi:10.1155/2012/842785 . the supplementary material contains ( i ) data on the cytotoxicity and binding affinity of free esa and ev for normal cells and for cancer cells ; and ( ii ) a comparison of the effect of free esa on the cell viabilities of osteosarcoma and carcinoma cells . this comparison indicates that the antiproliferative activity of free esa in sarcoma cells is higher than in carcinoma cells , which may be related to differences in the carbohydrate structure of the surface of the two cell types . we already reported that esa specifically binds to high mannose type sugar chains in the case of carcinoma cells , inducing apoptotic cell death . as shown in figure 4 of the flow cytometric measurements , it was confirmed that esa bound not only to carcinoma cells but also to sarcoma cells like ost cells and lm8 cells . moreover , pretreatment of ost cells with different types of glycosidases , which cleaved the sugar chains on the surface of the ost cells , significantly decreased the binding of esa to the cells ( figures 5 and 6 ) . these results provide evidence that binding of esa to the sarcoma cells occurs through specific interactions between esa and carbohydrate chains on the cell surface . esa exhibited higher affinity towards ost cells as compared to lm8 cells ( figure 4 ) . the reason for this may be due to differences in the carbohydrate structure in the two cell types . this point needs to be also investigated , however , before any clear conclusion about the cell specificity can be drawn . esa induces apoptosis in osteosarcoma cells as shown by using the double staining test for annexin - v and 7-add [ 2527 ] . at an elapsing time of 3 hours after adding esa , apoptosis in both ost cells and lm8 cells was obvious . moreover , almost all of the ost cells were dead after 24 hours incubation with esa ( 50 g / ml ) , as shown in figure 2(a ) . the number of lm8 cells appearing in the upper right region of the plot did not seem to increase ( see figure 2(b ) ) . this apparent failure in staining is related to the apoptotic progress of the cell , and the apoptosis could n't be correctly measured with the double staining method . in fact , in the analysis of the flow cytometry , in the lm8 cells often fragmented and therefore counted correctly , when incubated during 24 hours with esa ( data not shown ) . induction of apoptosis in ost cells by esa was demonstrated by measuring the expression of caspase-3 ( see figure 3 ) . it was shown that the addition of esa to ost cells led to apoptosis in cells of sarcoma , because the caspase-3 expression is known to be directly related to the apoptosis mechanism . thus , esa may be used as efficient tumor - targeting ligand and apoptosis inducer in a dds in a sarcoma therapy . as shown in our previous work , pegylated span 80 vesicles with immobilized esa ( abbreviated as epv ) are rather promising drug carriers for the treatment of carcinoma cancers . the ability of esa , and epv , as targeting unit and apoptosis inducer in the case of cells of sarcoma was examined further by flow cytometry as well as cell viability measurements , choosing ost cells as typical sarcoma cell type . as shown with the flow cytometric measurements in figure 6 , targeting of esa to ost cells in vitro was observed from the shift of the flow cytometric curve to the right hand side ( see figure 6 ) . furthermore , comparing epv with cv in figure 7 ( as mentioned in section 3.7 . ) , it was found that the macromolecular structure of peg on the vesicle surface did not hinder ost cell binding of esa which was localized on the vesicle surface together with peg . . it may be due to the high mobility of both esa and peg , because of the high membrane fluidity of span 80 vesicles , as mentioned previously [ 19 , 30 ] . in addition , epv showed anticancer activity against ost cells since after an elapsing time of about 48 hours after the addition of epv at an esa concentration of 2 g / ml , the ost cell viability was reduced to almost zero , as shown in figure 8 . it seems that the anticancer activities of esa against ost cells in the vesicle system ( figure 8) is stronger than those in free esa system ( figure 1 ) . however , the activities of the two systems can not be compared directly , because either the incubation time or the esa concentration was different in the two systems . for example , for a direct comparison of the activities of the two systems against ost cells , the time - course of the viability upon addition of free esa system ( figure 1(b ) ) should be measured at [ esa ] = 2 g / ml ; at this concentration and after an incubation time of 48 hours , the cells were no more viable if the vesicles system was used ( figure 8) . unfortunately , the data obtained from measurements with free esa at this low concentration showed great variations . on the other hand , we have already examined [ 4 , 6 ] the cytotoxicity of either esa or ev for various carcinoma cancer cells and normal cells , followed by examining the binding affinities of esa and ev to the cells . in these experiments , colo201 ( human colon adenocarcinoma ) , mcf-7 ( human breast adenocarcinoma ) , hela ( human cervix adenocarcinoma ) , and hb4c5 cells ( human hybridoma cell line ) were used as carcinoma cells , and mcf10 - 2a ( non - tumorigenic epitherial cell line ) and normal fibroblasts ( from the umbilical cord ) were also used as normal cells . esa and ev showed cytotoxicity against carcinoma cells but not against normal cells , see s-1 , supplementary material . figure 9 is a graphical imaginary view indicating the binding between carbohydrate chains of high mannose type on sarcoma membranes and esa on the pegylated span 80 vesicle . in the study presented , the following main results were obtained : ( i ) esa specifically binds to sarcoma cells and induces apoptotic death of the cells ; ( ii ) the antiproliferative activity of esa in sarcoma is higher than the activity in carcinoma ; ( iii ) esa immobilized onto pegylated span 80 vesicles ( epv ) shows antitumor activity against ost cells without any entrapped antitumor agents . furthermore , in a previous study , it was already revealed that esa and ev ( esa - immobilized on span 80 vesicles ) hardly bind to normal cells ( either mcf10 - 2a ( non - tumorigenic epithelial cells ) or normal fibroblasts from the umbilical cord ) ; and cytotoxicity caused by esa and ev was not observed for these normal cells . therefore , esa has considerable potential as novel type of targeting ligand against sarcoma . based on all these findings , we propose using epv as possible dds not only for the targeted treatment of carcinoma , but also for the targeted treatment of sarcoma . furthermore , the administration of pegylated span 80 vesicles with immobilized esa , in which anticancer drugs are encapsulated , is expected to express more effective antitumor activity against sarcoma as compared to empty epv . we already performed first in vivo experiments by using either ev or epv with entrapped anticancer drugs toward the development of a sarcoma therapy .

previously , we demonstrated that the novel lectin eucheuma serra agglutinin from a marine red alga ( esa ) induces apoptotic cell death in carcinoma . we now find that esa induces apoptosis also in the case of sarcoma cells . first , propidium iodide assays with ost cells and lm8 cells showed a decrease in cell viability after addition of esa . with 50 g / ml esa , the viabilities after 24 hours decreased to 54.7 11.4% in the case of ost cells and to 41.7 12.3% for lm8 cells . second , using fluorescently labeled esa and flow cytometric and fluorescence microscopic measurements , it could be shown that esa does not bind to cells that were treated with glycosidases , indicating importance of the carbohydrate chains on the surface of the cells for efficient esa - cell interactions . third , span 80 vesicles with surface - bound esa as active targeting ligand were shown to display sarcoma cell binding activity , leading to apoptosis and complete ost cell death after 48 hours at 2 g / ml esa . the findings indicate that span 80 vesicles with surface - bound esa are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma .

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

previously , we found that the novel lectin eucheuma serra agglutinin ( esa ) , which was successfully isolated by kawakubo et al . we also revealed , that the specific binding of esa to carcinoma cells is based on specific interactions between esa and the unique sugar chains of high mannose type on the surface of the carcinoma cells . furthermore , we successfully elaborated the basis for a novel type of drug delivery system ( dds ) for cancer therapy using esa ( i ) as targeting ligand to carcinoma tumors and ( ii ) as inducer of apoptosis due to specific esa binding to carcinoma cells . therefore , different therapeutic approaches have been employed for the treatment of sarcomas , if compared with the therapies applied for the treatment of carcinomas , except for the surgical treatment . on the other hand , the existence of cell surface - bound sugar chain structures , which are common among carcinomas and sarcomas , but not present in normal cells , has been suggested . therefore , on the basis of our previous in vitro and in vivo studies with esa bound to span 80 vesicles for targeting carcinoma cells , we found it worthwhile to investigate whether the lectin esa can also be applied in a therapeutic approach against osteosarcomas . aim of the work was to clarify the specificity of the binding of esa to either ost cells or lm8 cells , both being osteosarcoma cell lines . furthermore , the potential effectivity of esa as ligand on the surface of span 80 vesicles [ 6 , 14 , 18 , 19 , 21 , 22 ] with targeting function and as possible apoptosis - inducer for the treatment of osteosarcoma was also examined . in the work presented , the interactions between esa and ost cells and between esa and lm8 cells were examined by means of fluorescence microscopy and flow cytometry . as a result of our study , the evidence is presented that esa specifically binds to these two types of osteosarcoma cells , followed by induction of apoptosis due to this specific esa binding to the cells . furthermore , we could demonstrate that esa has a considerable potential as novel type of ligand immobilized onto pegylated span 80 vesicles , an important step towards the potential development of a therapy for the treatment of refractory osteosarcoma , as novel lipidic microcapsule drug - delivery system ( dds ) for transporting and delivering anticancer drugs for the treatment of cancer . eucheuma serra agglutinin ( esa ) was extracted from the red alga eucheuma serra , by means of ethanol precipitation , followed by purification with fast protein liquid chromatography ( fplc ) , using a 10 mmol / l sodium phosphate buffer ( ph = 7.4 ) . ost cells and lm8 cells were inoculated in 6-well culture plates at a cell density of 2.0 10 cells / ml suspended in d - mem with 10% fbs . after incubation during one day , the cell number and the viabilities of both types of cells were evaluated by means of the propidium iodide nucleic acid stain using flow cytometry . the viability assay of ost cells for epv was also performed by the same way as above . in a similar way , time - courses of the viability of both ost cells and lm8 cells were experimentally measured in medium with esa at a concentration of 50 g / ml . after 16 hours inoculation , the medium in each plate was exchanged with 10% fbs , d - mem containing 50 g / ml esa . ost cells were suspended at 2.0 10 cells / ml in d - mem with 10% fbs and then pipetted into 6-well culture plates . after 16 hours of incubation at 37c and 5% co2 , the medium in each plate was exchanged by 10% fbs , d - mem containing either 50 g / ml esa , or 50 g / ml esa + zvad - fmk ( = n - benzyloxycarbonyl - val - ala - asp(o - me ) fluoromethyl ketone ) which is a known caspase-3 inhibitor , or pbs as control . an amount of 1.22 mg / ml esa was fluorescently labeled by addition of 1 mg / ml rhodamine 6 g ( rh6 g ) in 0.15 m sodium carbonate buffer ( ph = 9.0 ) , followed by removal of free fitc by using a pd-10 column ( ge healthcare , ct , usa ) . ost cells and lm8 cells , suspended at a concentration of 2.0 10 cells / ml , were cultured in 10% fbs erdf medium . in a similar way , the binding activities of esa ( labeled with either rhodamine 6 g ( rh6 g ) or fitc ) to the sugar chains on the surface of ost cells were examined by incubation with -mannnosidase , or -mannnosidase , or endoglycosidase h for 2 hours before adding fluorescenctly labeled esa . in the present work , type 1 : span 80 vesicles with immobilized esa and immobilized peg ( epv ) , containing as inner aqueous solution pbs . type 3 : span 80 vesicles with immobilized esa ( ev ) containing encapsulated fitc . type 4 : span 80 vesicles with immobilized esa and immobilized peg ( epv ) containing encapsulated fitc . afterwards , the solvent was removed in a rotary evaporator at 28c under reduced pressure , yielding a water - lipid emulsion to which 6 ml of the esa - supe solution ( obtained as described above ) containing tween 80 ( 96 mg ) and dspe - peg2000 ( 14.2 mg / ml ) were added , followed by the second emulsification with the homogenizer for 2 min at 3500 rpm to obtain a heterogeneous span 80 vesicle suspension . the viabilities of ost cells and lm8 cells were measured in the concentration range from 10 g / ml to 50 g / ml to evaluate the possible anticancer activity of esa . addition of 50 g / ml esa , for example , decreased the cell viabilities of ost cells and lm8 cells to 54.7 11.4% and 41.7 12.3% , respectively . furthermore , figure 1(b ) the cell proliferation was inhibited completely by the addition of 50 g / ml esa after incubation for 48 hours . these experiments clearly demonstrate the anticancer activity of esa in the case of these osteosarcoma cells . the findings presented above about the inhibition of sarcoma cell proliferation ( see section 3.1 . ) therefore , apoptosis induction in either ost cells or lm8 cells by esa was examined by means of the double staining test for annexin v - pe and 7-add . as shown in figure 2(a ) and table 1 , the relative amount of cells in the lower right part of the diagram ( indicating early stages of apoptosis ) was 74.8% at an elapsing time of 3 hours after adding esa , while in the case of the control cells ( pbs - treated only , no esa ) , the amount of the cells was 14.2% in the same part . moreover , the amount of cells in the upper right part of the diagram ( indicating dead cells ) increased from 22.5% ( at 3 hours after esa addition ) to 71.0% ( at 24 hours ) . the amount of cells in the lower right part of the diagram increased from 19.8% ( control ) to 68.2% at an elapsing time of 3 hours after adding esa , being similar to the case of ost cells . from the results in sections 3.1 and 3.2 , it was found that esa specifically binds to ost cells and to lm8 cells , both being osteosarcoma cell lines , followed by induction of apoptosis . in the following investigations we mainly focused on ost cells , the activity of caspase-3 in ost cells was measured by using the caspase-3 assay in combination with the caspase-3 inhibitor zvad - fmk , as outlined in section 2.5 . on the other hand , the addition of zvad - fmk inhibited the expressed capase-3 to almost the same level as in the case of the control . these data indicate that esa induces apoptotic cell death in ost cells , which confirms the independent results presented in figure 2 . to investigate the binding of esa ( labeled with fitc ) to both ost cells and lm8 cells , flow if esa - fitc binding to cells occurs , a rightward shift of the flow cyotometric curve is expected . this , indeed , was observed in the experiments with ost cells and lm8 cells , as shown in figure 4 . the fluorescence intensity of the cells treated with esa - fitc increased significantly , as compared to the control cells ( treated with pbs only ) . the binding of esa to ost cells that were pretreated with glycosidases was investigated by labeling cell - bound esa with rhodamine 6 g ( rh6 g ) , see section 2.6 . first , the ost cells were pretreated with glycosidases to cleave sugar chains on the cell surface . then , the esa labeled with rh6 g was bound to the cells by incubating the cells for 1 hour , followed by a fluorescence microscopic observation of the labeled cells . as shown in figure 5 , non - treated ost cells ( as control ) displayed rh6 g fluorescence , but other ost cells that were pretreated with a glycosidases showed almost no fluorescence . this means that esa could not recognize the molecular structure of the sugar - chains on the surface of ost cell that were cleaved by glycosidases ; esa only recognized the native structure of the sugar - chains of the ost cells . thus , with these experiments it could be demonstrated that esa specifically binds to ost cells , through recognition of the sugar chains on the surface of the cells . to confirm the specific binding of esa to ost cells , a flow cytometric examination was also performed in a similar way as described in sections 3.4 and 3.5 . the results are shown in figure 6(a ) for cells treated with -mannosidase and -mannosidase , and in figure 6(b ) for cells treated with endoglycosidase h. in both cases , the decreases in fluorescence intensity in those cells that were treated with a glycosidase , if compared to untreated cells , were obvious . the intensity decrease in the case of treatment with -mannosidase seemed to be smaller than in the case of -mannosidase or endoglycosidase h. this is in good agreement with the images shown in figure 5 obtained with an independent analysis . to test whether esa could be used as osteosarcoma - targeting ligand on a vesicular dds , span 80 vesicles with surface bound esa were prepared , and the interaction of these vesicles with ost cells three types of span 80 vesicles were prepared and tested ( see section 2.9 ) : cv ( control vesicles , no esa ) , ev ( vesicles with immobilized esa ) , and epv ( pegylated vesicles with immobilized esa ) . the vesicles were then mixed with ost cells and incubated , as mentioned in section 2.9 . this means that both types of vesicles with surface bound esa , epv , and ev bind to ost cells stronger than cv does . furthermore , the fluorescence intensity of the cells treated with epv containing fitc was found to be almost equal to the fluorescence intensity of the cells that were treated with ev containing fitc . therefore , pegylation did not hinder the binding of esa to the sugar chains on the surface of the cells . thus , span 80 vesicles with immobilized esa may be well suited for the development of a dds for targeting osteosarcoma cells . in a final investigation the variation of the ost cells viability as a function of the concentration of added esa ( incubation time was 48 hours ) is shown in figure 8 . epv also clearly showed a strong anticancer activity against ost cells , inhibiting proliferation of ost cells completely in a culture medium that contained 2 g / ml esa . this result is promising as it shows that pegylated span 80 vesicles with immobilized esa are potentially useful as drug carrier system with endogenous antitumor activity against osteosarcoma . in the esa concentration range above about 2 g / ml complete death of the ost cells was observed , as shown in figure 8 . in this work , we report about our findings that esa has anticancer activity not only against carcinoma but also against sarcoma . this conclusion is based on the observation that both types of osteosarcoma cells , ost cells and lm8 cells , were significantly destroyed if incubated with esa at a concentration of 50 g / ml during a period of 24 hours , as shown in figure 1(a ) , and also destroyed completely if during 48 hours , as shown in figure 1(b ) . the effect of esa on the viabilities of osteosarcroma cells was compared with the effect of esa carcinoma cells studied previously , see s-2 , supplementary material , available online at doi:10.1155/2012/842785 . the supplementary material contains ( i ) data on the cytotoxicity and binding affinity of free esa and ev for normal cells and for cancer cells ; and ( ii ) a comparison of the effect of free esa on the cell viabilities of osteosarcoma and carcinoma cells . this comparison indicates that the antiproliferative activity of free esa in sarcoma cells is higher than in carcinoma cells , which may be related to differences in the carbohydrate structure of the surface of the two cell types . we already reported that esa specifically binds to high mannose type sugar chains in the case of carcinoma cells , inducing apoptotic cell death . as shown in figure 4 of the flow cytometric measurements , it was confirmed that esa bound not only to carcinoma cells but also to sarcoma cells like ost cells and lm8 cells . moreover , pretreatment of ost cells with different types of glycosidases , which cleaved the sugar chains on the surface of the ost cells , significantly decreased the binding of esa to the cells ( figures 5 and 6 ) . these results provide evidence that binding of esa to the sarcoma cells occurs through specific interactions between esa and carbohydrate chains on the cell surface . at an elapsing time of 3 hours after adding esa , apoptosis in both ost cells and lm8 cells was obvious . moreover , almost all of the ost cells were dead after 24 hours incubation with esa ( 50 g / ml ) , as shown in figure 2(a ) . the number of lm8 cells appearing in the upper right region of the plot did not seem to increase ( see figure 2(b ) ) . in fact , in the analysis of the flow cytometry , in the lm8 cells often fragmented and therefore counted correctly , when incubated during 24 hours with esa ( data not shown ) . it was shown that the addition of esa to ost cells led to apoptosis in cells of sarcoma , because the caspase-3 expression is known to be directly related to the apoptosis mechanism . as shown in our previous work , pegylated span 80 vesicles with immobilized esa ( abbreviated as epv ) are rather promising drug carriers for the treatment of carcinoma cancers . the ability of esa , and epv , as targeting unit and apoptosis inducer in the case of cells of sarcoma was examined further by flow cytometry as well as cell viability measurements , choosing ost cells as typical sarcoma cell type . as shown with the flow cytometric measurements in figure 6 , targeting of esa to ost cells in vitro was observed from the shift of the flow cytometric curve to the right hand side ( see figure 6 ) . , it was found that the macromolecular structure of peg on the vesicle surface did not hinder ost cell binding of esa which was localized on the vesicle surface together with peg . it may be due to the high mobility of both esa and peg , because of the high membrane fluidity of span 80 vesicles , as mentioned previously [ 19 , 30 ] . in addition , epv showed anticancer activity against ost cells since after an elapsing time of about 48 hours after the addition of epv at an esa concentration of 2 g / ml , the ost cell viability was reduced to almost zero , as shown in figure 8 . it seems that the anticancer activities of esa against ost cells in the vesicle system ( figure 8) is stronger than those in free esa system ( figure 1 ) . however , the activities of the two systems can not be compared directly , because either the incubation time or the esa concentration was different in the two systems . for example , for a direct comparison of the activities of the two systems against ost cells , the time - course of the viability upon addition of free esa system ( figure 1(b ) ) should be measured at [ esa ] = 2 g / ml ; at this concentration and after an incubation time of 48 hours , the cells were no more viable if the vesicles system was used ( figure 8) . on the other hand , we have already examined [ 4 , 6 ] the cytotoxicity of either esa or ev for various carcinoma cancer cells and normal cells , followed by examining the binding affinities of esa and ev to the cells . in the study presented , the following main results were obtained : ( i ) esa specifically binds to sarcoma cells and induces apoptotic death of the cells ; ( ii ) the antiproliferative activity of esa in sarcoma is higher than the activity in carcinoma ; ( iii ) esa immobilized onto pegylated span 80 vesicles ( epv ) shows antitumor activity against ost cells without any entrapped antitumor agents . furthermore , in a previous study , it was already revealed that esa and ev ( esa - immobilized on span 80 vesicles ) hardly bind to normal cells ( either mcf10 - 2a ( non - tumorigenic epithelial cells ) or normal fibroblasts from the umbilical cord ) ; and cytotoxicity caused by esa and ev was not observed for these normal cells . based on all these findings , we propose using epv as possible dds not only for the targeted treatment of carcinoma , but also for the targeted treatment of sarcoma . furthermore , the administration of pegylated span 80 vesicles with immobilized esa , in which anticancer drugs are encapsulated , is expected to express more effective antitumor activity against sarcoma as compared to empty epv .

lignin is a complex aromatic polymer , highly recalcitrant towards both chemical and biological degradation , characteristic of the cell wall of vascular plants ( fig . 1 ) . around 20% of the total carbon fixed by photosynthesis in land ecosystems is incorporated into lignin , being the second main constituent of plant biomass after cellulose . in addition of providing plant stems the rigidity required for growth on land , and waterproofing vascular tissues for sap circulation , a main role of lignin is to protect the cellulose polymer towards hydrolytic attack by most pathogen and saprophytic organisms . in spite of this , lignindegrading microbes evolved simultaneously with the colonization of land by vascular plants in the palaeozoic era , around 400 million year ago ( taylor and osborne , 1996 ) . microbial degradation of lignin ( martnez et al . , 2005 ; kersten and cullen , 2007 ) represents a key step for closing the carbon cycle , since removal of the lignin barrier enabled the subsequent use of plant carbohydrates by other microorganisms . three classical and two acylated lignin precursors or monolignols ( top ) , and structural model for gymnosperm lignin ( bottom ) . gymnosperms produce the simplest lignin type formed only by guaiacyl units derived from coniferyl alcohol ( 2 ) . in contrast , angiosperm lignin also include phydroxyphenyl and sinapyl units derived from pcoumaryl ( 1 ) and sinapyl ( 3 ) alcohols , as well as a variable amount of acylated lignin often derived from sinapyl alcohol esterified with acetic ( 4 ) , pcoumaric acid ( 5 ) or other organic acids ( ralph et al . , 2004 ; martnez et al . , carbon interunit linkages are formed during monolignol radical polymerization resulting in o4 ( a ) , phenylcoumaran ( b ) , pinoresinol ( c ) and dibenzodioxocin ( d ) substructures , among others . other minor structures ( in brackets ) include vanillin , coniferyl alcohol and dimethylcyclohexadienonetype units , the latter in new spirodienone substructures ( zhang et al . , 2006 ) lignin removal is also a central aspect in industrial uses of cellulosic biomass , such as bioethanol production and manufacture of cellulosebased chemicals and materials , including paper . in the plant cell wall , lignin concentrates in the middle lamella , its most external layer acting as a cementing agent between fibres ( fig . 2 , top ) . cellulose pulp manufacture basically consists in breaking down ( chemically or mechanically ) the middle lamella in such a way that wood fibres are individualized ( sixta , 2006 ) . although in lower concentration than plant carbohydrates ( cellulose and hemicelluloses ) lignin is also present in secondary wall , the thicker cellwall layer , where it is intimately associated to carbohydrates preventing their efficient hydrolysis in the production of bioethanol ( galbe and zacchi , 2007 ) . in the above industrial applications , biotechnology based on lignindegrading microbes and their enzymes can contribute to more efficient and environmentally sound use of renewable lignocellulosic feedstocks for sustainable production of materials , chemicals , biofuels and energy . pictorial scheme of the enzymatic degradation of plant cellwall lignin ( lcontaining circles represent the remaining lignin polymer ) by pleurotus vp , with contribution of extracellular flavooxidases ( such as aao ) generating hydrogen peroxide during redox cycling of nonphenolic aromatic aldehydes ( such as the fungal metabolite panisaldehyde ) with participation of intracellular arylaldehyde dehydrogenase . peroxidase oneelectron oxidation of lignin units ( the key step in the degradative process ) results in an unstable cation radical that experiences different reactions including breakdown of cc and c4ether linkages releasing the corresponding aromatic aldehydes ( vanillin in the case of guaiacyl units ) that can be intracellularly mineralized . in the case of p. chrysosporium lip , lignin attack requires the presence of veratryl alcohol , probably as an enzymebound mediator , and hydrogen peroxide is mainly generated by glyoxal oxidase . although lignin precursors , i.e. the three classical phydroxycinnamyl alcohols and their recently reported acylated forms ( ralph et al . , 2004 ; martnez et al . , 2008 ) , are phenolic compounds ( fig . 1 , top ) , the lignin polymer formed from these monolignols is basically nonphenolic ( fig . 1 , bottom ) . in the last step of lignin biosynthesis , plant peroxidases ( and maybe also laccases ) oxidize monolignol to their phenoxy radicals ( higuchi , 1997 ) . chemical coupling between the resonant forms of these radicals results in a variety of phenolic dimeric structures ( dilignols ) that can be enzymatically oxidized again , the process finally leading to lignin polymer formation . however , due to predominance of the corresponding radical forms and higher stability of the coupling products , ether linkages between the phenolic position ( c4 ) and a sidechain ( or aromatic ring ) carbon of the phydroxyphenylpropenoid precursors ( substructures a , b and d ) are strongly predominant in the growing polymer . due to the high frequency of these ether linkages , moreover , in contrast to cellulose formed by linear ( anhydroglucose ) chains and hemicelluloses that include short branches on a main polysaccharidic backbone , the lignin polymerization mechanism ( based on resonant radical coupling ) results in a complex threedimensional network ( fig . 1 , bottom ) due to both chain branching and inter / intrachain coupling during polymerization , as shown in updated lignin models ( gellerstedt and henriksson , 2008 ) . due to its nonphenolic aromatic nature , lignin units can not be oxidized by lowredoxpotential oxidoreductases , such as the plant peroxidases initiating the polymerization process . in fact , only a small group of highly specialized peroxidases secreted by ligninolytic fungi are able to degrade model compounds representing the main lignin substructures . the bulky nature of the heterogeneous lignin polymer forming a complex threedimensional network represents an additional limitation for biodegradation since the enzyme accessibility is strongly reduced . to overcome this difficulty , two main strategies have been developed by ligninolytic organisms based on : ( i ) presence of catalytic residues widely exposed at the surface of ligninolytic peroxidases , and ( ii ) use of redox mediators participating in the enzymatic attack the first lignin structural models were available in the 1970s ( nimz , 1974 ; adler , 1977 ) and new substructures are still being identified using modern analytical techniques ( karhunen et al . , 1995 ; zhang et al . , 2006 ; del ro et al . , 2007 ) . before this date no contrasted information on lignin structure was available , preventing studies on its microbial or enzymatic degradation . synthetic lignins and simple model compounds ( incorporating radioactive labelling ) were used to unravel the mechanisms of lignin attack by whiterot basidiomycetes , the only organisms that are able to extensively mineralize lignin ( eriksson et al . , 1990 ) . the complexity shown by the first lignin models and the variety of compounds identified in early lignocellulose biodegradation studies ( chen and chang , 1985 ) suggested a set of different degradative enzymes attacking the different lignin substructures and releasing different breakdown products . however , subsequent work using model compounds representative of the main substructures found in lignin revealed that lignindegrading organisms just adopted the opposite strategy . these studies , instead of revealing a variety of enzymes catalysing the different reactions observed , showed that the latter were the result of an unspecific oxidative attack on the benzenic rings of lignin units followed by different bond breakdown reactions due to the chemical instability of the cation radicals formed ( kirk and farrell , 1987 ) . moreover , using simple model compounds , it was possible to demonstrate that among the different oxidative enzymes produced by lignindegrading organisms , only a group of basidiomycete haemperoxidases could directly attack the nonphenolic lignin network ( martnez , 2002 ; hammel and cullen , 2008 ) . these enzymes include lignin peroxidase ( lip ) initially described in phanerochaete chrysosporium , the first basidiomycete whose genome was sequenced due to the interest on biological degradation of lignin ( martnez et al . , 2004 ) , and a versatile peroxidase ( vp ) more recently reported in pleurotus and bjerkandera species , the former genus including species being able to degrade lignin selectively ( martnez et al . , 1994 ) . vp is also able to oxidize mn , as reported for p. chrysosporium manganese peroxidase ( mnp ) . the mn resulting from the action of these two peroxidases oxidizes phenolic compounds but , in the presence of unsaturated lipids , it can also oxidize nonphenolic lignin via peroxidation radicals ( bao et al . , 1994 ) . the molecular evolution of ligninolytic peroxidases has been shown by a recent study ( morgenstern et al . , 2008 ) . taking into account the unique characteristics of the microbial attack to lignin , compared with hydrolytic attack to other natural polymers including plant carbohydrates , the process was described as an enzymatic combustion where enzymatically generated hydrogen peroxide oxidizes the lignin polymer in a reaction catalysed by the abovementioned highredoxpotential peroxidases ( kirk and farrell , 1987 ) ( fig . 2 ) . simultaneously to the discovery of ligninolytic peroxidases , several peroxidegenerating oxidases were described in ligninolytic fungi , such as glyoxal oxidase ( a copper radical enzyme ) , arylalcohol oxidase ( aao ) and pyranose2 oxidase ( two flavoenzymes ) ( martnez et al . , 2005 ) . in pleurotus species continuous production of hydrogen peroxide during redox cycling of anisaldehyde , an extracellular metabolite ( gutirrez et al . , 1994 ) is produced involving myceliumassociated arylalcohol dehydrogenase ( a nadph dehydrogenase ) working together with extracellular aao ( guilln and evans , 1994 ) ( fig . 2 ) . in p. chrysosporium , glyoxal oxidase uses products from lignocellulose degradation as the enzymereducing substrates for generating the hydrogen peroxide required by the ligninolytic peroxidases ( kersten and cullen , 2007 ) . the unstable cation radicals formed during peroxidase attack to lignin model compounds experience different chemical reactions including breakdown of cc and c4ether linkages resulting in the release of aromatic aldehydes , one of the main products found during enzymatic depolymerization of lignin ( fig . 2 ) , together with other reactions such as demethoxylation ( methanol release ) and aromaticring cleavage ( with formation of muconatetype structures ) ( martnez et al . , 2005 ) . the involvement of ligninolytic peroxidases in wood lignin degradation has been recently supported by comparison of the genomes of p. chrysosporium ( martnez et al . , 2004 ) , a model whiterot fungus characterized by its ability to remove wood lignin leaving a whitish cellulosic residue , and postia placenta ( martinez et al . , 2009 ) , a model brownrot fungus characterized by its ability to remove wood polysaccharides leaving a brown ligninenriched residue . this comparison showed a large set of ligninolytic peroxidase genes in the genome of p. chrysosporium , in agreement with previous studies ( cullen , 1997 ) . in contrast , the p. placenta genome includes genes of oxidases and other enzymes involved in cellulose attack via fenton chemistry , but contains an unique peroxidase gene related to the lowredoxpotential peroxidase of the nonligninolytic basidiomycete coprinopsis cinerea ( cip ) , and completely lacks ligninolytic peroxidase genes ( lip , vp or mnp ) . a third type of microbial oxidoreductases , laccases and related multicopper oxidases are produced by most ligninolytic basidiomycetes ( baldrian , 2006 ) as well as by eubacteria and actinomycetes growing on lignocellulosic materials ( sharma et al . , 2007 ) . however , they can degrade this and other recalcitrant compounds in the presence of redox mediators , as discussed below . laccases also play a variety of other physiological roles including mushroom morphogenesis , detoxification and humification , among others ( claus , 2004 ) . moreover , they seem involved in generation of lignocellulosedegrading hydroxyl radical ( via fenton chemistry ) in both whiterot ( guilln et al . , 2000 ) and at least some brownrot basidiomycetes , in agreement with the presence of laccase genes in the genome of p. placenta ( http://genome.jgipsf.org/pospl1/pospl1.home.html ) . all prokaryotic , fungal and plant haemperoxidases share a common folding and helical topology described in cytochrome c peroxidase ( ccp ) , the first peroxidase whose crystal structure was reported ( poulos et al . , 1980 ) and only more recently ( gajhede et al . , 1997 ) in the bestknown peroxidase , horseradish peroxidase ( hrp ) . it is noteworthy that lip , an enzyme that was completely unknown until 1983 ( glenn et al . , 1983 ; tien and kirk , 1983 ) , was the second peroxidase whose crystal structure was reported ( piontek et al . the rapid progresses of lip structure function studies , as well as the fact that two groups reported simultaneously its discovery and crystal structure , demonstrate the interest on highredoxpotential peroxidases . as mentioned above , all peroxidases require hydrogen peroxide , or other hydroperoxides , to activate the haem cofactor yielding the socalled compoundi in a common catalytic cycle ( dunford , 1999 ) ( fig . 3 ) . compoundi contains a reactive fe oxo complex with a cation radical at the porphyrin ring , formed by twoelectron oxidation of the fecontaining haem of the resting enzyme . oneelectron oxidation of one substrate molecule yields compoundii , where the porphyrin cation radical has been reduced . the remaining fe = o in compoundii oxidizes a second substrate molecule , and the enzyme returns to its ferric resting state to initiate a new catalytic cycle . the cycle includes twoelectron oxidation of the enzyme resting state ( rs , containing fe ) by hydroperoxide to yield compoundi ( ci ; containing feoxo and porphyrin cation radical ) , whose reduction in two oneelectron steps results in the intermediate compoundii ( cii ; containing fe = o after porphyrin reduction ) and then the resting form of the enzyme , with concomitant oxidation of two substrate molecules ( s ; which could be lowredoxpotential phenols and dyes , or mn in the cases of mnp and vp ) . the molecular structure of haemperoxidases includes two domains , probably originating from ancestral gene duplication , delimiting a central cavity where the haem cofactor is located ( li and poulos , 1994 ; banci , 1997 ) . in most cases , the access of both the enzymeoxidizing ( peroxide ) and reducing substrates to the haem cofactor is produced through a main access channel ( fig . 4 , left ) . taking into account the high reactivity of both compoundi and compoundii first , it prevents unspecific reduction of the activated enzyme by a variety of reductants different from its specific target substrate . second , it prevents intermolecular reaction resulting in oxidation of surface susceptible amino acids ( e.g. tyrosine residues ) leading to enzyme inactivation ( e.g. by dimerization reactions ) . oxidation of mn by basidiomycete mnp and vp is also produced through a specific access channel enabling entering of the cation to reach one of the haem propionates ( fig . 4 , left ) . the edge of this channel includes three acidic residues that bind mn specifically enabling its highefficiency oxidation . two different views of the solvent access surface in a ligninolytic peroxidase ( p. eryngii vp ; pdb entry 2boq ) revealing ( left ) the main haem access channel enabling hydrogen peroxide entrance for activation of the haem cofactor ( in yellow ) located in a central pocket ( lowredoxpotential phenols and dyes can also be oxidized at this channel albeit with low efficiency ) , and the mnoxidation channel formed by three acidic residues ( glu36 , glu40 and asp175 ) ; as well as an approximately 180 rotated view ( right ) of the same peroxidase showing the partially exposed sidechain ( yellow van der waals spheres including hydrogen atoms ) of the catalytic tryptophan ( trp164 ) involved in oxidation of highredoxpotential compounds , such as veratryl alcohol ( va ) and lignin models , as well as in highefficiency oxidation of some phenols and dyes , by longrange electron transfer ( lret ) to the haem cofactor ( surface colours correspond to electrostatic charge ) . a detail of the haem environment and other neighbour amino acid residues in a ligninolytic peroxidase ( pleurotus eryngii vp ) is shown in fig . these include two axial histidine residues , the socalled proximal ( his169 ) and distal ( his47 ) histidines , and other conserved residues ( martnez , 2002 ) . proximal histidine acts as the fifth ligand of the haem iron together with the four nitrogens of the tetrapyrrolic macrocycle , while distal histidine together with a conserved arginine ( vp arg43 ) contributes to iron reaction with hydrogen peroxide in compoundi formation ( hiner et al . , 2002 ) . two aromatic residues are also highly conserved at the proximal ( phe186 ) and distal ( phe46 ) sides of the haem of many peroxidases , in ccp being two tryptophan residues one of them playing a direct role in catalysis as mentioned below . two more residues at the proximal ( asp231 ) and distal ( asn78 ) sides of the haem establish hydrogen bonds with the two histidines . two structural ca ions are located in conserved binding sites at the two peroxidase domains contributing to folding . the abovementioned mnoxidation site of vp ( and mnp ) is shown at the right side of haem , being formed by three acidic residues positioning the cation near one of the haem propionates for direct electron transfer ( gold et al . , 2000 ; finally , a tryptophan residue ( trp164 ) is shown at the left side of the haem , being involved in oxidation of highredoxpotential aromatic compounds ( together with contiguous leu165 ) as described in the next section . the vp structure also includes several molecules of water , including those completing the characteristic coordinations of mn and ca ions . another water molecule ( w71 ) could act as the sixth ligand of the haem fe at a position close to that occupied by the fe = o oxygen of compoundi and compoundii , as shown for hrp ( berglund et al . , details of haem environment and other structurally and catalytically relevant residues in p. eryngii vp . his169 ( the fifth ligand of haem iron ) , phe186 and asp231 ( corresponding to his176 , phe193 and asp238 in liph8 ) are shown at the proximal side of the haem , while his47 , phe46 , arg43 and asn78 ( corresponding to his47 , phe46 and arg43 in liph8 ) are shown at the distal side . glu36 , glu40 and asp175 ( corresponding to ala36 , glu40 and asn182 in liph8 ) constitute the site of oxidation of mn ( red van der waals sphere ) near the internal propionate of haem , while trp164 ( corresponding to trp171 in liph8 ) is responsible for oxidation of lignin units and other aromatic compounds by lret ( red arrow ) to the activated haem cofactor via leu165 ( corresponding to leu172 in liph8 ) . finally , the ligands of the two structural ca ions ( green spheres ) are indicated at the proximal ( ser170 , asp187 , thr189 , val192 and asp194 ) and distal ( asp48 , gly60 , asp62 and ser64 ) sides ( corresponding , respectively , to ser177 , asp194 , thr196 , ile199 and asp201 ; and asp48 , gly66 , asp68 and ser70 in liph8 ) . several water molecules are also shown including those completing mn , ca and haem fe coordination . it has been shown that the redox potential of peroxidase compoundi and compoundii depends of the haem environment characteristics , as shown by hnmr spectra of the cyanide adduct of the enzyme ( banci et al . , 1991 ; 2003 ) . these included , as one of the most important factors , the position and more or less marked imidazolate character of the proximal histidine sidechain , whose n1 acts as the fifth ligand of the haem iron . the strength of this bond affects the electron deficiency of iron and , consequently , the reactivity of the activated enzyme . the characteristics of the proximal histidine sidechain that are affected by hbonding to other residues ( such as asp231 in fig . 5 ) ( banci et al . , 1995 ) and the position of helix f where this residue is located ( piontek et al . , 1993 ) affect the chemical shift of its h1 signal ( which was found at about 8 p.p.m . in lip , 16 p.p.m . in vp , 22 p.p.m . in ccp and 32 p.p.m . in hrp spectra ) in agreement with the reported higher redox potential of ligninolytic peroxidases compared with plant peroxidases ( millis et al . , 1989 ) . when the crystal structure of p. chrysosporium lip was reported , it was assumed that this peroxidase would oxidize highredoxpotential aromatic compounds at the main haem access channel . in fact , veratryl alcohol was modelled at the crystal structure channel to determine the eventual contacts ( poulos et al . , 1993 ) . this is the classical peroxidase substrate oxidation site , as shown in plant hrp and in the lowredoxpotential fungal cip ( smith and veitch , 1998 ) . however , the main haem access channel of lip is significantly narrower than those of cip or hrp , and the hypothesis of longrange electron transfer ( lret ) oxidation of lignin at the protein surface , initially proposed by du and colleagues ( 1992 ) and schoemaker and colleagues ( 1994 ) , was adopted by other authors . protein lret requires the existence of an amino acid residue susceptible to form a stable radical ( preferably a tyrosine , tryptophan or histidine residue ) located at the protein surface and adequately connected to the haem cofactor for electron transfer . the above authors suggested two residues ( trp171 and his82 ) as the origin of two lret pathways for oxidation of highredoxpotential aromatic compounds by lip . several years later a third pathway was proposed ( initiating at his239 ) ( johjima et al . , 1999 ) . no pathway starting at a tyrosine residue has been proposed since all ligninolytic peroxidases cloned up to date ( a total of near 50 sequences ) are free of tyrosine residues ( to prevent oxidative inactivation ) with the only exception of a trametes cervina lip discussed below . the same three putative lret pathways were identified in p. eryngii vp , and their eventual operation was investigated by sitedirected mutagenesis by prezboada and colleagues ( 2005 ) . this study definitively showed that only the pathway starting at the exposed tryptophan ( trp164 of isoenzyme vpl ) was operative for oxidation of highredoxpotential aromatic compounds including veratryl alcohol . the position of this residue at the vicinity of the haem cofactor is illustrated in fig . 5 , which also shows a leucine residue involved in the electron transfer ; and the solvent exposed aromatic sidechain of this tryptophan is shown in fig . 4 ( right ) . the catalytic role of a homologous tryptophan has also been shown in vp from other fungi ( tinoco et al . formation of the tryptophanylfree radical postulated by du and colleagues ( 1992 ) was confirmed by lowtemperature electron paramagnetic resonance ( epr ) of peroxideactivated vp ( pogni et al . , 2006 ) . the involvement of the homologous tryptophan residue of lip ( trp171 ) in oxidation of veratryl alcohol and , more interestingly , of a tetrameric lignin model compound was also confirmed by mutagenesis ( doyle et al . , 1998 ; although the lip tryptophanyl radical was not directly detected , as reported by vp epr , indirect evidence for its presence in the activated enzyme was obtained by adduct formation ( blodig et al . , 1999 ) . a difference between the two ligninolytic peroxidases concerns the surface environment of the catalytic tryptophan , which in lip has a partial negative charge , whereas in vp some acidic residues are substituted by basic residues ( fig . 4 , a noteworthy characteristic of vp is its ability to oxidize directly a series of highredoxpotential substrates that lip is able to oxidize only in the presence of veratryl alcohol ( see below ) . this fact is related to the catalytic tryptophan environment , and a charge inversion vp variant ( r257d ) was obtained that exhibited a liptype behaviour during oxidation of highredoxpotential dyes ( ruizdueas et al . , 2008 ) . after demonstrating the involvement of a protein radical in substrate oxidation by ligninolytic peroxidases , 6 ) that in general terms can be also applied to p. chrysosporium lip . in this cycle , compoundib containing fe = o and tryptophan radical , and compoundiib containing fe and tryptophan radical , are included together with normal compoundi and compoundii ( containing fe = o and porphyrin radical , and fe = o respectively ) that are now called compoundia and compoundiia . compoundib and compoundiib ( formed by lret to the activated haem cofactor ) represent a small percentage of the total activated enzyme ( pogni et al . , 2006 ) being in equilibrium with the corresponding compoundia and compoundiia . in addition to normal compoundi and compoundii of fig . 3 ( now cia and ciia ) , cib ( containing fe = o and tryptophan radical ) and ciib ( containing fe and tryptophan radical ) are included , being involved in oxidation of highredoxpotential compounds such as veratryl alcohol ( va ) and lignin units to their corresponding cation radicals . cib and ciib are formed by lret to the activated haem cofactor . adapted from prezboada and colleagues ( 2005 ) . a different lip form lacking a catalytic tryptophan residue has been reported in t. cervina ( miki et al . , 2006 ) . this unique peroxidase presents an exposed tyrosine at a different position of the molecule that seems to play the same role of the above tryptophan . interestingly , a plant peroxidase using a tyrosyl radical for oxidation of bulky phenols such as sinapyl alcohol ( structure 3 in fig . 1 ) has been recently reported ( sasaki et al . , 2008 ) . the use of small chemical oxidizers acting as redox mediators represents a second alternative to overcome the difficulties associated to the limited access of the bulky lignin polymer to the activated cofactor of peroxidases and other oxidoreductases . enzymemediator systems have been extensively investigated in the case of laccases after the work of bourbonnais and paice ( 1990 ) reporting that synthetic mediators expanded the application potential of these lowredoxpotential oxidoreductases enabling oxidation of nonphenolic lignin model compounds . these mediators are lowmolecularmass compounds that : ( i ) form stable free radicals oxidizing compounds that the enzyme alone is not able to oxidize , and ( ii ) diffuse away from the enzyme and can easily penetrate the lignocellulose matrix . it has been recently shown that some phenolic lignin precursors or degradation products can be used as natural laccase mediators in industrial processes , and suggested that some of them could play a similar role in nature ( camarero et al . , 2005 ) . however , the real significance of the laccasemediator systems in natural biodegradation of lignin is still to be demonstrated . the existence of an exposed protein radical transferring electrons to the haem via a lret pathway enables lip direct oxidation of veratryl alcohol and lignin model compounds , including nonphenolic tetramers ( mester et al . , 2001 ) . however , lip requires the presence of veratryl alcohol or other compounds forming aromatic cation radicals , to oxidize polymeric lignin and highredoxpotential dyes ( harvey et al . , 1986 ) . this contrasts with the veratryl alcoholindependent activity of vp oxidizing different compounds including lignin ( heinfling et al . 2001 ) . the role of veratryl alcohol cation radical as a real mediator in lip reactions has been matter of controversy , taking into account the short halflife of this species in aqueous media ( candeias and harvey , 1995 ) . however , the acidic environment surrounding lip trp171 could stabilize the cation radical that would act as an enzymebound mediator ( khindaria et al . , 1996 ) microbial oxidoreductases , including both peroxidases and laccases , have been investigated for biotechnological application including paper pulp bleaching in chlorinefree sequences ( paice et al . , ligninolytic peroxidases , in contrast to laccases and lowredoxpotential peroxidases that are used in several industrial sectors ( e.g. textiles , detergents , food and beverages , etc . ) , are not still commercially available due to different reasons including the low levels of enzyme obtained from natural and recombinant hosts . moreover , these oxidoreductases in most cases are not suitable biocatalysts as they are produced in nature . industrial processes often require enzymes recognizing specific substrates or proceeding under extreme conditions ( e.g. high hydrogen peroxide concentration or extreme ph and temperature ) . laccases have low redox potential and can degrade recalcitrant compounds only in the presence of redox mediators . however , the laccasemediator system has became extremely popular in biodegradation studies , as well as for processing and functionalization of lignocellulosic fibres ( riva , 2006 ; widsten and kandelbauer , 2008 ) . in spite of natural laccase mediators have been reported as potential substitutes of synthetic n(oh) compounds in different applications ( camarero et al . . , 2007 ) , economic issues related to the cost of the mediator , and environmental concerns related to the eventual release of toxic compounds during the enzymatic treatment are main drawbacks for the industrial implementation of this enzymatic system in the pulp and paper and other industrial sectors . in contrast to laccases , some ligninolytic peroxidases do not require mediators to degrade highredoxpotential compounds . indeed , they should be the enzymes of choice for removing lignin or transforming highredoxpotential aromatic compounds in different applications . the rapid acquisition of knowledge on the structure and function of these enzymes over the last years has been used to modulate their catalytic and operational properties using sitedirected mutagenesis in a variety of studies ( timofeevski et al . , 1999 ; reading and aust , 2000 ; 2003 ) . in those cases where the structural basis of the property to be improved is unknown , or too difficult to be predicted , directed evolution or , 1999 ; miyazakiimamura et al . , 2003 ; ryu et al . , considerable efforts have been devoted during the last years to improve the expression of ligninolytic and other peroxidases in different fungal hosts using a variety of strategies . among others , strong promoters , protease deficient strains , molecular chaperones and external sources of haem have been used with only partially successful results ( stewart et al . , 1996 ; , 2003 ; lchau et al . , 2004 ; wang et al . , 2004 ; eibes et al . , new strategies based on converting lowredoxpotential peroxidases , easily to express at levels of several grams per litre , into highredoxpotential peroxidases are being developed taking advantage of the structure function knowledge accumulated . in this sense , cip has been successfully modified and transformed into a liplike enzyme by introducing a catalytic tryptophan , at the same time that the acidity of the local environment of this residue was increased ( smith and doyle , 2006 ) . although the mutated enzyme was not so efficient as a true lip , it is possible to predict that additional modifications will yield an enzyme with the expected catalytic activity . hydrogen peroxide inactivate all peroxidases after several cycles of catalysis as a consequence of the competition between productive and unproductive electron sources ( including enzyme components ) in a process described as a suicide inactivation ( valderrama et al . , 2002 ) . different attempts aimed to improve hydrogen peroxide stability of ligninolytic peroxidases have been performed , mainly removing easily oxidizable and conformationally unstable amino acid residues located at the peroxidebinding side of haem with very promising results ( miyazaki and takahashi , 2001 ; miyazakiimamura et al . , 2003 ) . recently , a novel peroxidase from raphanus sativus has been identified and characterized as the only known case of a haemperoxidase intrinsically resistant to hydrogen peroxide ( gilrodrguez et al . , 2008 ) . indepth analysis of its structure will give the first structural evidences of peroxide stability to be used to increase the peroxide stability of ligninolytic and other peroxidases . ph and temperature inactivation of ligninolytic peroxidases is associated to the release of the two structural ca involved in stabilization of the molecular architecture ( sutherland and aust , 1996 ; george et al . , 1999 ; it has been described that losing these ions causes hexacoordination of the haem iron preventing lip and mnp activation by hydrogen peroxide , although more recently a decrease in redox potential has been suggested as the main inactivation cause in vp ( verdn et al . , 2006 ) . some authors have succeeded stabilizing peroxidases by avoiding the lose of ca by adding extra disulfide bridges ( reading and aust , 2000 ) . generation of the appropriate disulfide bridges could not only make these enzymes resistant to high ph and temperature , but it could also be used to increase their redox potential . mutations removing one of these interactions in cip improved its thermal stability by 134fold ( cherry et al . , 1999 ) . curiously these two residues are conserved in ligninolytic peroxidases ( lip , mnp and vp ) , and additional interactions between acidic residues can be observed in other regions of their molecular structures . it is expected that substitution of one amino acid residue of these acidic couples will promote enzyme stabilization towards high temperature , as described for cip . among ligninolytic peroxidases , vp presents especial biotechnological interest due to different reasons including : ( i ) catalytic versatility by combination of different substrate oxidation sites , and ( ii ) ability to degrade some compounds of interest that lip and mnp ( as well as cip ) are not able to oxidize directly . its catalytic versatility permits the application of vp in mnmediated or mnindependent reactions on both low and highredoxpotential substrates . the possibility to degrade directly a variety of recalcitrant compounds represents a considerable advantage compared with lip since the cost of veratryl alcohol required as mediator can be saved . among the different compounds of industrial and/or environmental interest that vp can transform , polycyclic aromatic hydrocarbons ( wang et al . , 2003 ) , phenolic and nonphenolic aromatic pollutants ( rodrguez et al . , 2004 ) , pesticides ( dvilavzquez et al . , 2005 ) and a variety of industrial dyes ( heinfling et al . , 1998b ) can be cited ( including , among others , reactive blue 38 and other azo dyes , reactive black 5 and other phthalocyanine dyes , anthracene and derivatives , benzo[a]pyrene , pyrene , 2,4dichlorophenol and pentachlorophenol ) . for some applications the use of vp in combination with redox mediators can also be considered ( tinoco et al . , 2007 ) . among them , the use of vp to reoxidize mncontaining polyoxometalates is an interesting possibility ( marques et al . , 2008 ) , since these highly promising catalysts for environmentally friendly delignification are very difficult to be chemically reoxidized . the promiscuity of ligninolytic fungi and their enzymes oxidizing aromatic xenobiotics and other recalcitrant compounds is due to the involvement of the lignindegrading enzymatic machinery in many of these reactions . to overcome the bulky nature and structural heterogeneity of the lignin polymer , these microorganisms have developed a highly unspecific extracellular system being able to subtract one electron directly from the benzenic rings of the different lignin units . highredoxpotential extracellular peroxidases , often forming catalytic radicals at the protein surface , are the key enzymes in this initial attack yielding partially oxidized products whose catabolism is finally completed intracellularly .

summarylignin is the second most abundant constituent of the cell wall of vascular plants , where it protects cellulose towards hydrolytic attack by saprophytic and pathogenic microbes . its removal represents a key step for carbon recycling in land ecosystems , as well as a central issue for industrial utilization of plant biomass . the lignin polymer is highly recalcitrant towards chemical and biological degradation due to its molecular architecture , where different nonphenolic phenylpropanoid units form a complex threedimensional network linked by a variety of ether and carbon carbon bonds . ligninolytic microbes have developed a unique strategy to handle lignin degradation based on unspecific oneelectron oxidation of the benzenic rings in the different lignin substructures by extracellular haemperoxidases acting synergistically with peroxidegenerating oxidases . these peroxidases posses two outstanding characteristics : ( i ) they have unusually high redox potential due to haem pocket architecture that enables oxidation of nonphenolic aromatic rings , and ( ii ) they are able to generate a protein oxidizer by electron transfer to the haem cofactor forming a catalytic tryptophanylfree radical at the protein surface , where it can interact with the bulky lignin polymer . the structure function information currently available is being used to build tailormade peroxidases and other oxidoreductases as industrial biocatalysts .

Interest of microbial degradation of lignin Chemical bases of lignin recalcitrance Ligninolytic organisms and enzymes General determinants of ligninolytic peroxidase structure and activity Direct peroxidase oxidation of lignin: longrange electron transfer (LRET) mechanism Redox mediators in lignin degradation Biotechnological interest and future trends

lignin is a complex aromatic polymer , highly recalcitrant towards both chemical and biological degradation , characteristic of the cell wall of vascular plants ( fig . around 20% of the total carbon fixed by photosynthesis in land ecosystems is incorporated into lignin , being the second main constituent of plant biomass after cellulose . in addition of providing plant stems the rigidity required for growth on land , and waterproofing vascular tissues for sap circulation , a main role of lignin is to protect the cellulose polymer towards hydrolytic attack by most pathogen and saprophytic organisms . in spite of this , lignindegrading microbes evolved simultaneously with the colonization of land by vascular plants in the palaeozoic era , around 400 million year ago ( taylor and osborne , 1996 ) . , 2005 ; kersten and cullen , 2007 ) represents a key step for closing the carbon cycle , since removal of the lignin barrier enabled the subsequent use of plant carbohydrates by other microorganisms . in contrast , angiosperm lignin also include phydroxyphenyl and sinapyl units derived from pcoumaryl ( 1 ) and sinapyl ( 3 ) alcohols , as well as a variable amount of acylated lignin often derived from sinapyl alcohol esterified with acetic ( 4 ) , pcoumaric acid ( 5 ) or other organic acids ( ralph et al . in the plant cell wall , lignin concentrates in the middle lamella , its most external layer acting as a cementing agent between fibres ( fig . although in lower concentration than plant carbohydrates ( cellulose and hemicelluloses ) lignin is also present in secondary wall , the thicker cellwall layer , where it is intimately associated to carbohydrates preventing their efficient hydrolysis in the production of bioethanol ( galbe and zacchi , 2007 ) . pictorial scheme of the enzymatic degradation of plant cellwall lignin ( lcontaining circles represent the remaining lignin polymer ) by pleurotus vp , with contribution of extracellular flavooxidases ( such as aao ) generating hydrogen peroxide during redox cycling of nonphenolic aromatic aldehydes ( such as the fungal metabolite panisaldehyde ) with participation of intracellular arylaldehyde dehydrogenase . peroxidase oneelectron oxidation of lignin units ( the key step in the degradative process ) results in an unstable cation radical that experiences different reactions including breakdown of cc and c4ether linkages releasing the corresponding aromatic aldehydes ( vanillin in the case of guaiacyl units ) that can be intracellularly mineralized . chemical coupling between the resonant forms of these radicals results in a variety of phenolic dimeric structures ( dilignols ) that can be enzymatically oxidized again , the process finally leading to lignin polymer formation . however , due to predominance of the corresponding radical forms and higher stability of the coupling products , ether linkages between the phenolic position ( c4 ) and a sidechain ( or aromatic ring ) carbon of the phydroxyphenylpropenoid precursors ( substructures a , b and d ) are strongly predominant in the growing polymer . due to the high frequency of these ether linkages , moreover , in contrast to cellulose formed by linear ( anhydroglucose ) chains and hemicelluloses that include short branches on a main polysaccharidic backbone , the lignin polymerization mechanism ( based on resonant radical coupling ) results in a complex threedimensional network ( fig . due to its nonphenolic aromatic nature , lignin units can not be oxidized by lowredoxpotential oxidoreductases , such as the plant peroxidases initiating the polymerization process . in fact , only a small group of highly specialized peroxidases secreted by ligninolytic fungi are able to degrade model compounds representing the main lignin substructures . the bulky nature of the heterogeneous lignin polymer forming a complex threedimensional network represents an additional limitation for biodegradation since the enzyme accessibility is strongly reduced . to overcome this difficulty , two main strategies have been developed by ligninolytic organisms based on : ( i ) presence of catalytic residues widely exposed at the surface of ligninolytic peroxidases , and ( ii ) use of redox mediators participating in the enzymatic attack the first lignin structural models were available in the 1970s ( nimz , 1974 ; adler , 1977 ) and new substructures are still being identified using modern analytical techniques ( karhunen et al . synthetic lignins and simple model compounds ( incorporating radioactive labelling ) were used to unravel the mechanisms of lignin attack by whiterot basidiomycetes , the only organisms that are able to extensively mineralize lignin ( eriksson et al . the complexity shown by the first lignin models and the variety of compounds identified in early lignocellulose biodegradation studies ( chen and chang , 1985 ) suggested a set of different degradative enzymes attacking the different lignin substructures and releasing different breakdown products . these studies , instead of revealing a variety of enzymes catalysing the different reactions observed , showed that the latter were the result of an unspecific oxidative attack on the benzenic rings of lignin units followed by different bond breakdown reactions due to the chemical instability of the cation radicals formed ( kirk and farrell , 1987 ) . these enzymes include lignin peroxidase ( lip ) initially described in phanerochaete chrysosporium , the first basidiomycete whose genome was sequenced due to the interest on biological degradation of lignin ( martnez et al . taking into account the unique characteristics of the microbial attack to lignin , compared with hydrolytic attack to other natural polymers including plant carbohydrates , the process was described as an enzymatic combustion where enzymatically generated hydrogen peroxide oxidizes the lignin polymer in a reaction catalysed by the abovementioned highredoxpotential peroxidases ( kirk and farrell , 1987 ) ( fig . in contrast , the p. placenta genome includes genes of oxidases and other enzymes involved in cellulose attack via fenton chemistry , but contains an unique peroxidase gene related to the lowredoxpotential peroxidase of the nonligninolytic basidiomycete coprinopsis cinerea ( cip ) , and completely lacks ligninolytic peroxidase genes ( lip , vp or mnp ) . however , they can degrade this and other recalcitrant compounds in the presence of redox mediators , as discussed below . laccases also play a variety of other physiological roles including mushroom morphogenesis , detoxification and humification , among others ( claus , 2004 ) . , 2000 ) and at least some brownrot basidiomycetes , in agreement with the presence of laccase genes in the genome of p. placenta ( http://genome.jgipsf.org/pospl1/pospl1.home.html ) . the rapid progresses of lip structure function studies , as well as the fact that two groups reported simultaneously its discovery and crystal structure , demonstrate the interest on highredoxpotential peroxidases . compoundi contains a reactive fe oxo complex with a cation radical at the porphyrin ring , formed by twoelectron oxidation of the fecontaining haem of the resting enzyme . oneelectron oxidation of one substrate molecule yields compoundii , where the porphyrin cation radical has been reduced . the cycle includes twoelectron oxidation of the enzyme resting state ( rs , containing fe ) by hydroperoxide to yield compoundi ( ci ; containing feoxo and porphyrin cation radical ) , whose reduction in two oneelectron steps results in the intermediate compoundii ( cii ; containing fe = o after porphyrin reduction ) and then the resting form of the enzyme , with concomitant oxidation of two substrate molecules ( s ; which could be lowredoxpotential phenols and dyes , or mn in the cases of mnp and vp ) . the molecular structure of haemperoxidases includes two domains , probably originating from ancestral gene duplication , delimiting a central cavity where the haem cofactor is located ( li and poulos , 1994 ; banci , 1997 ) . in most cases , the access of both the enzymeoxidizing ( peroxide ) and reducing substrates to the haem cofactor is produced through a main access channel ( fig . taking into account the high reactivity of both compoundi and compoundii first , it prevents unspecific reduction of the activated enzyme by a variety of reductants different from its specific target substrate . oxidation of mn by basidiomycete mnp and vp is also produced through a specific access channel enabling entering of the cation to reach one of the haem propionates ( fig . two different views of the solvent access surface in a ligninolytic peroxidase ( p. eryngii vp ; pdb entry 2boq ) revealing ( left ) the main haem access channel enabling hydrogen peroxide entrance for activation of the haem cofactor ( in yellow ) located in a central pocket ( lowredoxpotential phenols and dyes can also be oxidized at this channel albeit with low efficiency ) , and the mnoxidation channel formed by three acidic residues ( glu36 , glu40 and asp175 ) ; as well as an approximately 180 rotated view ( right ) of the same peroxidase showing the partially exposed sidechain ( yellow van der waals spheres including hydrogen atoms ) of the catalytic tryptophan ( trp164 ) involved in oxidation of highredoxpotential compounds , such as veratryl alcohol ( va ) and lignin models , as well as in highefficiency oxidation of some phenols and dyes , by longrange electron transfer ( lret ) to the haem cofactor ( surface colours correspond to electrostatic charge ) . a detail of the haem environment and other neighbour amino acid residues in a ligninolytic peroxidase ( pleurotus eryngii vp ) is shown in fig . proximal histidine acts as the fifth ligand of the haem iron together with the four nitrogens of the tetrapyrrolic macrocycle , while distal histidine together with a conserved arginine ( vp arg43 ) contributes to iron reaction with hydrogen peroxide in compoundi formation ( hiner et al . two aromatic residues are also highly conserved at the proximal ( phe186 ) and distal ( phe46 ) sides of the haem of many peroxidases , in ccp being two tryptophan residues one of them playing a direct role in catalysis as mentioned below . two more residues at the proximal ( asp231 ) and distal ( asn78 ) sides of the haem establish hydrogen bonds with the two histidines . the abovementioned mnoxidation site of vp ( and mnp ) is shown at the right side of haem , being formed by three acidic residues positioning the cation near one of the haem propionates for direct electron transfer ( gold et al . , 2000 ; finally , a tryptophan residue ( trp164 ) is shown at the left side of the haem , being involved in oxidation of highredoxpotential aromatic compounds ( together with contiguous leu165 ) as described in the next section . another water molecule ( w71 ) could act as the sixth ligand of the haem fe at a position close to that occupied by the fe = o oxygen of compoundi and compoundii , as shown for hrp ( berglund et al . his169 ( the fifth ligand of haem iron ) , phe186 and asp231 ( corresponding to his176 , phe193 and asp238 in liph8 ) are shown at the proximal side of the haem , while his47 , phe46 , arg43 and asn78 ( corresponding to his47 , phe46 and arg43 in liph8 ) are shown at the distal side . glu36 , glu40 and asp175 ( corresponding to ala36 , glu40 and asn182 in liph8 ) constitute the site of oxidation of mn ( red van der waals sphere ) near the internal propionate of haem , while trp164 ( corresponding to trp171 in liph8 ) is responsible for oxidation of lignin units and other aromatic compounds by lret ( red arrow ) to the activated haem cofactor via leu165 ( corresponding to leu172 in liph8 ) . it has been shown that the redox potential of peroxidase compoundi and compoundii depends of the haem environment characteristics , as shown by hnmr spectra of the cyanide adduct of the enzyme ( banci et al . these included , as one of the most important factors , the position and more or less marked imidazolate character of the proximal histidine sidechain , whose n1 acts as the fifth ligand of the haem iron . in hrp spectra ) in agreement with the reported higher redox potential of ligninolytic peroxidases compared with plant peroxidases ( millis et al . this is the classical peroxidase substrate oxidation site , as shown in plant hrp and in the lowredoxpotential fungal cip ( smith and veitch , 1998 ) . however , the main haem access channel of lip is significantly narrower than those of cip or hrp , and the hypothesis of longrange electron transfer ( lret ) oxidation of lignin at the protein surface , initially proposed by du and colleagues ( 1992 ) and schoemaker and colleagues ( 1994 ) , was adopted by other authors . protein lret requires the existence of an amino acid residue susceptible to form a stable radical ( preferably a tyrosine , tryptophan or histidine residue ) located at the protein surface and adequately connected to the haem cofactor for electron transfer . the position of this residue at the vicinity of the haem cofactor is illustrated in fig . this fact is related to the catalytic tryptophan environment , and a charge inversion vp variant ( r257d ) was obtained that exhibited a liptype behaviour during oxidation of highredoxpotential dyes ( ruizdueas et al . compoundib and compoundiib ( formed by lret to the activated haem cofactor ) represent a small percentage of the total activated enzyme ( pogni et al . the use of small chemical oxidizers acting as redox mediators represents a second alternative to overcome the difficulties associated to the limited access of the bulky lignin polymer to the activated cofactor of peroxidases and other oxidoreductases . enzymemediator systems have been extensively investigated in the case of laccases after the work of bourbonnais and paice ( 1990 ) reporting that synthetic mediators expanded the application potential of these lowredoxpotential oxidoreductases enabling oxidation of nonphenolic lignin model compounds . these mediators are lowmolecularmass compounds that : ( i ) form stable free radicals oxidizing compounds that the enzyme alone is not able to oxidize , and ( ii ) diffuse away from the enzyme and can easily penetrate the lignocellulose matrix . the existence of an exposed protein radical transferring electrons to the haem via a lret pathway enables lip direct oxidation of veratryl alcohol and lignin model compounds , including nonphenolic tetramers ( mester et al . however , the laccasemediator system has became extremely popular in biodegradation studies , as well as for processing and functionalization of lignocellulosic fibres ( riva , 2006 ; widsten and kandelbauer , 2008 ) . , 2007 ) , economic issues related to the cost of the mediator , and environmental concerns related to the eventual release of toxic compounds during the enzymatic treatment are main drawbacks for the industrial implementation of this enzymatic system in the pulp and paper and other industrial sectors . the rapid acquisition of knowledge on the structure and function of these enzymes over the last years has been used to modulate their catalytic and operational properties using sitedirected mutagenesis in a variety of studies ( timofeevski et al . , considerable efforts have been devoted during the last years to improve the expression of ligninolytic and other peroxidases in different fungal hosts using a variety of strategies . , new strategies based on converting lowredoxpotential peroxidases , easily to express at levels of several grams per litre , into highredoxpotential peroxidases are being developed taking advantage of the structure function knowledge accumulated . in this sense , cip has been successfully modified and transformed into a liplike enzyme by introducing a catalytic tryptophan , at the same time that the acidity of the local environment of this residue was increased ( smith and doyle , 2006 ) . although the mutated enzyme was not so efficient as a true lip , it is possible to predict that additional modifications will yield an enzyme with the expected catalytic activity . ph and temperature inactivation of ligninolytic peroxidases is associated to the release of the two structural ca involved in stabilization of the molecular architecture ( sutherland and aust , 1996 ; george et al . , 1999 ; it has been described that losing these ions causes hexacoordination of the haem iron preventing lip and mnp activation by hydrogen peroxide , although more recently a decrease in redox potential has been suggested as the main inactivation cause in vp ( verdn et al . generation of the appropriate disulfide bridges could not only make these enzymes resistant to high ph and temperature , but it could also be used to increase their redox potential . among ligninolytic peroxidases , vp presents especial biotechnological interest due to different reasons including : ( i ) catalytic versatility by combination of different substrate oxidation sites , and ( ii ) ability to degrade some compounds of interest that lip and mnp ( as well as cip ) are not able to oxidize directly . the possibility to degrade directly a variety of recalcitrant compounds represents a considerable advantage compared with lip since the cost of veratryl alcohol required as mediator can be saved . the promiscuity of ligninolytic fungi and their enzymes oxidizing aromatic xenobiotics and other recalcitrant compounds is due to the involvement of the lignindegrading enzymatic machinery in many of these reactions . to overcome the bulky nature and structural heterogeneity of the lignin polymer , these microorganisms have developed a highly unspecific extracellular system being able to subtract one electron directly from the benzenic rings of the different lignin units . highredoxpotential extracellular peroxidases , often forming catalytic radicals at the protein surface , are the key enzymes in this initial attack yielding partially oxidized products whose catabolism is finally completed intracellularly .

dendritic cells ( dcs ) consist of a network of immunomodulatory cells with the ability to present antigen to t lymphocytes and to induce powerful antigen - specific primary immune responses . dcs may exert an anti - neoplastic effect by processing and presenting tumor antigen to autologous effector lymphocytes and thereby stimulating tumor - specific cytotoxicity . the presence of increased numbers of intratumoral dcs has been associated with improved clinical outcomes in human patients with non - small cell lung cancer and colorectal cancer , among other malignancies.2 , 3 however , dcs can also exert an opposing , immunosuppressive , and tumor - sustaining effect in the context of malignancy , either by increased surface expression of immune checkpoint proteins such as pd-1 or by the elaboration of tolerogenic substances such as indoleamine 2,3-dioxygenase . in this sense , dcs also participate in the fundamental immune tolerance of many tumors , as they are eventually able to evade the regulatory function of the immune system and grow without restriction . the field of cancer vaccines seeks to reverse such tumor - mediated immune tolerance by fostering the development of clinically meaningful tumor - specific immunity . the use of dcs as a platform for cancer vaccine development is based on their unique potency as antigen presenting cells with the capacity to induce a primary immune response . mature dcs constitutively express co - stimulatory molecules , such as cd80 and cd86 , which facilitate t lymphocyte immunoreactivity . additionally , dcs have the ability to participate in cross - presentation , in which exogenous antigen is presented to cd8-positive cytotoxic t lymphocytes via the major histocompatibility complex ( mhc ) class i molecule . by preferentially engaging in cross - presentation , dcs are able to exert a direct cytotoxic effect on exogenous antigens expressed by tumor cells . as a result , fundamental issues regarding optimization of the dc model for tumor vaccination include : ( 1 ) choosing ideal single tumor antigen targets , ( 2 ) selecting the appropriate strategy for loading of single tumor antigens onto dcs , and ( 3 ) determining the role for multiple tumor antigens and/or whole - cell approaches . each of these aspects of dc - tumor vaccine production will be discussed briefly below . the selection of an appropriate target single tumor antigen is critical for the development of a vaccine strategy that preserves tumor specificity and immunologic efficacy . common shared tumor antigens that have been explored in this setting include proteins / peptides otherwise expressed only during embryonic development ( e.g. , cancer testis antigens such as ny - eso-1 and sp17),8 , 9 peptides aberrantly or preferentially expressed by malignant cells ( e.g. , muc1 in acute myelogenous leukemia and multiple myeloma or bcma , which is selectively expressed by b - lymphocytes and plasma cells ) , or antigens truly unique to the tumor cell , such as the idiotype protein arising from the variable region of the immunoglobulin gene . additionally , intense research has recently focused on the use of tumor neoantigens those generated by somatic alterations in the genomes of cancer cells as they acquire neoplastic characteristics as an antigenic source for dc - tumor vaccination . cancer testis antigens serve as attractive platforms for dc - tumor vaccine creation because of their limited expression on normal tissues and high expression by malignant hematologic cells , as well as the ease with which their mrna can be incorporated into autologous dcs via electroporation . sp17 , prame , csage , pasd1 , cage / ddx53 , ctage1 , hage / ddx43 , and plu-1/jard1b is expressed across numerous human b- and t cell lymphoma cell lines . a similar observation has been made regarding the expression of cancer testis antigens of the mage and ssx families in bone marrow biopsy specimens of human patients with multiple myeloma.15 , 16 , 17 expression of such cancer testis antigens may vary across different disease states . for example , expression of the cancer testis antigens ny - eso-1 and magea3/a6 increases on leukemic blasts following treatment with the hypomethylating agent decitabine in human patients with acute myelogenous leukemia . likewise , expression of the cancer testis antigen nxf2 is increased on neoplastic lymphocytes after the administration of decitabine in patients with chronic lymphocytic leukemia . such variability in cancer testis antigen expression over time and following specific therapy has important implications not only for the choice of which of these antigens are best suited for incorporation into a dc - tumor vaccine , but also for the most appropriate timing of such vaccination . several cancer testis antigen vaccines have already been tested in clinical trials for the hematologic malignancies , with variable degrees of immunologic and clinical success ( table 1 ) . additional cancer testis antigen - dc vaccines are currently in development ( table 2 ) . loading strategies of single tumor antigens for dc - tumor vaccine production in the treatment of hematologic malignancies consist of in vivo and ex vivo methods . in vivo dc antigen loading for tumor - specific vaccination has been accomplished by using nanoparticles coated with antibodies specific to dc surface markers as the vehicle by which to carry tumor antigen and adjuvant immunostimulatory molecules to dcs.20 , 21 this approach has several advantages compared to ex vivo manipulation , including decreased cost , improved ease of administration , and applicability in a variety of clinical practice environments , as well as the potential to target more dcs and different dc subsets than those available during ex vivo production . by contrast , the ex vivo method of dc - tumor vaccine generation allows for more standardization and control of antigen loading onto dcs , as well as the ability to ensure that autologous dcs have fully matured prior to vaccination and thus have reached their complete immunogenic capability . dc maturation can be stimulated ex vivo by exposing cd14-positive peripheral blood monocytes in culture to various cocktails of cytokines , including interleukin ( il)-1 , il-6 , tumor necrosis factor alpha ( tnf- ) , and pge2 , or , more commonly , granulocyte - macrophage colony - stimulating factor ( gm - csf ) , il-4 , and tnf-. for this reason , monocyte derived precursor populations have been the most common source for autologous dc tumor vaccine production . however , the ontogeny of human dcs is complex and has yet to be elucidated fully . myeloid / classic dc , plasmacytoid dc , monocyte - related dc , and langerhans cell subtypes exert divergent biological effects as antigen presenting cells , including differential capacity for cross presentation , cytokine production , and polarization toward th1 , th2 , th17 , or regulatory t cell ( treg ) immunophenotypes under the influence of various microbiological and biochemical stimuli.25 , 26 , 27 , 28 each of these alternative dc subtypes , including langerhans cells,29 , 30 , 31 dcs derived from cd34-positive umbilical cord blood progenitor cells,32 , 33 , 34 , 35 and plasmacytoid dcs36 , 37 , 38 , 39 have been explored for use in dc vaccine development in different solid and hematologic malignancies . further pre - clinical and clinical studies are necessary to determine precisely how the choice of dc subtype for vaccine production will impact vaccine yield and tumor - specific immunogenicity . a potential limitation of single antigen dc vaccines is their susceptibility to immune evasion due to the downregulation of antigen expression . alternatively , loading of antigen derived from autologous whole tumor cells allows for the induction of a polyvalent response that can potentially target shared and patient - unique neoantigens . whole - cell techniques for vaccine production include creation of dc - tumor fusion cells , as well as loading of dcs with cellular lysates,40 , 41 apoptotic bodies,42 , 43 tumor exosomes,44 , 45 or whole - cell dna or rna . have determined that dc - tumor whole - cell fusions induce a more potent cytotoxic t - lymphocyte response against human acute myelogenous leukemia ( aml ) cells in the in vitro setting than those dc vaccines created with tumor cell lysates or apoptotic bodies . nevertheless , further research into the most appropriate whole - cell approach for tumor antigen loading remains necessary . our group has focused on using a whole - cell dc - tumor fusion approach for the creation of autologous dc vaccines in aml and multiple myeloma ( figure 1 ) . using this technique , patient - derived tumor cells are fused to autologous ex vivo - generated dcs by co - culture in the presence of polyethylene glycol . these dc - tumor cell fusions have several distinct immunologic advantages for vaccination in that they present numerous shared antigens and tumor neoantigens to immune effector cells and do so through both the mhc class i / cd8 ( cytotoxic t lymphocyte ) and mhc class ii / cd4 ( helper t lymphocyte ) pathways . this vaccine platform has proved to be successful in early phase clinical trials for aml and multiple myeloma , with larger phase iii clinical trials to be performed in the near future . dc - tumor vaccination has been explored in clinical trials for several hematologic malignancies , including indolent non - hodgkin s lymphoma , chronic myelogenous leukemia ( cml ) , chronic lymphocytic leukemia ( cll ) , adult t cell leukemia / lymphoma ) , multiple myeloma , and aml ( table 1 ) . while biologic potency has been demonstrated in many instances , the clinical efficacy of the dc - tumor vaccine treatment strategy for the hematologic malignancies continues to be investigated in ongoing studies ( table 2 ) . in an initial trial of a dc vaccine pulsed with tumor - specific idiotype for follicular lymphoma , a subset of patients developed a measurable anti - tumor immune response to the vaccine , and one of the patients experienced complete tumor regression . in a subsequent larger study by the same group , 35 patients with follicular lymphoma underwent dc vaccination using the tumor idiotype vaccine platform following standard chemotherapy . immunologic response and regression of residual disease were noted in 65% and 22% of patients , respectively . of note , 70% of patients in this study remained without tumor progression at a median follow - up of 43 months . idiotype - based vaccines subsequently proved successful in a number of phase i and ii clinical trials for follicular lymphoma,51 , 52 , 53 , 54 , 55 but did not reach pre - specified clinical efficacy endpoints in three separate large phase iii studies.56 , 57 , 58 importantly , however , all of these larger trials administered tumor idiotype alone as their method of vaccination , without autologous dcs . therefore , further development and clinical study of idiotype vaccines against follicular lymphoma using a dc platform may still be warranted . whole tumor cell techniques of antigen loading have also been studied in clinical trials of follicular lymphoma . for example , a pilot study of 18 patients with relapsed indolent follicular lymphoma demonstrated that vaccination with dcs loaded with autologous tumor cells that were heat - shocked and irradiated was associated with objective clinical response and stable disease in 6 ( 33% ) and 8 ( 44% ) patients , respectively . clinical responses were significantly associated with a reduction in tregs and an increase in natural killer ( nk ) cells . this whole - cell approach to dc vaccine antigen loading against follicular lymphoma , therefore , also merits further clinical study . because of the success of immunologic approaches to the treatment of cml , namely the striking effectiveness of donor lymphocyte infusion following allogeneic hematopoietic stem cell transplantation , it was hypothesized that additional immunotherapeutic techniques may prove to be beneficial in inducing disease control for patients with cml . in 2003 , ossenkoppele et al . reported the results of a pilot study of three patients with cml who were administered dcs that were obtained from autologous peripheral blood mononuclear cells . there were two of these patients that developed delayed type hypersensitivity reactions to cml - derived dcs , and one patient ( 33% ) was found to have a sustained , leukemia - specific response after 20 months of follow up . there were six patients that were subsequently studied in a larger phase i trial of a dc - based vaccine for cml . although no clinical responses were noted in this study , an increase in t cell immunogenicity to cml was observed . this spurred a further trial of dc vaccination , in which ten patients with chronic - phase cml were treated with dcs that had been harvested from autologous peripheral blood monocytes . in this series , three patients ( 30% ) were found to have an expansion of t lymphocytes with specificity for leukemia - specific antigens and cytogenetic / molecular response was noted in four patients ( 40% ) . dc - based vaccination strategies therefore may represent a promising treatment option for cml in the future , particularly for patients with minimal residual disease following treatment with a potent bcr - abl tyrosine kinase inhibitor . because the clonal lymphocyte population in cll represents the overwhelming preponderance of circulating nucleated cells , harvesting of dcs via leukapheresis of peripheral blood mononuclear cells can be technically challenging in patients with this disease . it is for this reason that allogeneic dcs as opposed to autologous dcs , which have been used in the vast majority of other studies of dc - tumor vaccines for hematologic malignancies were first used for the creation of a dc - based tumor vaccine against cll . in a small pilot study of nine patients with early stage cll ( rai 0 - 1 ) , allogeneic dcs were obtained from healthy , unrelated donors and were then exposed to the study subjects cll tumor lysate and tumor apoptotic bodies . administration of the resulting allogeneic dc - tumor vaccine led to a decrease in the amount of circulating cll cells across all of the patients . furthermore , one patient ( 11.1% ) developed expansion of cytotoxic t lymphocytes directed against a leukemia - associated antigen . autologous dc - based vaccination against chronic lymphocytic leukemia was subsequently reported by hus et al . in 2008 . in this study of 12 patients with early stage cll ( rai stage 02 ) , dc vaccine was produced by isolation of peripheral blood mononuclear cells , which were then co - cultured with leukemia cell lysates . following vaccination , five patients ( 41.7% ) demonstrated a decrease in the number of peripheral blood leukemia cells , three patients ( 25% ) demonstrated stable disease , and four patients ( 33% ) experienced disease progression . there were four patients ( 33% ) that were found to have increased numbers of cd8-positive t lymphocytes directed against leukemia - specific antigens , and patients with clinical response were noted to have increased levels of the immunostimulatory cytokine il-12 , as well as decreased numbers of immunosuppressive tregs . subsequently reported a cohort of 15 patients with cll who were treated with a dc vaccine that was produced with tumor apoptotic bodies . although no objective clinical responses were noted in this study , ten patients ( 66% ) developed leukemia - specific immune responses . the encouraging results of these early - phase trials indicate that dc - based tumor vaccination strategies may prove to have a role in the therapeutic armamentarium against cll in the future . in 2015 , suehiro et al . reported an early phase clinical trial of a dc - tumor vaccine for patients with atll . for this study , a dc - tumor vaccine was created by pulsing autologous dcs with tax , a peptide product of human t cell leukemia virus type - i ( htlv-1 ) , the causative viral agent of atll . tax - specific cytotoxic t lymphocyte responses were identified in all three patients in this trial . two of three patients experienced a partial remission within the first 8 weeks , and one of these patients converted to a complete remission subsequently . these two patients remained in remission 24 and 19 months after vaccination , with no need for further chemotherapy . the encouraging clinical outcome in this trial indicates the need for further study of this promising immunotherapy for atll . the initial feasibility study of an idiotype - pulsed dc vaccine for the treatment of patients with multiple myeloma following autologous peripheral blood stem cell transplantation was reported by reichardt et al . from stanford university in 1999 . in this series , 12 patients were administered two intravenous infusions of idiotype - pulsed autologous dcs , with 5 monthly subcutaneous boosters of idiotype , keyhole limpet hemocyanin ( klh ) , and immune adjuvant . there were two patients ( 16.7% ) that developed an idiotype - specific cellular immune response , and these patients remained in complete remission at a minimum follow - up of 16 months . similar results were subsequently reported by groups in italy , wales , australia , germany , and arkansas , as well as within a larger cohort of patients from stanford . an idiotype - pulsed dc vaccine has also been demonstrated to lead to idiotype - specific t lymphocyte expansion in patients with earlier stages of myeloma . in a subsequent phase ii study performed at the mayo clinic , 27 patients with multiple myeloma who underwent consolidation therapy with autologous hematopoietic stem cell transplantation received idiotype - pulsed dcs ( apc8020 , mylovenge ) as post - transplantation adjunctive therapy between july 1998 and june 2001 . these patients were compared to a non - randomized , parallel group of control patients who underwent autologous hematopoietic stem cell transplantation for myeloma during that time , but were not vaccinated with apc8020 . comparison of these two groups revealed a statistically and clinically significant improvement in overall survival in the vaccine group ( 5.3 years ) compared to the unvaccinated group ( 3.4 years , p = 0.02 ) . others have reported a cancer testis antigen mrna antigen - loading strategy for dc - myeloma vaccination . in a phase i study , hobo et al . obtained autologous monocyte - derived dcs from 12 patients with multiple myeloma who were treated with induction chemotherapy and high - dose melphalan with autologous hematopoietic stem cell transplantation . these dcs were pulsed with klh and were electroporated with mrna from the cancer testis antigen mage3 , as well as survivin and b cell maturation antigen ( bcma ) . the dc - mrna - loaded vaccines were then re - administered to the patients intravenously and intradermally . two patients ( 16.7% ) in this study developed vaccine - specific t lymphocyte responses . our group has developed a dc - tumor fusion vaccine model , whereby patient - derived myeloma cells are fused to ex vivo - generated dcs . in a phase i study of 18 patients with advanced disease , vaccination resulted in the expansion of cd4-positive and cd8-positive myeloma - reactive t lymphocytes in 11 of 15 evaluable patients , with the majority of patients experiencing disease stabilization . in a phase ii trial of 36 subjects evaluating the dc - myeloma vaccine following autologous hematopoietic stem cell transplantation , 47% and 78% of patients experienced a complete response / near complete response and complete response / very good partial response , respectively . importantly , 24% of patients with a partial response following autologous hematopoietic stem cell transplantation were converted to complete response after dc - tumor fusion cell administration in the absence of any other therapy , consistent with the possibility that the vaccine targeted post - transplant residual disease . based on these data , a randomized trial of dc - tumor fusion vaccination for multiple myeloma with lenalidomide maintenance versus lenalidomide maintenance alone is being conducted under the auspices of the ctn cooperative group ( clinicaltrials.gov identifier : nct02728102 ) . dc - myeloma fusion vaccination is also being studied in conjunction with blockade of pd-1 , in an effort to augment vaccine effectiveness by immune checkpoint inhibition . several other trials of dc - myeloma vaccines are currently open to enrollment ( table 2 ) , including those using an adenovirus vector to load the survivin antigen onto autologous dcs ( clinicaltrials.gov identifier : nct02851056 ) , and those using langerhans cells electroporated with the mrna of the cancer testis antigens ct7 and mage - a3 , as well as wt1 , for antigen loading ( clinicaltrials.gov identifier : nct01995708 ) . various methods of antigen selection and loading have been employed for the creation of dc vaccines for therapeutic use in aml . these have included whole tumor cell - dc fusions , mrna coding for particular tumor antigens , apoptotic tumor bodies , and differentiation of leukemic blasts into autologous dcs . developed a dc vaccine against aml by electroporating autologous dcs with the mrna of wt1 , an oncogene that is expressed in most cases of aml . in a phase i / ii study of ten patients with aml , administration of this vaccine led to increases in wt1-specific t lymphocyte proliferation and wt1-specific interferon--producing cd8-positive t lymphocytes . furthermore , molecular remission was induced in five of these patients ( 50% ) , and two patients who were in a partial remission were converted to complete remission after vaccination . several clinical trials using electroporation of specific mrnas as the method of dc vaccine construction against aml are currently ongoing ( table 2).86 , 87 , 88 in a study of 17 patients with aml who were not candidates for allogeneic hematopoietic stem cell transplantation and achieved first complete remission after induction chemotherapy , our group demonstrated that vaccination with autologous dc / aml whole fusion cells induced the expansion of leukemia specific cd4-positive and cd8-positive t lymphocytes . remarkably , 12 of the patients ( 71% ) remained alive without aml recurrence after a median follow - up of 57 months . the encouraging results of this trial will lead to a randomized , phase iii trial of this dc - aml whole - cell fusion vaccine in the near future . in spite of the therapeutic excitement of dc - tumor vaccines against the hematologic malignancies , significant challenges to their widespread adoption in clinical practice remain . from a technical perspective , standardization of dc - tumor vaccine preparation while increasingly sophisticated strategies to identify patient specific mutations / antigens for dc - tumor vaccine production have evolved , determining the immunologic relevance of such individual shared and neoantigens will be crucial . furthermore , these newly identified antigens may show distinctive patterns of expression in different disease settings , with important implications for the use and timing of dc - tumor vaccines directed against those antigens . improvements in elucidating the complex interactions of the tumor microenvironment that induce effector t cell dysfunction and compromise migration and cytotoxic t lymphocyte mediated killing of tumor cells at the tumor bed are critical . while an individual disease setting may be dominated by a particular perturbation of immunity , hematologic malignancies typically manifest multiple areas of dysfunction that necessitate repair . dc vaccines may play a critical role in stimulating expansion of tumor - reactive lymphocytes , but other areas of immune dysregulation will need to be addressed in order for these cells to be maximally effective at the site of disease . efforts to augment dc vaccine potency are now focusing on reversing these critical elements of the immunosuppressive milieu , including blockade of immunosuppressive accessory cells and reversal of alterations of biologic pathways that foster immune tolerance . combining vaccination with immunomodulatory drugs such as lenalidomide or immune checkpoint blockade is being explored.91 , 92 future research efforts in this realm are also likely to include exploration of combinations of dc - tumor vaccination with various novel immunotherapy strategies , including chimeric antigen receptor ( car ) t lymphocyte therapy , myeloid - derived suppressor cell ( mdsc ) inhibitors , and therapies that deplete tregs , as well as lentviral or retroviral gene therapy techniques for improved induction of anti - tumor immune response.96 , 97 while the current generation of dc - based vaccinations has demonstrated promise against the hematologic malignancies , further refinements in vaccine strategies are clearly needed to develop this promising area of investigation into a clinically meaningful therapy for patients with blood cancers .

dendritic cells ( dcs ) are potent antigen - presenting cells that constitute a major component of the immune system s role in the recognition , elimination , and tolerance of cancer . the unique immunologic capabilities of dcs have recently been harnessed for therapeutic use with the creation of dc - based anti - tumor vaccines , several of which have moved into testing in clinical trials for hematologic malignancies . this review summarizes how treatment strategies using dc - based anti - tumor vaccines are advancing immunotherapeutic options for these diseases .

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dendritic cells ( dcs ) consist of a network of immunomodulatory cells with the ability to present antigen to t lymphocytes and to induce powerful antigen - specific primary immune responses . dcs may exert an anti - neoplastic effect by processing and presenting tumor antigen to autologous effector lymphocytes and thereby stimulating tumor - specific cytotoxicity . the presence of increased numbers of intratumoral dcs has been associated with improved clinical outcomes in human patients with non - small cell lung cancer and colorectal cancer , among other malignancies.2 , 3 however , dcs can also exert an opposing , immunosuppressive , and tumor - sustaining effect in the context of malignancy , either by increased surface expression of immune checkpoint proteins such as pd-1 or by the elaboration of tolerogenic substances such as indoleamine 2,3-dioxygenase . in this sense , dcs also participate in the fundamental immune tolerance of many tumors , as they are eventually able to evade the regulatory function of the immune system and grow without restriction . the field of cancer vaccines seeks to reverse such tumor - mediated immune tolerance by fostering the development of clinically meaningful tumor - specific immunity . the use of dcs as a platform for cancer vaccine development is based on their unique potency as antigen presenting cells with the capacity to induce a primary immune response . additionally , dcs have the ability to participate in cross - presentation , in which exogenous antigen is presented to cd8-positive cytotoxic t lymphocytes via the major histocompatibility complex ( mhc ) class i molecule . as a result , fundamental issues regarding optimization of the dc model for tumor vaccination include : ( 1 ) choosing ideal single tumor antigen targets , ( 2 ) selecting the appropriate strategy for loading of single tumor antigens onto dcs , and ( 3 ) determining the role for multiple tumor antigens and/or whole - cell approaches . each of these aspects of dc - tumor vaccine production will be discussed briefly below . , cancer testis antigens such as ny - eso-1 and sp17),8 , 9 peptides aberrantly or preferentially expressed by malignant cells ( e.g. , muc1 in acute myelogenous leukemia and multiple myeloma or bcma , which is selectively expressed by b - lymphocytes and plasma cells ) , or antigens truly unique to the tumor cell , such as the idiotype protein arising from the variable region of the immunoglobulin gene . additionally , intense research has recently focused on the use of tumor neoantigens those generated by somatic alterations in the genomes of cancer cells as they acquire neoplastic characteristics as an antigenic source for dc - tumor vaccination . cancer testis antigens serve as attractive platforms for dc - tumor vaccine creation because of their limited expression on normal tissues and high expression by malignant hematologic cells , as well as the ease with which their mrna can be incorporated into autologous dcs via electroporation . sp17 , prame , csage , pasd1 , cage / ddx53 , ctage1 , hage / ddx43 , and plu-1/jard1b is expressed across numerous human b- and t cell lymphoma cell lines . a similar observation has been made regarding the expression of cancer testis antigens of the mage and ssx families in bone marrow biopsy specimens of human patients with multiple myeloma.15 , 16 , 17 expression of such cancer testis antigens may vary across different disease states . for example , expression of the cancer testis antigens ny - eso-1 and magea3/a6 increases on leukemic blasts following treatment with the hypomethylating agent decitabine in human patients with acute myelogenous leukemia . likewise , expression of the cancer testis antigen nxf2 is increased on neoplastic lymphocytes after the administration of decitabine in patients with chronic lymphocytic leukemia . such variability in cancer testis antigen expression over time and following specific therapy has important implications not only for the choice of which of these antigens are best suited for incorporation into a dc - tumor vaccine , but also for the most appropriate timing of such vaccination . several cancer testis antigen vaccines have already been tested in clinical trials for the hematologic malignancies , with variable degrees of immunologic and clinical success ( table 1 ) . additional cancer testis antigen - dc vaccines are currently in development ( table 2 ) . loading strategies of single tumor antigens for dc - tumor vaccine production in the treatment of hematologic malignancies consist of in vivo and ex vivo methods . in vivo dc antigen loading for tumor - specific vaccination has been accomplished by using nanoparticles coated with antibodies specific to dc surface markers as the vehicle by which to carry tumor antigen and adjuvant immunostimulatory molecules to dcs.20 , 21 this approach has several advantages compared to ex vivo manipulation , including decreased cost , improved ease of administration , and applicability in a variety of clinical practice environments , as well as the potential to target more dcs and different dc subsets than those available during ex vivo production . by contrast , the ex vivo method of dc - tumor vaccine generation allows for more standardization and control of antigen loading onto dcs , as well as the ability to ensure that autologous dcs have fully matured prior to vaccination and thus have reached their complete immunogenic capability . dc maturation can be stimulated ex vivo by exposing cd14-positive peripheral blood monocytes in culture to various cocktails of cytokines , including interleukin ( il)-1 , il-6 , tumor necrosis factor alpha ( tnf- ) , and pge2 , or , more commonly , granulocyte - macrophage colony - stimulating factor ( gm - csf ) , il-4 , and tnf-. myeloid / classic dc , plasmacytoid dc , monocyte - related dc , and langerhans cell subtypes exert divergent biological effects as antigen presenting cells , including differential capacity for cross presentation , cytokine production , and polarization toward th1 , th2 , th17 , or regulatory t cell ( treg ) immunophenotypes under the influence of various microbiological and biochemical stimuli.25 , 26 , 27 , 28 each of these alternative dc subtypes , including langerhans cells,29 , 30 , 31 dcs derived from cd34-positive umbilical cord blood progenitor cells,32 , 33 , 34 , 35 and plasmacytoid dcs36 , 37 , 38 , 39 have been explored for use in dc vaccine development in different solid and hematologic malignancies . further pre - clinical and clinical studies are necessary to determine precisely how the choice of dc subtype for vaccine production will impact vaccine yield and tumor - specific immunogenicity . whole - cell techniques for vaccine production include creation of dc - tumor fusion cells , as well as loading of dcs with cellular lysates,40 , 41 apoptotic bodies,42 , 43 tumor exosomes,44 , 45 or whole - cell dna or rna . have determined that dc - tumor whole - cell fusions induce a more potent cytotoxic t - lymphocyte response against human acute myelogenous leukemia ( aml ) cells in the in vitro setting than those dc vaccines created with tumor cell lysates or apoptotic bodies . our group has focused on using a whole - cell dc - tumor fusion approach for the creation of autologous dc vaccines in aml and multiple myeloma ( figure 1 ) . using this technique , patient - derived tumor cells are fused to autologous ex vivo - generated dcs by co - culture in the presence of polyethylene glycol . these dc - tumor cell fusions have several distinct immunologic advantages for vaccination in that they present numerous shared antigens and tumor neoantigens to immune effector cells and do so through both the mhc class i / cd8 ( cytotoxic t lymphocyte ) and mhc class ii / cd4 ( helper t lymphocyte ) pathways . this vaccine platform has proved to be successful in early phase clinical trials for aml and multiple myeloma , with larger phase iii clinical trials to be performed in the near future . dc - tumor vaccination has been explored in clinical trials for several hematologic malignancies , including indolent non - hodgkin s lymphoma , chronic myelogenous leukemia ( cml ) , chronic lymphocytic leukemia ( cll ) , adult t cell leukemia / lymphoma ) , multiple myeloma , and aml ( table 1 ) . while biologic potency has been demonstrated in many instances , the clinical efficacy of the dc - tumor vaccine treatment strategy for the hematologic malignancies continues to be investigated in ongoing studies ( table 2 ) . in an initial trial of a dc vaccine pulsed with tumor - specific idiotype for follicular lymphoma , a subset of patients developed a measurable anti - tumor immune response to the vaccine , and one of the patients experienced complete tumor regression . idiotype - based vaccines subsequently proved successful in a number of phase i and ii clinical trials for follicular lymphoma,51 , 52 , 53 , 54 , 55 but did not reach pre - specified clinical efficacy endpoints in three separate large phase iii studies.56 , 57 , 58 importantly , however , all of these larger trials administered tumor idiotype alone as their method of vaccination , without autologous dcs . whole tumor cell techniques of antigen loading have also been studied in clinical trials of follicular lymphoma . for example , a pilot study of 18 patients with relapsed indolent follicular lymphoma demonstrated that vaccination with dcs loaded with autologous tumor cells that were heat - shocked and irradiated was associated with objective clinical response and stable disease in 6 ( 33% ) and 8 ( 44% ) patients , respectively . because of the success of immunologic approaches to the treatment of cml , namely the striking effectiveness of donor lymphocyte infusion following allogeneic hematopoietic stem cell transplantation , it was hypothesized that additional immunotherapeutic techniques may prove to be beneficial in inducing disease control for patients with cml . there were two of these patients that developed delayed type hypersensitivity reactions to cml - derived dcs , and one patient ( 33% ) was found to have a sustained , leukemia - specific response after 20 months of follow up . there were six patients that were subsequently studied in a larger phase i trial of a dc - based vaccine for cml . this spurred a further trial of dc vaccination , in which ten patients with chronic - phase cml were treated with dcs that had been harvested from autologous peripheral blood monocytes . dc - based vaccination strategies therefore may represent a promising treatment option for cml in the future , particularly for patients with minimal residual disease following treatment with a potent bcr - abl tyrosine kinase inhibitor . because the clonal lymphocyte population in cll represents the overwhelming preponderance of circulating nucleated cells , harvesting of dcs via leukapheresis of peripheral blood mononuclear cells can be technically challenging in patients with this disease . it is for this reason that allogeneic dcs as opposed to autologous dcs , which have been used in the vast majority of other studies of dc - tumor vaccines for hematologic malignancies were first used for the creation of a dc - based tumor vaccine against cll . administration of the resulting allogeneic dc - tumor vaccine led to a decrease in the amount of circulating cll cells across all of the patients . autologous dc - based vaccination against chronic lymphocytic leukemia was subsequently reported by hus et al . following vaccination , five patients ( 41.7% ) demonstrated a decrease in the number of peripheral blood leukemia cells , three patients ( 25% ) demonstrated stable disease , and four patients ( 33% ) experienced disease progression . there were four patients ( 33% ) that were found to have increased numbers of cd8-positive t lymphocytes directed against leukemia - specific antigens , and patients with clinical response were noted to have increased levels of the immunostimulatory cytokine il-12 , as well as decreased numbers of immunosuppressive tregs . the encouraging results of these early - phase trials indicate that dc - based tumor vaccination strategies may prove to have a role in the therapeutic armamentarium against cll in the future . reported an early phase clinical trial of a dc - tumor vaccine for patients with atll . for this study , a dc - tumor vaccine was created by pulsing autologous dcs with tax , a peptide product of human t cell leukemia virus type - i ( htlv-1 ) , the causative viral agent of atll . two of three patients experienced a partial remission within the first 8 weeks , and one of these patients converted to a complete remission subsequently . in this series , 12 patients were administered two intravenous infusions of idiotype - pulsed autologous dcs , with 5 monthly subcutaneous boosters of idiotype , keyhole limpet hemocyanin ( klh ) , and immune adjuvant . there were two patients ( 16.7% ) that developed an idiotype - specific cellular immune response , and these patients remained in complete remission at a minimum follow - up of 16 months . similar results were subsequently reported by groups in italy , wales , australia , germany , and arkansas , as well as within a larger cohort of patients from stanford . comparison of these two groups revealed a statistically and clinically significant improvement in overall survival in the vaccine group ( 5.3 years ) compared to the unvaccinated group ( 3.4 years , p = 0.02 ) . others have reported a cancer testis antigen mrna antigen - loading strategy for dc - myeloma vaccination . the dc - mrna - loaded vaccines were then re - administered to the patients intravenously and intradermally . our group has developed a dc - tumor fusion vaccine model , whereby patient - derived myeloma cells are fused to ex vivo - generated dcs . in a phase i study of 18 patients with advanced disease , vaccination resulted in the expansion of cd4-positive and cd8-positive myeloma - reactive t lymphocytes in 11 of 15 evaluable patients , with the majority of patients experiencing disease stabilization . in a phase ii trial of 36 subjects evaluating the dc - myeloma vaccine following autologous hematopoietic stem cell transplantation , 47% and 78% of patients experienced a complete response / near complete response and complete response / very good partial response , respectively . importantly , 24% of patients with a partial response following autologous hematopoietic stem cell transplantation were converted to complete response after dc - tumor fusion cell administration in the absence of any other therapy , consistent with the possibility that the vaccine targeted post - transplant residual disease . based on these data , a randomized trial of dc - tumor fusion vaccination for multiple myeloma with lenalidomide maintenance versus lenalidomide maintenance alone is being conducted under the auspices of the ctn cooperative group ( clinicaltrials.gov identifier : nct02728102 ) . dc - myeloma fusion vaccination is also being studied in conjunction with blockade of pd-1 , in an effort to augment vaccine effectiveness by immune checkpoint inhibition . several other trials of dc - myeloma vaccines are currently open to enrollment ( table 2 ) , including those using an adenovirus vector to load the survivin antigen onto autologous dcs ( clinicaltrials.gov identifier : nct02851056 ) , and those using langerhans cells electroporated with the mrna of the cancer testis antigens ct7 and mage - a3 , as well as wt1 , for antigen loading ( clinicaltrials.gov identifier : nct01995708 ) . various methods of antigen selection and loading have been employed for the creation of dc vaccines for therapeutic use in aml . these have included whole tumor cell - dc fusions , mrna coding for particular tumor antigens , apoptotic tumor bodies , and differentiation of leukemic blasts into autologous dcs . developed a dc vaccine against aml by electroporating autologous dcs with the mrna of wt1 , an oncogene that is expressed in most cases of aml . several clinical trials using electroporation of specific mrnas as the method of dc vaccine construction against aml are currently ongoing ( table 2).86 , 87 , 88 in a study of 17 patients with aml who were not candidates for allogeneic hematopoietic stem cell transplantation and achieved first complete remission after induction chemotherapy , our group demonstrated that vaccination with autologous dc / aml whole fusion cells induced the expansion of leukemia specific cd4-positive and cd8-positive t lymphocytes . remarkably , 12 of the patients ( 71% ) remained alive without aml recurrence after a median follow - up of 57 months . the encouraging results of this trial will lead to a randomized , phase iii trial of this dc - aml whole - cell fusion vaccine in the near future . in spite of the therapeutic excitement of dc - tumor vaccines against the hematologic malignancies , significant challenges to their widespread adoption in clinical practice remain . from a technical perspective , standardization of dc - tumor vaccine preparation while increasingly sophisticated strategies to identify patient specific mutations / antigens for dc - tumor vaccine production have evolved , determining the immunologic relevance of such individual shared and neoantigens will be crucial . furthermore , these newly identified antigens may show distinctive patterns of expression in different disease settings , with important implications for the use and timing of dc - tumor vaccines directed against those antigens . improvements in elucidating the complex interactions of the tumor microenvironment that induce effector t cell dysfunction and compromise migration and cytotoxic t lymphocyte mediated killing of tumor cells at the tumor bed are critical . while an individual disease setting may be dominated by a particular perturbation of immunity , hematologic malignancies typically manifest multiple areas of dysfunction that necessitate repair . dc vaccines may play a critical role in stimulating expansion of tumor - reactive lymphocytes , but other areas of immune dysregulation will need to be addressed in order for these cells to be maximally effective at the site of disease . efforts to augment dc vaccine potency are now focusing on reversing these critical elements of the immunosuppressive milieu , including blockade of immunosuppressive accessory cells and reversal of alterations of biologic pathways that foster immune tolerance . combining vaccination with immunomodulatory drugs such as lenalidomide or immune checkpoint blockade is being explored.91 , 92 future research efforts in this realm are also likely to include exploration of combinations of dc - tumor vaccination with various novel immunotherapy strategies , including chimeric antigen receptor ( car ) t lymphocyte therapy , myeloid - derived suppressor cell ( mdsc ) inhibitors , and therapies that deplete tregs , as well as lentviral or retroviral gene therapy techniques for improved induction of anti - tumor immune response.96 , 97 while the current generation of dc - based vaccinations has demonstrated promise against the hematologic malignancies , further refinements in vaccine strategies are clearly needed to develop this promising area of investigation into a clinically meaningful therapy for patients with blood cancers .

vitamin d has been a hot topic in the medical world for the past 10 years . concerns about vitamin d have resurfaced in medical and scientific literature owing to its multiple effects on human health . we are beginning to learn that it plays a much wider role in health and disease prevention . the classical and non - classical pathways of this hormone affect calcium metabolism , the immune system , cell proliferation and differentiation , infection , and cancer . the question scientists have been working on for almost a decade is why and how vitamin d is affecting conception , pregnancy and the health of the newborn . also the media has been taking increasing interest , and public expectations have been raised regarding the enhanced roles for vitamin d in pregnancy . it is an unrecognized epidemic common among children , adults and pregnant women throughout the world , across ethnicity and season . there are increasing studies worldwide reporting poor vitamin d status , including those in tropical countries . the prevalence of vitamin d deficiency has been reported to range from 15% to 80% . it is now recognized that everyone is at risk for vitamin d deficiency . skin color and modern practices of cosmetic dermatology ( sun tan lotions and creams , etc . ) modern day cosmetology and our urbanization has eclipsed the sunlight and it is impossible is to fulfill daily intake via diet as a large amount of vitamin d needs to be consumed . vitamin d deficiency is prevalent in india , a finding that is unexpected in a tropical country with abundant sunshine . there are few data from india about the prevalence of hypovitaminosis d in pregnancy and in the newborn . the increasing prevalence of disorders linked to vitamin d deficiency is reflected in the several hundred children with rickets treated each year . however , these children represent a small proportion of the individuals with a suboptimal vitamin d status in the population . the daily dietary calcium intake of both the urban and rural populations was low compared with the recommended dietary allowances issued by the indian council of medical research . low dietary calcium intake and 25(oh ) d concentrations were associated with deleterious effects on bone mineral homeostasis . in the last 3 years , an increasing amount of research suggests that some of the damage done by vitamin d deficiency is done in - utero , while the fetus is developing . much of that damage may be permanent , that is , it can not be fully reversed by taking vitamin d after birth . the 4-carbon ring backbone of this molecule makes it more of a steroid hormone than a vitamin . it is structurally similar to estrogen , testosterone , progesterone and all the steroid hormones . because of its steroid structure and function , vitamin d plays an important role in priming cells for other hormones to do their action . vitamin d itself is devoid of any biological activity , but enzymatic conversion to 1,25-dihydroxyvitamin d [ 1,25(oh ) 2d ] generates the hormonal form with diverse biological activities . the active form of vitamin d ( 1,25-dihydroxyvitamin d3 , 1,25[oh ] 2d3 ) has well - established effects on bone metabolism and mineral homeostasis . however , recently it has become clear that 1,25(oh ) 2d3 has potent anti - proliferative and immunomodulatory actions that are not immediately linked to its role as a skeletal regulator . the actions of 1,25(oh ) 2d are mediated through specific , high affinity binding to the vitamin d receptor ( vdr ) , which is present in multiple tissues . important changes occur in the maternal concentration of vitamin d and in calcium metabolism to provide the calcium needed for fetal bone mineral accretion during pregnancy . approximately 25 - 30 g of calcium are transferred to the fetal skeleton by the end of pregnancy , most of which is transferred during the last trimester . the requirement for vitamin d in maintaining normal calcium metabolism throughout pregnancy and lactation in mothers , fetuses and newborn infants is still controversial . it is clear , however , that vitamin d requirements are increased in mothers during pregnancy and lactation . established as the chorioallantoic placenta at the end of the first trimester , villous tissues secrete multiple hormones that maintain pregnancy and regulate placental physiology . the human endometrial decidua makes 1,25(oh ) 2d and 24,25(oh ) 2d and the placenta synthesizes only 24,25(oh ) 2d . data suggest that 1,25(oh ) 2d aids implantation and maintains normal pregnancy , supports fetal growth through delivery of calcium , controls secretion of multiple placental hormones , and limits production of proinflammatory cytokines . notably , the 24,25(oh ) 2d synthesized by the placenta accumulates in bone and may be involved in ossification of the fetal skeleton . in rats , the placenta transports 25(oh ) 2d and 24,25(oh ) 2d but not 1,25(oh ) 2d . although transplacental transport has not been studied in humans , vitamin d passage from the mother to the fetus would be facilitated by serum concentrations of 1,25(oh ) 2d being higher in the maternal compared to the fetal circulations . roles of vitamin d are to maintain skeletal calcium balance by promoting calcium absorption in the intestines , promoting bone resobption by increasing osteoclast number , maintaining calcium and phosphate levels for bone formation , and allowing proper functioning of parathyroid hormone to maintain serum calcium levels . vitamin d deficiency can result in lower bone mineral density and an increased risk of bone loss ( osteoporosis ) or bone fracture because a lack of vitamin d alters mineral metabolism in the body . the most important source of vitamin d is the skin 's synthesis of the vitamin from sunlight . use of sun blocks , increased coverage of clothing and time spent indoors increase the risk of vitamin d deficiency . it has been estimated that exposure to sunlight for usually no more than 5 - 15 min / d between 10 am and 3 pm , in the spring , summer , and fall at latitudes above and below 35 ( and all year near the equator ) to exposed parts of the body involving arms , legs and face provides the body with its required 1000 iu of cholecalciferol . sunshine in not adequate in many parts of world . in the united states staple foods like milk , breakfast cereals and margarines are artificially fortified with vitamin d. at present , the number and variety of vitamin d fortified foods available on the market differs significantly between countries and is attributed to the country - specific policies on food fortification which are not yet unified . vitamin d is present in a small number of foods , although , for an average person food will only supply about 10% of the amount needed . dietary sources of vitamin d include : fatty fish species , such as catfish , salmon , mackerel , tuna etc . , although liver and cod liver oil contain vitamin d , they are not recommended in pregnancy as they also contain too much vitamin a. vitamin d is a unique nutrient because its requirement can be met by both endogenous production from sunlight as well as exogenous dietary sources , which complicates determining the body 's daily nutritional requirements . methods are currently available to quantify the contribution of endogenous vitamin d synthesis resulting from sun exposure , but serious limitations remain in accurately estimating dietary vitamin d intake because of the incompleteness of nutrient databases for both vitamin d - fortified food and vitamin d supplements . thus , increasing vitamin d intake from vitamin d fortified foods , and vitamin d supplements , in combination with sensible sun exposure , maximize a person 's vitamin d status to promote good health . women with darker skin fitzpatrick class 6 are more prone to vitamin d deficiency as they need four to ten times sunlight than fitzpatrick class 1 - 4 skinned people for adequate vitamin d synthesis . thus , daily vitamin requirement varies with ethnicity , and this is relevant in case of indian population . pre - pregnancy obesity is also associated with significant increases in the odds of maternal and neonatal vitamin d deficiency , independent of other factors such as ethnicity . this deficiency is because body fat stores much of the vitamin d made in the skin , making it less available to the body . certain medications like steroids , anti - epileptic medications , cholesterol - lowering drugs , and some diuretics reduce absorption of vitamin d from the intestines . women who have intestinal malabsorption diseases like celiac disease and crohn 's disease or partial removal or bypass of the stomach or intestines absorb less of both dietary and supplemental vitamin d are at increased risk of deficiency . there is controversy concerning levels of nutrient intake , and at times the concept that more is better emerges . it is said that serum 25-hydroxyvitamin d concentrations above 75 nmol / l ( 30 ng / ml ) are not consistently associated with increased benefit . for vitamin d , there are still underlying questions : how much is the daily requirement and how much is too much ? new research suggests that women who take high doses of vitamin d during pregnancy have a greatly reduced risk of complications , including gestational diabetes , preterm birth , and infection . this recommendation may be controversial because very high doses of vitamin d have long been believed to cause birth defects . w epidemiological analyses implicated high dietary vitamin d intake during pregnancy results in birth of syndromic babies . in the early 1960s , williams , et al . described a syndrome of supravalvular aortic stenosis , peripheral pulmonic stenosis , and body features indistinguishable from those in survivors of the syndrome of idiopathic hypercalcemia of infancy . friedman and colleagues developed a model for this disorder in rabbits by administering near - fatal amounts of ergocalciferol during pregnancy . based on these accumulated observations , the uncertain teratogenic potential of high doses of calciferol analogs or of the associated disturbances in mineral homeostasis continues to cause concern . various human studies are done so far involving pharmacological doses of vitamin d during pregnancy . a study in human subjects involved the administration of 100,000 iu vitamin d per day ( 2.5 mg / day ) throughout pregnancy to hypoparathyroid women to maintain serum calcium with no fatal outcome . goodenday , et al . reported that as hypoparathyroid patients have completed many pregnancies while receiving ergocalciferol , it is unlikely that material vitamin d , 25(oh ) d , or 24,25(oh ) 2d per se are teratogens . most prenatal vitamins have around 400 iu of vitamin d , and most health groups recommend taking no more than 2,000 iu of the vitamin in supplement form daily . eventually , as circulating 25(oh ) d increases to toxic concentrations , the classic situation of hypercalciuria , hypercalcemia , and , finally , extraskeletal calcification becomes evident . hypercalciuria due to excessive vitamin d intakes is always accompanied by circulating 25(oh ) d concentrations > 250nmol / l ( 100 ng / ml ) . to attain circulating 25(oh ) d concentrations that exceed 250 nmol / l ( 100 ng / ml ) , a daily vitamin d intake well in excess of 10,000 iu / d ( 250 g / d ) for several months would be required . however , hypervitaminosis d has never occurred when physiologic amounts of vitamin d are ingested . in addition , no case of hypervitaminosis d from sun exposure has ever been reported . this is supported by the recent finding from a randomized , double - blind , placebo - controlled trial examining the effects of a single annual megadose of vitamin d3 ( 500,000 iu , equivalent to approximately 1370 iu / d ) on fall and fracture outcomes in community - dwelling elderly women with a history of fall or fracture . adequate vitamin d intake is essential for maternal and fetal health during pregnancy , and prevention of adverse outcomes . recent work emphasizes the importance of non - classical roles of vitamin d in pregnancy and the placenta . vitamin d deficiency during pregnancy is associated with the non - classical actions of this hormone , being linked with preeclampsia , insulin resistance , gestational diabetes mellitus , bacterial vaginosis , and an increased risk for caesarean section delivery . women who have vitamin d deficiency do not usually feel any different but in some may have muscle weakness and weakened bones . pregnancy does not exacerbate hypocalcaemia and secondary hyperparathyroidism in people with pre - existing vitamin d deficiency . a new study finds that women who develop severe preeclampsia tend to have lower blood levels of vitamin d than healthy pregnant women raising the possibility that the vitamin plays a role in the complication . preeclampsia rates are elevated during winter months , when sunlight - dependent 25(oh ) d productions are reduced . preeclampsia is associated with low circulating levels of igf - i and 1,25(oh ) 2d and , in vitro , igf-1 increases 1,25(oh ) 2d production by primary human syncytiotrophoblasts from placentas from normal pregnancies but not from preeclamptic pregnancies . studies by other groups have reported abnormal expression of 1-hydroxylase , a vitamin d activating enzyme in preeclamptic pregnancies , revealing a potential role for 1,25(oh ) 2d3 as a regulator of placentation . induction of the 1-hydroxylase in early gestation might provide a mechanism by which environmental or dietary vitamin d can influence fetal - placental development . two clinical trials support a potential role of vitamin d in the prevention of preeclampsia , although neither of these treated with vitamin d supplements alone . in an uncontrolled trial , supplementation with a multivitamin / mineral supplement and halibut liver oil ( containing 900 iu / d vitamin d ) provided at 20 wk gestation reduced the odds of preeclampsia by 32% ( 95% ci , 11 - 47% ) . vitamin d supplementation in early pregnancy should be explored for preventing preeclampsia and promoting neonatal well - being . 1,25(oh ) 2d regulates insulin secretion by pancreatic -cells and thereby affects circulating glucose levels . as expected , low concentration of 25(oh ) d is a risk factor for insulin resistance , glucose intolerance , and features of metabolic syndrome in normoglycemic subjects . vitamin d deficiency during early pregnancy significantly increases the risk for gestational diabetes in later pregnancy . low 25(oh ) d levels are correlated with increased bacterial vaginosis in the first trimester . bacterial vaginosis is more prevalent in black women , who typically have lower serum 25(oh ) d concentrations and have a six - fold higher chance of vitamin d deficiency , compared with white women . vitamin d has effects on the immune system , cytokines , and antibacterial peptides that are likely to regulate the bacterial flora . nutritional vitamin d status has very recently been linked to the human innate immune system and its ability to contain mycobacterium tuberculosis . serum 25(oh ) d levels are inversely related to primary cesarean section in nulliparous women , an unexpected and unexplained maternal outcome recently identified . the risk was four - fold higher in women with serum 25(oh ) d levels below 37.5 conversely , vitamin d deficiency results in proximal muscle weakness and decreased lower extremity muscle function perhaps contributing to the risk for cesarean section . the cochrane library issued a review of vitamin d supplementation during pregnancy and identified 7 relevant studies . the cochrane review concluded that there is not enough evidence to evaluate the requirements and effects of vitamin d supplementation during pregnancy . data from three trials involving 463 women show a trend for women who receive vitamin d supplementation during pregnancy to more frequently have a baby with a birth weight below 2500 grams than those women receiving no treatment or placebo , although the statistical significance was borderline . animal models of vitamin d deficiency have shown just how important adequate nutritional intakes of vitamin d are to skeletal , cardiovascular , and neurologic development in experimental animals . weishaar and simpson showed that lengthy periods of vitamin d deficiency in rats are associated with profound changes in cardiovascular function , including increases in cardiac and vascular muscle contractile function . a recent study provides evidence with reference to the consequences of vitamin d deficiency on the neurodevelopment of the fetus during pregnancy in a rat model . a study on rodents showed that hypovitaminosis d trabecular bone loss , and concluded that vitamin d is indispensable for normal bone mineralization during the reproductive period in rats . research has found direct connections between vitamin d and the genes known to be involved in ms , but exact pathology and whether vitamin d supplements during pregnancy or childhood can lessen the likelihood of the child developing ms later in life is not known . while there is interest in the role of vitamin d in the prevention of multiple sclerosis , following epidemiological studies demonstrating an association between vitamin d supplementation and reduced prevalence of the disease , future research , including randomized controlled trials in pregnant or nonpregnant individuals , is awaited to confirm or refute such benefit . because the poor vitamin d stores of the mother may impair vitamin d state in the infant , it is important to know whether rickets can be prevented in breast fed infants by supplementation of the mother . the canadian pediatric society recommended 2000 iu of vitamin d3 for pregnant and lactating mothers with periodic blood tests to check levels of 25(oh ) d and calcium . the american academy of pediatrics recommendations focus on supplementing the infant and make no specific recommendations about universally supplementing breastfeeding mothers . a sufficient supply of vitamin d to the breast fed infant is achieved only by increasing the maternal supplementation up to 2000 iu / day . as such a dose is far higher than the daily dietary allowance recommended for lactating mothers its safety over prolonged periods is not known and should be examined . other suggests vitamin d supplementation of 400 iu / day to breast fed infants is the most secure way of preventing rickets in infants . fetal vitamin d concentrations are mainly dependent on maternal concentration , and maternal deficiency may lead to adverse outcomes in offspring . vitamin d - deficiency in mothers have significantly increased risk of infantile rickets due to inadequate maternal fetal transfer of 25-hydroxyvitamin d. recent retrospective studies found a significant and previously undetected association of maternal vitamin d deficiency with rickets - associated infant heart failure and with acute lower respiratory tract infection , a serious complication often associated with sepsis without clinical signs of rickets . while vitamin d supplementation in pregnancy has previously been associated with reduced risk of wheezing and type 1 diabetes . a few studies have observed that maternal vitamin d concentrations are related to offspring birth weight and growth during the postnatal years . lower maternal vitamin d status was associated with lower bone mineral concentration and impaired glucose homeostasis in newborn infants . maternal vitamin d deficiency also has been associated with craniotabes , a softening of skull bones that is one of the earliest signs of vitamin d deficiency , in a case study with neonatal seizures of a hypocalcemic infant and with impaired skeletal development in utero . interestingly , vitamin d deficiency during pregnancy is also associated with risks of health problems later in childhood , including improper bone development at 9 yrs of age , asthma , dental cavities , schizophrenia , and type i diabetes.[3739 ] the concept that maternal nutritional status influences the risk of chronic disorders in the offspring has attracted interest over the past 2 decades . however , very few studies have been in position to examine this association directly in animals . women of indian origin , especially pregnant women , are known to have a high prevalence of vitamin d deficiency . in indian women calcium intakes are low and the demands on calcium economy are high because of repeated cycles of pregnancy and lactation . a study in pregnant women in south india assessed maternal vitamin d status by measuring serum 25-hydroxyvitamin d in stored serum samples . more than 60% of the women of the women had low 25(oh ) d concentration ( < 50 nmol / l ) at 30-week gestation . although there was no association between maternal vitamin d status and offspring birth size . at present , vitamin d supplementation is not a part of antenatal care programs in india . women of reproductive age are assumed to be able to obtain the recommended intake for almost all vitamins without the use of supplements , and no national organization recommends routine vitamin d supplementation during pregnancy unless a woman is at nutritional risk . the us preventive services task force does not comment for or against routine screening for vitamin d deficiency in pregnant women . one approach is to consider serum testing in patients at high risk for vitamin d deficiency but treating without testing those at lower risk . the basis for these recommendations was made before it was possible to measure the circulating concentration of 25-hydroxyvitamin d [ 25(oh ) d ] , the indicator of nutritional vitamin d status . endocrine society issues practice guideline on vitamin d and the guideline recommend that clinicians screen for vitamin d deficiency in people at risk for deficiency , including obese individuals , blacks , pregnant and lactating women , and patients with malabsorption syndromes . if electing to test vitamin d status , serum 25-hydroxyvitamin d is the accepted biomarker to be offered early in pregnancy . although 1,25(oh ) 2d is the active circulating form of vitamin d , measuring this level is not helpful because it is quickly and tightly regulated by the kidney . true deficiency would be evident only by measuring 25(oh ) d. of note , questions have been raised regarding the need for standardization of assays . a large laboratory ( quest diagnostics ) recently reported the possibility of thousands of incorrect vitamin d level results . there is no consensus about the optimal 25(oh ) d level , but many experts accept a range 75nmol / l ( 30 ng / ml ) as optimal . controversy exists regarding the optimum concentration of serum 25-hydroxyvitamin d for defining vitamin d deficiency , especially in pregnancy . most experts agree that serum vitamin d levels below 50 nmol / l ( 20 ng / ml ) represent deficiency . however this current practice is based on the skeletal actions of the vitamin and may not be applicable for its non - classic actions . as was recently pointed out in a cochrane review , the reality is that the actual vitamin d requirement during pregnancy is not known . for that matter there is no dietary recommended intake ( dri ) for vitamin d. what is known today is that for a pregnant woman , the adequate intake for vitamin d is 200 iu per day . however this recommended level , which was largely arbitrarily set , seems to be less helpful to improve the nutritional vitamin d status of pregnant women . national osteoporosis foundation 's ( nof ) recommends 400 - 800iu vitamin d for pregnant women . a recent systematic review concluded that antenatal vitamin d supplementation is effective in improving the vitamin d status of asian and white women , improves growth in the first year of life in south asian babies and therefore may contribute to reducing the incidence of rickets in this latter group , without evidence of harm . current nice guidance states clearly that pregnant women are informed , at their first antenatal booking , of the importance of adequate vitamin d during pregnancy and after , to maintain their own and their baby 's health . these women are advised to take 10 micrograms per day in the form of a multivitamin supplement . it is crucial to ensure that at - risk women are aware of this need . those identified as at - risk include : women from black and ethnic minorities who are socially - excluded , women with limited exposure to sunlight , especially those who are housebound and obese women with prepregnancy bmi > 30 . current u.s . . however , research in recent years has been challenging those ideas on what is enough , and what is too much . the u.s . guidelines are currently under review . for now , though , 600 iu in prenatal vitamins remains the recommended daily intake for pregnant women . however , getting 25(oh ) d levels consistently above 75 nmol / l ( 30 ng / ml ) may require at least 1500 - 2000 iu / day of vitamin d. if a mother is vitamin d deficient , breast milk is not a good source of vitamin d , so infants need to be given vitamin d supplementation until they are weaned . also women are encouraged to continue to take vitamin d supplements after pregnancy to help protect against health problems such as osteoporosis . it was recently shown that a maternal supplementation of 2100 iu vitamin d / day was needed , when administered during the period of lactation , in order to observe an increase in serum levels of 25(oh ) d in the breast - fed infants comparable to that observed in children given 400 iu / day . the most important source of vitamin d is the skin 's synthesis of the vitamin from sunlight . use of sun blocks , increased coverage of clothing and time spent indoors increase the risk of vitamin d deficiency . it has been estimated that exposure to sunlight for usually no more than 5 - 15 min / d between 10 am and 3 pm , in the spring , summer , and fall at latitudes above and below 35 ( and all year near the equator ) to exposed parts of the body involving arms , legs and face provides the body with its required 1000 iu of cholecalciferol . staple foods like milk , breakfast cereals and margarines are artificially fortified with vitamin d. at present , the number and variety of vitamin d fortified foods available on the market differs significantly between countries and is attributed to the country - specific policies on food fortification which are not yet unified . vitamin d is present in a small number of foods , although , for an average person food will only supply about 10% of the amount needed . dietary sources of vitamin d include : fatty fish species , such as catfish , salmon , mackerel , tuna etc . , egg , beef and fish liver oils . although liver and cod liver oil contain vitamin d , they are not recommended in pregnancy as they also contain too much vitamin a. vitamin d is a unique nutrient because its requirement can be met by both endogenous production from sunlight as well as exogenous dietary sources , which complicates determining the body 's daily nutritional requirements . methods are currently available to quantify the contribution of endogenous vitamin d synthesis resulting from sun exposure , but serious limitations remain in accurately estimating dietary vitamin d intake because of the incompleteness of nutrient databases for both vitamin d - fortified food and vitamin d supplements . thus , increasing vitamin d intake from vitamin d fortified foods , and vitamin d supplements , in combination with sensible sun exposure , maximize a person 's vitamin d status to promote good health . women with darker skin fitzpatrick class 6 are more prone to vitamin d deficiency as they need four to ten times sunlight than fitzpatrick class 1 - 4 skinned people for adequate vitamin d synthesis . thus , daily vitamin requirement varies with ethnicity , and this is relevant in case of indian population . pre - pregnancy obesity is also associated with significant increases in the odds of maternal and neonatal vitamin d deficiency , independent of other factors such as ethnicity . this deficiency is because body fat stores much of the vitamin d made in the skin , making it less available to the body . certain medications like steroids , anti - epileptic medications , cholesterol - lowering drugs , and some diuretics reduce absorption of vitamin d from the intestines . women who have intestinal malabsorption diseases like celiac disease and crohn 's disease or partial removal or bypass of the stomach or intestines absorb less of both dietary and supplemental vitamin d are at increased risk of deficiency . there is controversy concerning levels of nutrient intake , and at times the concept that more is better emerges . it is said that serum 25-hydroxyvitamin d concentrations above 75 nmol / l ( 30 ng / ml ) are not consistently associated with increased benefit . for vitamin d , there are still underlying questions : how much is the daily requirement and how much is too much ? new research suggests that women who take high doses of vitamin d during pregnancy have a greatly reduced risk of complications , including gestational diabetes , preterm birth , and infection . this recommendation may be controversial because very high doses of vitamin d have long been believed to cause birth defects . w epidemiological analyses implicated high dietary vitamin d intake during pregnancy results in birth of syndromic babies . in the early 1960s , williams , et al . described a syndrome of supravalvular aortic stenosis , peripheral pulmonic stenosis , and body features indistinguishable from those in survivors of the syndrome of idiopathic hypercalcemia of infancy . friedman and colleagues developed a model for this disorder in rabbits by administering near - fatal amounts of ergocalciferol during pregnancy . based on these accumulated observations , the uncertain teratogenic potential of high doses of calciferol analogs or of the associated disturbances in mineral homeostasis continues to cause concern . various human studies are done so far involving pharmacological doses of vitamin d during pregnancy . a study in human subjects involved the administration of 100,000 iu vitamin d per day ( 2.5 mg / day ) throughout pregnancy to hypoparathyroid women to maintain serum calcium with no fatal outcome . goodenday , et al . reported that as hypoparathyroid patients have completed many pregnancies while receiving ergocalciferol , it is unlikely that material vitamin d , 25(oh ) d , or 24,25(oh ) 2d per se are teratogens . most prenatal vitamins have around 400 iu of vitamin d , and most health groups recommend taking no more than 2,000 iu of the vitamin in supplement form daily . eventually , as circulating 25(oh ) d increases to toxic concentrations , the classic situation of hypercalciuria , hypercalcemia , and , finally , extraskeletal calcification becomes evident . hypercalciuria due to excessive vitamin d intakes is always accompanied by circulating 25(oh ) d concentrations > 250nmol / l ( 100 ng / ml ) . to attain circulating 25(oh ) d concentrations that exceed 250 nmol / l ( 100 ng / ml ) , a daily vitamin d intake well in excess of 10,000 iu / d ( 250 g / d ) for several months would be required . however , hypervitaminosis d has never occurred when physiologic amounts of vitamin d are ingested . in addition , no case of hypervitaminosis d from sun exposure has ever been reported . this is supported by the recent finding from a randomized , double - blind , placebo - controlled trial examining the effects of a single annual megadose of vitamin d3 ( 500,000 iu , equivalent to approximately 1370 iu / d ) on fall and fracture outcomes in community - dwelling elderly women with a history of fall or fracture . adequate vitamin d intake is essential for maternal and fetal health during pregnancy , and prevention of adverse outcomes . recent work emphasizes the importance of non - classical roles of vitamin d in pregnancy and the placenta . vitamin d deficiency during pregnancy is associated with the non - classical actions of this hormone , being linked with preeclampsia , insulin resistance , gestational diabetes mellitus , bacterial vaginosis , and an increased risk for caesarean section delivery . women who have vitamin d deficiency do not usually feel any different but in some may have muscle weakness and weakened bones . pregnancy does not exacerbate hypocalcaemia and secondary hyperparathyroidism in people with pre - existing vitamin d deficiency . a new study finds that women who develop severe preeclampsia tend to have lower blood levels of vitamin d than healthy pregnant women raising the possibility that the vitamin plays a role in the complication . preeclampsia rates are elevated during winter months , when sunlight - dependent 25(oh ) d productions are reduced . preeclampsia is associated with low circulating levels of igf - i and 1,25(oh ) 2d and , in vitro , igf-1 increases 1,25(oh ) 2d production by primary human syncytiotrophoblasts from placentas from normal pregnancies but not from preeclamptic pregnancies . studies by other groups have reported abnormal expression of 1-hydroxylase , a vitamin d activating enzyme in preeclamptic pregnancies , revealing a potential role for 1,25(oh ) 2d3 as a regulator of placentation . induction of the 1-hydroxylase in early gestation might provide a mechanism by which environmental or dietary vitamin d can influence fetal - placental development . two clinical trials support a potential role of vitamin d in the prevention of preeclampsia , although neither of these treated with vitamin d supplements alone . in an uncontrolled trial , supplementation with a multivitamin / mineral supplement and halibut liver oil ( containing 900 iu / d vitamin d ) provided at 20 wk gestation reduced the odds of preeclampsia by 32% ( 95% ci , 11 - 47% ) . vitamin d supplementation in early pregnancy should be explored for preventing preeclampsia and promoting neonatal well - being . 1,25(oh ) 2d regulates insulin secretion by pancreatic -cells and thereby affects circulating glucose levels . as expected , low concentration of 25(oh ) d is a risk factor for insulin resistance , glucose intolerance , and features of metabolic syndrome in normoglycemic subjects . vitamin d deficiency during early pregnancy significantly increases the risk for gestational diabetes in later pregnancy . low 25(oh ) d levels are correlated with increased bacterial vaginosis in the first trimester . bacterial vaginosis is more prevalent in black women , who typically have lower serum 25(oh ) d concentrations and have a six - fold higher chance of vitamin d deficiency , compared with white women . vitamin d has effects on the immune system , cytokines , and antibacterial peptides that are likely to regulate the bacterial flora . nutritional vitamin d status has very recently been linked to the human innate immune system and its ability to contain mycobacterium tuberculosis . serum 25(oh ) d levels are inversely related to primary cesarean section in nulliparous women , an unexpected and unexplained maternal outcome recently identified . the risk was four - fold higher in women with serum 25(oh ) d levels below 37.5 nmol / l ( 15ng / ml ) controlling for multiple confounding factors . conversely , vitamin d deficiency results in proximal muscle weakness and decreased lower extremity muscle function perhaps contributing to the risk for cesarean section . the cochrane library issued a review of vitamin d supplementation during pregnancy and identified 7 relevant studies . the cochrane review concluded that there is not enough evidence to evaluate the requirements and effects of vitamin d supplementation during pregnancy . data from three trials involving 463 women show a trend for women who receive vitamin d supplementation during pregnancy to more frequently have a baby with a birth weight below 2500 grams than those women receiving no treatment or placebo , although the statistical significance was borderline . animal models of vitamin d deficiency have shown just how important adequate nutritional intakes of vitamin d are to skeletal , cardiovascular , and neurologic development in experimental animals . weishaar and simpson showed that lengthy periods of vitamin d deficiency in rats are associated with profound changes in cardiovascular function , including increases in cardiac and vascular muscle contractile function . a recent study provides evidence with reference to the consequences of vitamin d deficiency on the neurodevelopment of the fetus during pregnancy in a rat model . a study on rodents showed that hypovitaminosis d trabecular bone loss , and concluded that vitamin d is indispensable for normal bone mineralization during the reproductive period in rats . research has found direct connections between vitamin d and the genes known to be involved in ms , but exact pathology and whether vitamin d supplements during pregnancy or childhood can lessen the likelihood of the child developing ms later in life is not known . while there is interest in the role of vitamin d in the prevention of multiple sclerosis , following epidemiological studies demonstrating an association between vitamin d supplementation and reduced prevalence of the disease , future research , including randomized controlled trials in pregnant or nonpregnant individuals , is awaited to confirm or refute such benefit . because the poor vitamin d stores of the mother may impair vitamin d state in the infant , it is important to know whether rickets can be prevented in breast fed infants by supplementation of the mother . the canadian pediatric society recommended 2000 iu of vitamin d3 for pregnant and lactating mothers with periodic blood tests to check levels of 25(oh ) d and calcium . the american academy of pediatrics recommendations focus on supplementing the infant and make no specific recommendations about universally supplementing breastfeeding mothers . a sufficient supply of vitamin d to the breast fed infant is achieved only by increasing the maternal supplementation up to 2000 iu / day . as such a dose is far higher than the daily dietary allowance recommended for lactating mothers its safety over prolonged periods is not known and should be examined . other suggests vitamin d supplementation of 400 iu / day to breast fed infants is the most secure way of preventing rickets in infants . fetal vitamin d concentrations are mainly dependent on maternal concentration , and maternal deficiency may lead to adverse outcomes in offspring . vitamin d - deficiency in mothers have significantly increased risk of infantile rickets due to inadequate maternal fetal transfer of 25-hydroxyvitamin d. recent retrospective studies found a significant and previously undetected association of maternal vitamin d deficiency with rickets - associated infant heart failure and with acute lower respiratory tract infection , a serious complication often associated with sepsis without clinical signs of rickets . while vitamin d supplementation in pregnancy has previously been associated with reduced risk of wheezing and type 1 diabetes . a few studies have observed that maternal vitamin d concentrations are related to offspring birth weight and growth during the postnatal years . lower maternal vitamin d status was associated with lower bone mineral concentration and impaired glucose homeostasis in newborn infants . maternal vitamin d deficiency also has been associated with craniotabes , a softening of skull bones that is one of the earliest signs of vitamin d deficiency , in a case study with neonatal seizures of a hypocalcemic infant and with impaired skeletal development in utero . interestingly , vitamin d deficiency during pregnancy is also associated with risks of health problems later in childhood , including improper bone development at 9 yrs of age , asthma , dental cavities , schizophrenia , and type i diabetes.[3739 ] the concept that maternal nutritional status influences the risk of chronic disorders in the offspring has attracted interest over the past 2 decades . however , very few studies have been in position to examine this association directly in animals . women of indian origin , especially pregnant women , are known to have a high prevalence of vitamin d deficiency . in indian women calcium intakes are low and the demands on calcium economy are high because of repeated cycles of pregnancy and lactation . a study in pregnant women in south india assessed maternal vitamin d status by measuring serum 25-hydroxyvitamin d in stored serum samples . more than 60% of the women of the women had low 25(oh ) d concentration ( < 50 nmol / l ) at 30-week gestation . although there was no association between maternal vitamin d status and offspring birth size . at present , vitamin d supplementation is not a part of antenatal care programs in india . women of reproductive age are assumed to be able to obtain the recommended intake for almost all vitamins without the use of supplements , and no national organization recommends routine vitamin d supplementation during pregnancy unless a woman is at nutritional risk . the us preventive services task force does not comment for or against routine screening for vitamin d deficiency in pregnant women . one approach is to consider serum testing in patients at high risk for vitamin d deficiency but treating without testing those at lower risk . the basis for these recommendations was made before it was possible to measure the circulating concentration of 25-hydroxyvitamin d [ 25(oh ) d ] , the indicator of nutritional vitamin d status . endocrine society issues practice guideline on vitamin d and the guideline recommend that clinicians screen for vitamin d deficiency in people at risk for deficiency , including obese individuals , blacks , pregnant and lactating women , and patients with malabsorption syndromes . if electing to test vitamin d status , serum 25-hydroxyvitamin d is the accepted biomarker to be offered early in pregnancy . although 1,25(oh ) 2d is the active circulating form of vitamin d , measuring this level is not helpful because it is quickly and tightly regulated by the kidney . true deficiency would be evident only by measuring 25(oh ) d. of note , questions have been raised regarding the need for standardization of assays . a large laboratory ( quest diagnostics ) there is no consensus about the optimal 25(oh ) d level , but many experts accept a range 75nmol / l ( 30 ng / ml ) as optimal . controversy exists regarding the optimum concentration of serum 25-hydroxyvitamin d for defining vitamin d deficiency , especially in pregnancy . most experts agree that serum vitamin d levels below 50 nmol / l ( 20 ng / ml ) represent deficiency . however this current practice is based on the skeletal actions of the vitamin and may not be applicable for its non - classic actions . as was recently pointed out in a cochrane review , the reality is that the actual vitamin d requirement during pregnancy is not known . for that matter there is no dietary recommended intake ( dri ) for vitamin d. what is known today is that for a pregnant woman , the adequate intake for vitamin d is 200 iu per day . however this recommended level , which was largely arbitrarily set , seems to be less helpful to improve the nutritional vitamin d status of pregnant women . national osteoporosis foundation 's ( nof ) recommends 400 - 800iu vitamin d for pregnant women . a recent systematic review concluded that antenatal vitamin d supplementation is effective in improving the vitamin d status of asian and white women , improves growth in the first year of life in south asian babies and therefore may contribute to reducing the incidence of rickets in this latter group , without evidence of harm . current nice guidance states clearly that pregnant women are informed , at their first antenatal booking , of the importance of adequate vitamin d during pregnancy and after , to maintain their own and their baby 's health . these women are advised to take 10 micrograms per day in the form of a multivitamin supplement . it is crucial to ensure that at - risk women are aware of this need . those identified as at - risk include : women from black and ethnic minorities who are socially - excluded , women with limited exposure to sunlight , especially those who are housebound and obese women with prepregnancy bmi > 30 . current u.s . . however , research in recent years has been challenging those ideas on what is enough , and what is too much . the u.s . guidelines are currently under review . for now , though , 600 iu in prenatal vitamins remains the recommended daily intake for pregnant women . however , getting 25(oh ) d levels consistently above 75 nmol / l ( 30 ng / ml ) may require at least 1500 - 2000 iu / day of vitamin d. if a mother is vitamin d deficient , breast milk is not a good source of vitamin d , so infants need to be given vitamin d supplementation until they are weaned . also women are encouraged to continue to take vitamin d supplements after pregnancy to help protect against health problems such as osteoporosis . it was recently shown that a maternal supplementation of 2100 iu vitamin d / day was needed , when administered during the period of lactation , in order to observe an increase in serum levels of 25(oh ) d in the breast - fed infants comparable to that observed in children given 400 iu / day . vitamin d has emerged as something of a wonder supplement , according to the claims of dozens of studies published in the past few years . the current lack of evidence of benefit for women at lower risk of vitamin d deficiency points to the need for further research into vitamin d supplementation in pregnant women with clinical neonatal and infant end - points under scrutiny . there is a similar gap in the knowledge base for optimal dosing , as there is little empirical robust evidence to support 600 iu / day . further research is required , particularly to establish the dose needed to supplement pregnant women with pre - existing deficiency and the optimal gestation at which vitamin d supplementation should be started . recommendations should be made on informing women of the importance of maintaining adequate vitamin d stores in pregnancy , particularly for those at greatest risk of vitamin d deficiency . sensible sun exposure and education of the public about the beneficial effects of some limited sun exposure to satisfy their body 's vitamin d requirements should be implemented . future studies are essential to determine the true vitamin d requirement during pregnancy not only for maternal skeletal preservation and fetal skeletal formation , but also for fetal imprinting " that may affect neurodevelopment , immune function and chronic disease susceptibility soon after birth as well as later in life .

vitamin d deficiency is a preventable health problem . vitamin d deficiency among pregnant women is frequent in many populations over the world . research indicates that adequate vitamin d intake in pregnancy is optimal for maternal , fetal and child health . adverse health outcomes during pregnancy are preeclampsia ; gestational diabetes mellitus and caesarean section . consequences in newborns are low birth weight , neonatal rickets , a risk of neonatal hypocalcaemia , asthma and/or type 1 diabetes . vitamin d deficiency during pregnancy is the origin for a host of future perils for the child , especially effect on neurodevelopment and immune system . some of this damage done by maternal vitamin d deficiency gets evident after many years . therefore , prevention of vitamin d deficiency among pregnant women is essential . the currently recommended supplementation amount of vitamin d is not sufficient to maintain a value of 25 hydroxy vitamin d above 30 ng / ml , during pregnancy . studies are underway to establish the recommended daily doses of vitamin d in pregnant women . clearly , further investigation is required into the effects of vitamin d , of vitamin d supplementation , and of vitamin d analogs for improvement in human health generally and mothers and children specifically . this review discusses vitamin d metabolism , dietary requirements and recommendations and implications of vitamin d deficiency during pregnancy and lactation .

I V Sources of vitamin D Overdose Role of vitamin D in pregnant women Role of vitamin D in newborn and infant Testing and treatment for vitamin D deficiency C

the classical and non - classical pathways of this hormone affect calcium metabolism , the immune system , cell proliferation and differentiation , infection , and cancer . also the media has been taking increasing interest , and public expectations have been raised regarding the enhanced roles for vitamin d in pregnancy . the prevalence of vitamin d deficiency has been reported to range from 15% to 80% . modern day cosmetology and our urbanization has eclipsed the sunlight and it is impossible is to fulfill daily intake via diet as a large amount of vitamin d needs to be consumed . vitamin d deficiency is prevalent in india , a finding that is unexpected in a tropical country with abundant sunshine . the increasing prevalence of disorders linked to vitamin d deficiency is reflected in the several hundred children with rickets treated each year . in the last 3 years , an increasing amount of research suggests that some of the damage done by vitamin d deficiency is done in - utero , while the fetus is developing . the requirement for vitamin d in maintaining normal calcium metabolism throughout pregnancy and lactation in mothers , fetuses and newborn infants is still controversial . it is clear , however , that vitamin d requirements are increased in mothers during pregnancy and lactation . roles of vitamin d are to maintain skeletal calcium balance by promoting calcium absorption in the intestines , promoting bone resobption by increasing osteoclast number , maintaining calcium and phosphate levels for bone formation , and allowing proper functioning of parathyroid hormone to maintain serum calcium levels . vitamin d deficiency can result in lower bone mineral density and an increased risk of bone loss ( osteoporosis ) or bone fracture because a lack of vitamin d alters mineral metabolism in the body . the most important source of vitamin d is the skin 's synthesis of the vitamin from sunlight . use of sun blocks , increased coverage of clothing and time spent indoors increase the risk of vitamin d deficiency . , although liver and cod liver oil contain vitamin d , they are not recommended in pregnancy as they also contain too much vitamin a. vitamin d is a unique nutrient because its requirement can be met by both endogenous production from sunlight as well as exogenous dietary sources , which complicates determining the body 's daily nutritional requirements . thus , increasing vitamin d intake from vitamin d fortified foods , and vitamin d supplements , in combination with sensible sun exposure , maximize a person 's vitamin d status to promote good health . for vitamin d , there are still underlying questions : how much is the daily requirement and how much is too much ? new research suggests that women who take high doses of vitamin d during pregnancy have a greatly reduced risk of complications , including gestational diabetes , preterm birth , and infection . w epidemiological analyses implicated high dietary vitamin d intake during pregnancy results in birth of syndromic babies . various human studies are done so far involving pharmacological doses of vitamin d during pregnancy . most prenatal vitamins have around 400 iu of vitamin d , and most health groups recommend taking no more than 2,000 iu of the vitamin in supplement form daily . hypercalciuria due to excessive vitamin d intakes is always accompanied by circulating 25(oh ) d concentrations > 250nmol / l ( 100 ng / ml ) . to attain circulating 25(oh ) d concentrations that exceed 250 nmol / l ( 100 ng / ml ) , a daily vitamin d intake well in excess of 10,000 iu / d ( 250 g / d ) for several months would be required . this is supported by the recent finding from a randomized , double - blind , placebo - controlled trial examining the effects of a single annual megadose of vitamin d3 ( 500,000 iu , equivalent to approximately 1370 iu / d ) on fall and fracture outcomes in community - dwelling elderly women with a history of fall or fracture . adequate vitamin d intake is essential for maternal and fetal health during pregnancy , and prevention of adverse outcomes . recent work emphasizes the importance of non - classical roles of vitamin d in pregnancy and the placenta . vitamin d deficiency during pregnancy is associated with the non - classical actions of this hormone , being linked with preeclampsia , insulin resistance , gestational diabetes mellitus , bacterial vaginosis , and an increased risk for caesarean section delivery . a new study finds that women who develop severe preeclampsia tend to have lower blood levels of vitamin d than healthy pregnant women raising the possibility that the vitamin plays a role in the complication . two clinical trials support a potential role of vitamin d in the prevention of preeclampsia , although neither of these treated with vitamin d supplements alone . as expected , low concentration of 25(oh ) d is a risk factor for insulin resistance , glucose intolerance , and features of metabolic syndrome in normoglycemic subjects . vitamin d deficiency during early pregnancy significantly increases the risk for gestational diabetes in later pregnancy . bacterial vaginosis is more prevalent in black women , who typically have lower serum 25(oh ) d concentrations and have a six - fold higher chance of vitamin d deficiency , compared with white women . vitamin d has effects on the immune system , cytokines , and antibacterial peptides that are likely to regulate the bacterial flora . the cochrane library issued a review of vitamin d supplementation during pregnancy and identified 7 relevant studies . the cochrane review concluded that there is not enough evidence to evaluate the requirements and effects of vitamin d supplementation during pregnancy . data from three trials involving 463 women show a trend for women who receive vitamin d supplementation during pregnancy to more frequently have a baby with a birth weight below 2500 grams than those women receiving no treatment or placebo , although the statistical significance was borderline . animal models of vitamin d deficiency have shown just how important adequate nutritional intakes of vitamin d are to skeletal , cardiovascular , and neurologic development in experimental animals . weishaar and simpson showed that lengthy periods of vitamin d deficiency in rats are associated with profound changes in cardiovascular function , including increases in cardiac and vascular muscle contractile function . a recent study provides evidence with reference to the consequences of vitamin d deficiency on the neurodevelopment of the fetus during pregnancy in a rat model . a study on rodents showed that hypovitaminosis d trabecular bone loss , and concluded that vitamin d is indispensable for normal bone mineralization during the reproductive period in rats . research has found direct connections between vitamin d and the genes known to be involved in ms , but exact pathology and whether vitamin d supplements during pregnancy or childhood can lessen the likelihood of the child developing ms later in life is not known . while there is interest in the role of vitamin d in the prevention of multiple sclerosis , following epidemiological studies demonstrating an association between vitamin d supplementation and reduced prevalence of the disease , future research , including randomized controlled trials in pregnant or nonpregnant individuals , is awaited to confirm or refute such benefit . vitamin d - deficiency in mothers have significantly increased risk of infantile rickets due to inadequate maternal fetal transfer of 25-hydroxyvitamin d. recent retrospective studies found a significant and previously undetected association of maternal vitamin d deficiency with rickets - associated infant heart failure and with acute lower respiratory tract infection , a serious complication often associated with sepsis without clinical signs of rickets . while vitamin d supplementation in pregnancy has previously been associated with reduced risk of wheezing and type 1 diabetes . a few studies have observed that maternal vitamin d concentrations are related to offspring birth weight and growth during the postnatal years . maternal vitamin d deficiency also has been associated with craniotabes , a softening of skull bones that is one of the earliest signs of vitamin d deficiency , in a case study with neonatal seizures of a hypocalcemic infant and with impaired skeletal development in utero . interestingly , vitamin d deficiency during pregnancy is also associated with risks of health problems later in childhood , including improper bone development at 9 yrs of age , asthma , dental cavities , schizophrenia , and type i diabetes. women of indian origin , especially pregnant women , are known to have a high prevalence of vitamin d deficiency . in indian women calcium intakes are low and the demands on calcium economy are high because of repeated cycles of pregnancy and lactation . a study in pregnant women in south india assessed maternal vitamin d status by measuring serum 25-hydroxyvitamin d in stored serum samples . women of reproductive age are assumed to be able to obtain the recommended intake for almost all vitamins without the use of supplements , and no national organization recommends routine vitamin d supplementation during pregnancy unless a woman is at nutritional risk . the us preventive services task force does not comment for or against routine screening for vitamin d deficiency in pregnant women . endocrine society issues practice guideline on vitamin d and the guideline recommend that clinicians screen for vitamin d deficiency in people at risk for deficiency , including obese individuals , blacks , pregnant and lactating women , and patients with malabsorption syndromes . if electing to test vitamin d status , serum 25-hydroxyvitamin d is the accepted biomarker to be offered early in pregnancy . although 1,25(oh ) 2d is the active circulating form of vitamin d , measuring this level is not helpful because it is quickly and tightly regulated by the kidney . controversy exists regarding the optimum concentration of serum 25-hydroxyvitamin d for defining vitamin d deficiency , especially in pregnancy . most experts agree that serum vitamin d levels below 50 nmol / l ( 20 ng / ml ) represent deficiency . as was recently pointed out in a cochrane review , the reality is that the actual vitamin d requirement during pregnancy is not known . current nice guidance states clearly that pregnant women are informed , at their first antenatal booking , of the importance of adequate vitamin d during pregnancy and after , to maintain their own and their baby 's health . however , getting 25(oh ) d levels consistently above 75 nmol / l ( 30 ng / ml ) may require at least 1500 - 2000 iu / day of vitamin d. if a mother is vitamin d deficient , breast milk is not a good source of vitamin d , so infants need to be given vitamin d supplementation until they are weaned . the most important source of vitamin d is the skin 's synthesis of the vitamin from sunlight . use of sun blocks , increased coverage of clothing and time spent indoors increase the risk of vitamin d deficiency . although liver and cod liver oil contain vitamin d , they are not recommended in pregnancy as they also contain too much vitamin a. vitamin d is a unique nutrient because its requirement can be met by both endogenous production from sunlight as well as exogenous dietary sources , which complicates determining the body 's daily nutritional requirements . thus , increasing vitamin d intake from vitamin d fortified foods , and vitamin d supplements , in combination with sensible sun exposure , maximize a person 's vitamin d status to promote good health . women with darker skin fitzpatrick class 6 are more prone to vitamin d deficiency as they need four to ten times sunlight than fitzpatrick class 1 - 4 skinned people for adequate vitamin d synthesis . certain medications like steroids , anti - epileptic medications , cholesterol - lowering drugs , and some diuretics reduce absorption of vitamin d from the intestines . it is said that serum 25-hydroxyvitamin d concentrations above 75 nmol / l ( 30 ng / ml ) are not consistently associated with increased benefit . for vitamin d , there are still underlying questions : how much is the daily requirement and how much is too much ? new research suggests that women who take high doses of vitamin d during pregnancy have a greatly reduced risk of complications , including gestational diabetes , preterm birth , and infection . w epidemiological analyses implicated high dietary vitamin d intake during pregnancy results in birth of syndromic babies . various human studies are done so far involving pharmacological doses of vitamin d during pregnancy . a study in human subjects involved the administration of 100,000 iu vitamin d per day ( 2.5 mg / day ) throughout pregnancy to hypoparathyroid women to maintain serum calcium with no fatal outcome . most prenatal vitamins have around 400 iu of vitamin d , and most health groups recommend taking no more than 2,000 iu of the vitamin in supplement form daily . hypercalciuria due to excessive vitamin d intakes is always accompanied by circulating 25(oh ) d concentrations > 250nmol / l ( 100 ng / ml ) . to attain circulating 25(oh ) d concentrations that exceed 250 nmol / l ( 100 ng / ml ) , a daily vitamin d intake well in excess of 10,000 iu / d ( 250 g / d ) for several months would be required . this is supported by the recent finding from a randomized , double - blind , placebo - controlled trial examining the effects of a single annual megadose of vitamin d3 ( 500,000 iu , equivalent to approximately 1370 iu / d ) on fall and fracture outcomes in community - dwelling elderly women with a history of fall or fracture . adequate vitamin d intake is essential for maternal and fetal health during pregnancy , and prevention of adverse outcomes . recent work emphasizes the importance of non - classical roles of vitamin d in pregnancy and the placenta . vitamin d deficiency during pregnancy is associated with the non - classical actions of this hormone , being linked with preeclampsia , insulin resistance , gestational diabetes mellitus , bacterial vaginosis , and an increased risk for caesarean section delivery . two clinical trials support a potential role of vitamin d in the prevention of preeclampsia , although neither of these treated with vitamin d supplements alone . as expected , low concentration of 25(oh ) d is a risk factor for insulin resistance , glucose intolerance , and features of metabolic syndrome in normoglycemic subjects . vitamin d deficiency during early pregnancy significantly increases the risk for gestational diabetes in later pregnancy . bacterial vaginosis is more prevalent in black women , who typically have lower serum 25(oh ) d concentrations and have a six - fold higher chance of vitamin d deficiency , compared with white women . the cochrane library issued a review of vitamin d supplementation during pregnancy and identified 7 relevant studies . the cochrane review concluded that there is not enough evidence to evaluate the requirements and effects of vitamin d supplementation during pregnancy . data from three trials involving 463 women show a trend for women who receive vitamin d supplementation during pregnancy to more frequently have a baby with a birth weight below 2500 grams than those women receiving no treatment or placebo , although the statistical significance was borderline . animal models of vitamin d deficiency have shown just how important adequate nutritional intakes of vitamin d are to skeletal , cardiovascular , and neurologic development in experimental animals . weishaar and simpson showed that lengthy periods of vitamin d deficiency in rats are associated with profound changes in cardiovascular function , including increases in cardiac and vascular muscle contractile function . a recent study provides evidence with reference to the consequences of vitamin d deficiency on the neurodevelopment of the fetus during pregnancy in a rat model . a study on rodents showed that hypovitaminosis d trabecular bone loss , and concluded that vitamin d is indispensable for normal bone mineralization during the reproductive period in rats . research has found direct connections between vitamin d and the genes known to be involved in ms , but exact pathology and whether vitamin d supplements during pregnancy or childhood can lessen the likelihood of the child developing ms later in life is not known . while there is interest in the role of vitamin d in the prevention of multiple sclerosis , following epidemiological studies demonstrating an association between vitamin d supplementation and reduced prevalence of the disease , future research , including randomized controlled trials in pregnant or nonpregnant individuals , is awaited to confirm or refute such benefit . vitamin d - deficiency in mothers have significantly increased risk of infantile rickets due to inadequate maternal fetal transfer of 25-hydroxyvitamin d. recent retrospective studies found a significant and previously undetected association of maternal vitamin d deficiency with rickets - associated infant heart failure and with acute lower respiratory tract infection , a serious complication often associated with sepsis without clinical signs of rickets . while vitamin d supplementation in pregnancy has previously been associated with reduced risk of wheezing and type 1 diabetes . a few studies have observed that maternal vitamin d concentrations are related to offspring birth weight and growth during the postnatal years . maternal vitamin d deficiency also has been associated with craniotabes , a softening of skull bones that is one of the earliest signs of vitamin d deficiency , in a case study with neonatal seizures of a hypocalcemic infant and with impaired skeletal development in utero . interestingly , vitamin d deficiency during pregnancy is also associated with risks of health problems later in childhood , including improper bone development at 9 yrs of age , asthma , dental cavities , schizophrenia , and type i diabetes. women of indian origin , especially pregnant women , are known to have a high prevalence of vitamin d deficiency . in indian women calcium intakes are low and the demands on calcium economy are high because of repeated cycles of pregnancy and lactation . a study in pregnant women in south india assessed maternal vitamin d status by measuring serum 25-hydroxyvitamin d in stored serum samples . at present , vitamin d supplementation is not a part of antenatal care programs in india . women of reproductive age are assumed to be able to obtain the recommended intake for almost all vitamins without the use of supplements , and no national organization recommends routine vitamin d supplementation during pregnancy unless a woman is at nutritional risk . the us preventive services task force does not comment for or against routine screening for vitamin d deficiency in pregnant women . endocrine society issues practice guideline on vitamin d and the guideline recommend that clinicians screen for vitamin d deficiency in people at risk for deficiency , including obese individuals , blacks , pregnant and lactating women , and patients with malabsorption syndromes . if electing to test vitamin d status , serum 25-hydroxyvitamin d is the accepted biomarker to be offered early in pregnancy . although 1,25(oh ) 2d is the active circulating form of vitamin d , measuring this level is not helpful because it is quickly and tightly regulated by the kidney . controversy exists regarding the optimum concentration of serum 25-hydroxyvitamin d for defining vitamin d deficiency , especially in pregnancy . most experts agree that serum vitamin d levels below 50 nmol / l ( 20 ng / ml ) represent deficiency . as was recently pointed out in a cochrane review , the reality is that the actual vitamin d requirement during pregnancy is not known . for that matter there is no dietary recommended intake ( dri ) for vitamin d. what is known today is that for a pregnant woman , the adequate intake for vitamin d is 200 iu per day . current nice guidance states clearly that pregnant women are informed , at their first antenatal booking , of the importance of adequate vitamin d during pregnancy and after , to maintain their own and their baby 's health . for now , though , 600 iu in prenatal vitamins remains the recommended daily intake for pregnant women . however , getting 25(oh ) d levels consistently above 75 nmol / l ( 30 ng / ml ) may require at least 1500 - 2000 iu / day of vitamin d. if a mother is vitamin d deficient , breast milk is not a good source of vitamin d , so infants need to be given vitamin d supplementation until they are weaned . the current lack of evidence of benefit for women at lower risk of vitamin d deficiency points to the need for further research into vitamin d supplementation in pregnant women with clinical neonatal and infant end - points under scrutiny . further research is required , particularly to establish the dose needed to supplement pregnant women with pre - existing deficiency and the optimal gestation at which vitamin d supplementation should be started . recommendations should be made on informing women of the importance of maintaining adequate vitamin d stores in pregnancy , particularly for those at greatest risk of vitamin d deficiency . future studies are essential to determine the true vitamin d requirement during pregnancy not only for maternal skeletal preservation and fetal skeletal formation , but also for fetal imprinting " that may affect neurodevelopment , immune function and chronic disease susceptibility soon after birth as well as later in life .

we compared symptoms and laboratory results collected for children participating in the teddy study ( 17 ) diagnosed with type 1 diabetes between 1 january 2004 and 31 december 2010 with data from the population - based search study and population based registries ( swediabkids , finnish pediatric diabetes register , and german dpv register ) from all participating countries in teddy for the same incident years ( when available ) . in the teddy study , children with genetic risk of type 1 diabetes are followed intensively from 3 months of age . teddy is a multicenter study , with sites in sweden , finland , germany , and the u.s . all children are screened at birth for type 1 diabetes hla risk genotypes , and children at risk are followed . the aim of teddy is to determine environmental factors associated with the development of islet autoantibodies and type 1 diabetes in children with increased genetic risk of the disease . participating children are followed every third month from 3 months of age to measure height and weight , collect blood and stool samples , and obtain responses to a series of questionnaires . blood samples at each visit are analyzed for autoantibodies against gad , insulinoma associated protein 2 , and insulin . in antibody - positive children , additionally , autoantibody - positive children > 3 years of age undergo ogtts every 6 months . follow - up is intended to continue until subjects reach 15 years of age . at diagnosis of type 1 diabetes , data are collected from the treating physician using a standardized case report form requiring documentation that american diabetes association criteria for the diagnosis have been met ( 18 ) . data collection includes information on symptoms and parameters of metabolic decompensation , such as ph , urine ketones , blood ketones ( betahydroxybutyrate ) , and electrolytes . because routine care for children with type 1 diabetes varies considerably from site to site , the diagnosis form includes a free text area allowing study staff to extract information about the child s clinical status . this is critical for determining likely dka status in children where no laboratory evaluation was performed . in some countries ( i.e. , finland and sweden ) , nearly all children diagnosed with type 1 diabetes have blood obtained for assessment of acid - base balance and are admitted to a hospital for inpatient care . in other countries , children without clinical signs of dka and mild symptoms are preferentially treated as outpatients , and blood for evaluation of acid - base balance is only taken if the pediatrician suspects dka or if the child is critically ill . for comparative analyses of dka at onset , we obtained data from national incidence studies being performed in the same countries as the teddy study ( for the european registers during 20042009 and for the u.s . multicenter study aimed at identifying prevalent and incident cases of diabetes among individuals < 20 years of age at diagnosis . for these analyses , we included children diagnosed with type 1 diabetes in 2004 and 2005 . dka was defined in the search protocol using medical abstraction to identify bicarbonate < 15 mmol / l or venous ph < 7.25 ( arterial / capillary ph < 7.30 ) , international classification of diseases , ninth revision , code 250.1 , or physician s diagnosis of dka . our analysis included 275 search subjects diagnosed with type 1 diabetes when < 5 years of age . swediabkids is the swedish pediatric national quality registry for type 1 diabetes , covering 100% of all children diagnosed in sweden . the incidence register is part of this national online follow - up quality register for pediatric patients , where all pediatric diabetes clinic visits are recorded . in this report , we include data from 651 swedish children diagnosed with type 1 diabetes between 2005 and 2009 and < 5 years of age at diagnosis . of those , 44 children were excluded from this analysis due to missing ph or bicarbonate values . dka was defined as arterial ph < 7.30 and severe if arterial ph was < 7.10 . the finnish pediatric diabetes register was established in 2002 and collects data on all children in finland diagnosed with type 1 diabetes . diabetes was diagnosed according to world health organization criteria , and dka was diagnosed as arterial ph < 7.30 and considered severe if arterial ph was < 7.10 . in children without arterial ph or bicarbonate data , negative urine ketones , negative blood ketones , or lack of symptoms ( polyuria , polydipsia , and polyphagia ) were used to exclude dka . our analysis includes 737 children aged < 5 years from the finnish diabetes register diagnosed with type 1 diabetes from 2005 to 2009 . the german / austrian diabetes documentation and quality management system ( dpv ) is a continuous diabetes data acquisition system providing data from 53,485 type 1 diabetes patients throughout germany and austria . dka is defined in the dpv as arterial ph < 7.30 and severe dka as arterial ph < 7.10 . in patients without biochemical data , chart abstraction and physician attestation of the patient s condition at diagnosis our analysis includes 1,812 dpv children from the dpv diagnosed with diabetes from 2005 to 2009 . because of differences in data collection and management of newly diagnosed patients within teddy , search , and the population - based registries , we used two definitions of dka to ensure appropriate comparisons ( table 2 ) . when comparing teddy data to the swedish register , strict criteria for dka were used and included arterial ph < 7.30 ( venous ph < 7.25 ) or standardized bicarbonate < 15 mmol / l . severe dka was defined as arterial ph < 7.10 or standardized bicarbonate < 5 mmol / l . for comparisons with search , german - dpv , and finnish registers , broad criteria for dka were used to include the strict criteria and , in cases where ph or bicarbonate values were missing , betahydroxybutyrate > 3.0 mmol / l , urine ketones > 40 mg / dl , or physician s diagnosis . data were analyzed using the statistical analysis system software ( version 9.2 ; sas institute , cary , nc ) . prevalence estimates and 95% confidence intervals were calculated as the proportion presenting with dka at the time of diagnosis by age - group ( < 2 and < 5 years ) based on appropriate definition ( strict or broad ) . rates were compared using statistics or fisher exact test ( for small samples , less than or equal to five per cell ) to assess overall differences in prevalence estimates by study . we compared symptoms and laboratory results collected for children participating in the teddy study ( 17 ) diagnosed with type 1 diabetes between 1 january 2004 and 31 december 2010 with data from the population - based search study and population based registries ( swediabkids , finnish pediatric diabetes register , and german dpv register ) from all participating countries in teddy for the same incident years ( when available ) . in the teddy study , children with genetic risk of type 1 diabetes are followed intensively from 3 months of age . teddy is a multicenter study , with sites in sweden , finland , germany , and the u.s . all children are screened at birth for type 1 diabetes hla risk genotypes , and children at risk are followed . the aim of teddy is to determine environmental factors associated with the development of islet autoantibodies and type 1 diabetes in children with increased genetic risk of the disease . participating children are followed every third month from 3 months of age to measure height and weight , collect blood and stool samples , and obtain responses to a series of questionnaires . blood samples at each visit are analyzed for autoantibodies against gad , insulinoma associated protein 2 , and insulin . in antibody - positive children , additionally , autoantibody - positive children > 3 years of age undergo ogtts every 6 months . at diagnosis of type 1 diabetes , data are collected from the treating physician using a standardized case report form requiring documentation that american diabetes association criteria for the diagnosis have been met ( 18 ) . data collection includes information on symptoms and parameters of metabolic decompensation , such as ph , urine ketones , blood ketones ( betahydroxybutyrate ) , and electrolytes . because routine care for children with type 1 diabetes varies considerably from site to site , the diagnosis form includes a free text area allowing study staff to extract information about the child s clinical status . this is critical for determining likely dka status in children where no laboratory evaluation was performed . in some countries ( i.e. , finland and sweden ) , nearly all children diagnosed with type 1 diabetes have blood obtained for assessment of acid - base balance and are admitted to a hospital for inpatient care . in other countries , children without clinical signs of dka and mild symptoms are preferentially treated as outpatients , and blood for evaluation of acid - base balance is only taken if the pediatrician suspects dka or if the child is critically ill . for comparative analyses of dka at onset , we obtained data from national incidence studies being performed in the same countries as the teddy study ( for the european registers during 20042009 and for the u.s . study during 20042005 ) . multicenter study aimed at identifying prevalent and incident cases of diabetes among individuals < 20 years of age at diagnosis . for these analyses , we included children diagnosed with type 1 diabetes in 2004 and 2005 . dka was defined in the search protocol using medical abstraction to identify bicarbonate < 15 mmol / l or venous ph < 7.25 ( arterial / capillary ph < 7.30 ) , international classification of diseases , ninth revision , code 250.1 , or physician s diagnosis of dka . our analysis included 275 search subjects diagnosed with type 1 diabetes when < 5 years of age . swediabkids is the swedish pediatric national quality registry for type 1 diabetes , covering 100% of all children diagnosed in sweden . the incidence register is part of this national online follow - up quality register for pediatric patients , where all pediatric diabetes clinic visits are recorded . in this report , we include data from 651 swedish children diagnosed with type 1 diabetes between 2005 and 2009 and < 5 years of age at diagnosis . of those , 44 children were excluded from this analysis due to missing ph or bicarbonate values . dka was defined as arterial ph < 7.30 and severe if arterial ph was < 7.10 . the finnish pediatric diabetes register was established in 2002 and collects data on all children in finland diagnosed with type 1 diabetes . diabetes was diagnosed according to world health organization criteria , and dka was diagnosed as arterial ph < 7.30 and considered severe if arterial ph was < 7.10 . in children without arterial ph or bicarbonate data , negative urine ketones , negative blood ketones , or lack of symptoms ( polyuria , polydipsia , and polyphagia ) were used to exclude dka . our analysis includes 737 children aged < 5 years from the finnish diabetes register diagnosed with type 1 diabetes from 2005 to 2009 . the german / austrian diabetes documentation and quality management system ( dpv ) is a continuous diabetes data acquisition system providing data from 53,485 type 1 diabetes patients throughout germany and austria . dka is defined in the dpv as arterial ph < 7.30 and severe dka as arterial ph < 7.10 . in patients without biochemical data , chart abstraction and physician attestation of the patient s condition at diagnosis is considered sufficient to rule out dka . our analysis includes 1,812 dpv children from the dpv diagnosed with diabetes from 2005 to 2009 . multicenter study aimed at identifying prevalent and incident cases of diabetes among individuals < 20 years of age at diagnosis . for these analyses , we included children diagnosed with type 1 diabetes in 2004 and 2005 . dka was defined in the search protocol using medical abstraction to identify bicarbonate < 15 mmol / l or venous ph < 7.25 ( arterial / capillary ph < 7.30 ) , international classification of diseases , ninth revision , code 250.1 , or physician s diagnosis of dka . our analysis included 275 search subjects diagnosed with type 1 diabetes when < 5 years of age . swediabkids is the swedish pediatric national quality registry for type 1 diabetes , covering 100% of all children diagnosed in sweden . the incidence register is part of this national online follow - up quality register for pediatric patients , where all pediatric diabetes clinic visits are recorded . in this report , we include data from 651 swedish children diagnosed with type 1 diabetes between 2005 and 2009 and < 5 years of age at diagnosis . of those , 44 children were excluded from this analysis due to missing ph or bicarbonate values . dka was defined as arterial ph < 7.30 and severe if arterial ph was < 7.10 . the finnish pediatric diabetes register was established in 2002 and collects data on all children in finland diagnosed with type 1 diabetes . diabetes was diagnosed according to world health organization criteria , and dka was diagnosed as arterial ph < 7.30 and considered severe if arterial ph was < 7.10 . in children without arterial ph or bicarbonate data , negative urine ketones , negative blood ketones , or lack of symptoms ( polyuria , polydipsia , and polyphagia ) were used to exclude dka . our analysis includes 737 children aged < 5 years from the finnish diabetes register diagnosed with type 1 diabetes from 2005 to 2009 . the german / austrian diabetes documentation and quality management system ( dpv ) is a continuous diabetes data acquisition system providing data from 53,485 type 1 diabetes patients throughout germany and austria . dka is defined in the dpv as arterial ph < 7.30 and severe dka as arterial ph < 7.10 . in patients without biochemical data , chart abstraction and physician attestation of the patient s condition at diagnosis is considered sufficient to rule out dka . our analysis includes 1,812 dpv children from the dpv diagnosed with diabetes from 2005 to 2009 . because of differences in data collection and management of newly diagnosed patients within teddy , search , and the population - based registries , we used two definitions of dka to ensure appropriate comparisons ( table 2 ) . when comparing teddy data to the swedish register , strict criteria for dka were used and included arterial ph < 7.30 ( venous ph < 7.25 ) or standardized bicarbonate < 15 mmol / l . severe dka was defined as arterial ph < 7.10 or standardized bicarbonate < 5 mmol / l . for comparisons with search , german - dpv , and finnish registers , broad criteria for dka were used to include the strict criteria and , in cases where ph or bicarbonate values were missing , betahydroxybutyrate > 3.0 mmol / l , urine ketones > 40 mg / dl , or physician s diagnosis . data were analyzed using the statistical analysis system software ( version 9.2 ; sas institute , cary , nc ) . prevalence estimates and 95% confidence intervals were calculated as the proportion presenting with dka at the time of diagnosis by age - group ( < 2 and < 5 years ) based on appropriate definition ( strict or broad ) . rates were compared using statistics or fisher exact test ( for small samples , less than or equal to five per cell ) to assess overall differences in prevalence estimates by study . teddy screened 424,788 children for type 1 diabetes hla risk , identified 21,588 eligible subjects , and enrolled 8,677 children . as of 31 december 2010 , 80 teddy subjects were diagnosed with type 1 diabetes ( supplementary fig . 40 children were < 2 years of age and 79 were < 5 years of age at diagnosis . the mean ( range ) hba1c ( using national glycohemoglobin standardization program standards ) at diagnosis was 7.8% ( 5.013.3 ) . forty - three of the newly diagnosed children were males , and the median ( range ) age in months was 23.8 ( 855 ) . despite the relatively young age of the new onset cohort , 24 of 79 ( 30% ) were asymptomatic at diagnosis . of the asymptomatic children , 16 were from the general population ( representing 29% of the newly diagnosed general population subjects ) whereas 8 were from first degree relative families ( representing 33% of newly diagnosed first - degree relatives ) . in children < 2 years of age at diagnosis , 6 of 40 ( 15% ) had dka under the broad definition of dka whereas 5 of 31 ( 16.1% ) had dka under the strict definition of dka . teddy children diagnosed with type 1 diabetes before 2 years of age had lower rates of mild and total dka when compared with population - based studies and registries ( search , p < 0.0001 ; german dpv register , p < 0.0001 ; swediabkids , p = 0.02 ; finnish register , p = 0.0005 ) ( table 3 ) . dka rates in children aged < 5 years and < 2 years at diagnosis na , data not available . * * n cases / sample population denominator , prevalence ( 95% confidence intervals ) . * < 5 years of age at diagnosis , 9 of 79 ( 11.3% ) had dka at diagnosis of diabetes under the broad definition of dka . using the strict definition of dka and excluding those with insufficient data , 8 of 61 ( 13.1% ) < 5 years of age had dka at diagnosis of diabetes . total dka ( mild and severe dka ) at diagnosis was significantly less common in teddy participants ( 11.3% ) when compared with search ( 36.4% ) ( p < 0.0001 ) and german dpv ( 25.3% ) ( p 0.0001 ) children , respectively , but were not statistically different from dka at diagnosis for patients from the swedish ( 16.9% ) ( p = 0.45 ) or finnish ( 18.7% ) ( p = 0.11 ) registers . although numbers were small , the rate of severe dka ( arterial ph < 7.10 ) did not differ significantly between children diagnosed within the teddy study and those reported from national registries in any age - group . notably , all three teddy children diagnosed with severe dka were < 2 years of age ( 8 , 10 , and 14 months , respectively ) and had developed islet autoantibodies before clinical presentation of disease . in children < 2 years of age at diagnosis , 6 of 40 ( 15% ) had dka under the broad definition of dka whereas 5 of 31 ( 16.1% ) had dka under the strict definition of dka . teddy children diagnosed with type 1 diabetes before 2 years of age had lower rates of mild and total dka when compared with population - based studies and registries ( search , p < 0.0001 ; german dpv register , p < 0.0001 ; swediabkids , p = 0.02 ; finnish register , p = 0.0005 ) ( table 3 ) . dka rates in children aged < 5 years and < 2 years at diagnosis na , data not available . * * n cases / sample population denominator , prevalence ( 95% confidence intervals ) . * all comparisons made to teddy strict definition . in children < 5 years of age at diagnosis , 9 of 79 ( 11.3% ) had dka at diagnosis of diabetes under the broad definition of dka . using the strict definition of dka and excluding those with insufficient data , 8 of 61 ( 13.1% ) < 5 years of age had dka at diagnosis of diabetes . total dka ( mild and severe dka ) at diagnosis was significantly less common in teddy participants ( 11.3% ) when compared with search ( 36.4% ) ( p < 0.0001 ) and german dpv ( 25.3% ) ( p 0.0001 ) children , respectively , but were not statistically different from dka at diagnosis for patients from the swedish ( 16.9% ) ( p = 0.45 ) or finnish ( 18.7% ) ( p = 0.11 ) registers . although numbers were small , the rate of severe dka ( arterial ph < 7.10 ) did not differ significantly between children diagnosed within the teddy study and those reported from national registries in any age - group . notably , all three teddy children diagnosed with severe dka were < 2 years of age ( 8 , 10 , and 14 months , respectively ) and had developed islet autoantibodies before clinical presentation of disease . in this study we compared the prevalence of dka in children participating in teddy and diagnosed with type 1 diabetes before the age of 5 years with national population - based registers . our analyses demonstrate that teddy subjects diagnosed with type 1 diabetes before the age of 2 years experienced significantly less dka at diagnosis ( 15% ) when compared with new - onset patients reported in national diabetes registers from countries participating in teddy and the search study ( 3550% ) . similarly , teddy children diagnosed with type 1 diabetes before the age of 5 years experienced less dka at onset when compared with children in the search study and the german dpv registry , but not compared with children from the finnish or swedish registries . although the relatively high proportion of teddy subjects who were first - degree relatives of type 1 diabetes patients may have confounded our analysis , dka rates within the teddy cohort were similar among children recruited from the general population and those with a first - degree relative with type 1 diabetes , indicating that knowledge of diabetes risk and close longitudinal follow - up may be associated with reduced dka risk regardless of family history . although our analysis is strengthened by the inclusion of data from a national incidence study and registries performed in each of the countries participating in teddy , we should note that children with incident diabetes from colorado and washington could have simultaneously been included in both the search and teddy databases , and the teddy participants in sweden , finland , and germany could have been included in the national registries . while such an occurrence could have biased our data , the effect ( given lower rates of dka noted in teddy ) would have been to mask the differences observed . as we were still able to document significant reductions in dka when comparing teddy to search and the national registries , the effect of any potential bias was likely minimal . unfortunately , we were unable to perform an analysis where participants in both teddy and a population - based study or registry were excluded . perhaps the most challenging issue related to inclusion of data from multiple national incidence registries was that of interpreting data with an appreciation of the different definitions of dka used in each study ( table 2 ) . the search study used a composite definition of dka that includes standard cutoffs for ph and bicarbonate but also allows for nonbiochemical clinical documentation to account for dka status . the finnish and dpv registries used standard biochemical data , but excluded dka in children with negative urine ketones , negative blood ketones , or lack of symptoms . for the purposes of this analysis , teddy defined dka using both strict criteria ( standard cutoffs for ph and bicarbonate ) and broad criteria ( allowing us to document dka status on the basis of urine / blood ketones and clinician documentation ) . when comparing overall dka rates in teddy participants with children reported in the swedish and finnish registry , no significant differences were seen in children diagnosed before 5 years of age . however , rates of dka in teddy participants aged < 5 years were significantly lower when compared with children from the u.s . these observations are concordant with reports that countries with a high incidence of type 1 diabetes ( finland and sweden ) report reduced frequencies of dka when compared with countries with lower incidence of type 1 diabetes . decreased frequencies of dka at onset of disease have been reported from finland between 1982 and 2001 , with a decrease from 22.4 to 15.2% in children 015 years of age and a decrease from 32.1 to 17.7% in children with onset before the age of 5 years ( 6 ) . that said , the rate of dka in children < 2 years of age at onset remained unacceptably high ( 39.1% ) ( 6 ) . on the contrary , german data from the dpv register did not reveal reduced frequency of dka in any age - group between 1995 and 2007 ( 8) . in a swedish study , the incidence of dka at onset of type 1 diabetes in children diagnosed from 2000 to 2004 was 16% , with no difference reported between age - groups . however , children < 2 years of age were not specifically analyzed ( 15 ) . the youngest children with new onset type 1 diabetes ( < 2 years of age ) remain at high risk of dka ( table 1 ) ( 6 ) . more specifically , these young children are prone to severe dka at onset . of the children in teddy diagnosed with severe dka ( ph < 7.10 ) , all three were < 2 years of age ( 8 , 10 , and 14 months , respectively ) . although all three were antibody positive prior to diagnosis , we should note that because of the lag time from antibody testing to reporting of results , the parents of these children were likely not informed of the antibody status prior to the diagnosis . as such , rapid analysis and reporting of antibody status in high - risk children could further aid in reducing dka rates . unfortunately , our analysis lacked the power to determine if participation in teddy reduced the risk of severe dka in children < 2 years of age . that said , the observation of reduction in overall dka in the youngest patients with new - onset diabetes suggests that participation in a longitudinal natural history study with provision of updated autoantibody risk information , frequent follow - up , and scheduled laboratory evaluations ( autoantibodies , hba1c , glucose , and ogtt ) to diagnose asymptomatic cases is associated with reduced dka risk . although potentially cost prohibitive outside the constraints of a research protocol , combined approaches to educate communities on signs and symptoms of dka ( as in the parma , italy experiments ) and to provide genetic or antibody screening to further identify high - risk subjects may eventually be required to achieve the goal of entirely preventing dka in children . dka is a serious condition with high morbidity and risk of developing cerebral edema ( for a review see levin ) . an early diagnosis of diabetes would therefore be directly beneficial to the child and indirectly beneficial to society through reduction of morbidity , mortality , and cost at onset of disease . additionally , early diagnosis and aggressive management of diabetes may be beneficial in allowing for the presence of a larger functioning -cell mass , easier metabolic control , and possible reductions in long - term complication risk ( 12,20 ) . children diagnosed in an early stage of disease may also be eligible to participate in intervention trials aimed at protecting remaining -cell mass . in conclusion , intensive longitudinal follow - up and continuous education regarding diabetes risk , as provided in diabetes prediction studies such as teddy , may yield direct benefit to young children diagnosed with type 1 diabetes through early diagnosis and reduction of dka risk . ongoing efforts to reduce and eliminate dka at diagnosis in young children are urgently needed .

objectiveyoung children have an unacceptably high prevalence of diabetic ketoacidosis ( dka ) at the clinical diagnosis of type 1 diabetes . the aim of this study was to determine whether knowledge of genetic risk and close follow - up for development of islet autoantibodies through participation in the environmental determinants of diabetes in the young ( teddy ) study results in lower prevalence of dka at diabetes onset in children aged < 2 and < 5 years compared with population - based incidence studies and registries.research design and methodssymptoms and laboratory data collected on teddy participants diagnosed with type 1 diabetes between 2004 and 2010 were compared with data collected during the similar periods from studies and registries in all teddy - participating countries ( u.s . , search for diabetes in youth study ; sweden , swediabkids ; finland , finnish pediatric diabetes register ; and germany , diabetes patienten verlaufsdokumenation [ dpv ] register).resultsa total of 40 children younger than age 2 years and 79 children younger than age 5 years were diagnosed with type 1 diabetes in teddy as of december 2010 . in children < 2 years of age at onset , dka prevalence in teddy participants was significantly lower than in all comparative registries ( german dpv register , p < 0.0001 ; swediabkids , p = 0.02 ; search , p < 0.0001 ; finnish register , p < 0.0001 ) . the prevalence of dka in teddy children diagnosed at < 5 years of age ( 13.1% ) was significantly lower compared with search ( 36.4% ) ( p < 0.0001 ) and the german dpv register ( 32.2% ) ( p < 0.0001 ) but not compared with swediabkids or the finnish register.conclusionsparticipation in the teddy study is associated with reduced risk of dka at diagnosis of type 1 diabetes in young children .

RESEARCH DESIGN AND METHODS Participants Collection of data at diagnosis of type 1 diabetes National registries for comparison SEARCH for Diabetes in Youth (SEARCH). Swediabkids. Finnish Diabetes Register. DPV. Definition of DKA Statistical analysis RESULTS DKA in children <2 years of age DKA in children <5 years of age Severe DKA CONCLUSIONS Supplementary Material

we compared symptoms and laboratory results collected for children participating in the teddy study ( 17 ) diagnosed with type 1 diabetes between 1 january 2004 and 31 december 2010 with data from the population - based search study and population based registries ( swediabkids , finnish pediatric diabetes register , and german dpv register ) from all participating countries in teddy for the same incident years ( when available ) . in the teddy study , children with genetic risk of type 1 diabetes are followed intensively from 3 months of age . teddy is a multicenter study , with sites in sweden , finland , germany , and the u.s . all children are screened at birth for type 1 diabetes hla risk genotypes , and children at risk are followed . the aim of teddy is to determine environmental factors associated with the development of islet autoantibodies and type 1 diabetes in children with increased genetic risk of the disease . in antibody - positive children , additionally , autoantibody - positive children > 3 years of age undergo ogtts every 6 months . follow - up is intended to continue until subjects reach 15 years of age . at diagnosis of type 1 diabetes , data are collected from the treating physician using a standardized case report form requiring documentation that american diabetes association criteria for the diagnosis have been met ( 18 ) . because routine care for children with type 1 diabetes varies considerably from site to site , the diagnosis form includes a free text area allowing study staff to extract information about the child s clinical status . , finland and sweden ) , nearly all children diagnosed with type 1 diabetes have blood obtained for assessment of acid - base balance and are admitted to a hospital for inpatient care . for comparative analyses of dka at onset , we obtained data from national incidence studies being performed in the same countries as the teddy study ( for the european registers during 20042009 and for the u.s . multicenter study aimed at identifying prevalent and incident cases of diabetes among individuals < 20 years of age at diagnosis . for these analyses , we included children diagnosed with type 1 diabetes in 2004 and 2005 . dka was defined in the search protocol using medical abstraction to identify bicarbonate < 15 mmol / l or venous ph < 7.25 ( arterial / capillary ph < 7.30 ) , international classification of diseases , ninth revision , code 250.1 , or physician s diagnosis of dka . our analysis included 275 search subjects diagnosed with type 1 diabetes when < 5 years of age . swediabkids is the swedish pediatric national quality registry for type 1 diabetes , covering 100% of all children diagnosed in sweden . the incidence register is part of this national online follow - up quality register for pediatric patients , where all pediatric diabetes clinic visits are recorded . in this report , we include data from 651 swedish children diagnosed with type 1 diabetes between 2005 and 2009 and < 5 years of age at diagnosis . the finnish pediatric diabetes register was established in 2002 and collects data on all children in finland diagnosed with type 1 diabetes . our analysis includes 737 children aged < 5 years from the finnish diabetes register diagnosed with type 1 diabetes from 2005 to 2009 . the german / austrian diabetes documentation and quality management system ( dpv ) is a continuous diabetes data acquisition system providing data from 53,485 type 1 diabetes patients throughout germany and austria . in patients without biochemical data , chart abstraction and physician attestation of the patient s condition at diagnosis our analysis includes 1,812 dpv children from the dpv diagnosed with diabetes from 2005 to 2009 . because of differences in data collection and management of newly diagnosed patients within teddy , search , and the population - based registries , we used two definitions of dka to ensure appropriate comparisons ( table 2 ) . for comparisons with search , german - dpv , and finnish registers , broad criteria for dka were used to include the strict criteria and , in cases where ph or bicarbonate values were missing , betahydroxybutyrate > 3.0 mmol / l , urine ketones > 40 mg / dl , or physician s diagnosis . prevalence estimates and 95% confidence intervals were calculated as the proportion presenting with dka at the time of diagnosis by age - group ( < 2 and < 5 years ) based on appropriate definition ( strict or broad ) . we compared symptoms and laboratory results collected for children participating in the teddy study ( 17 ) diagnosed with type 1 diabetes between 1 january 2004 and 31 december 2010 with data from the population - based search study and population based registries ( swediabkids , finnish pediatric diabetes register , and german dpv register ) from all participating countries in teddy for the same incident years ( when available ) . in the teddy study , children with genetic risk of type 1 diabetes are followed intensively from 3 months of age . teddy is a multicenter study , with sites in sweden , finland , germany , and the u.s . all children are screened at birth for type 1 diabetes hla risk genotypes , and children at risk are followed . the aim of teddy is to determine environmental factors associated with the development of islet autoantibodies and type 1 diabetes in children with increased genetic risk of the disease . in antibody - positive children , additionally , autoantibody - positive children > 3 years of age undergo ogtts every 6 months . at diagnosis of type 1 diabetes , data are collected from the treating physician using a standardized case report form requiring documentation that american diabetes association criteria for the diagnosis have been met ( 18 ) . because routine care for children with type 1 diabetes varies considerably from site to site , the diagnosis form includes a free text area allowing study staff to extract information about the child s clinical status . , finland and sweden ) , nearly all children diagnosed with type 1 diabetes have blood obtained for assessment of acid - base balance and are admitted to a hospital for inpatient care . for comparative analyses of dka at onset , we obtained data from national incidence studies being performed in the same countries as the teddy study ( for the european registers during 20042009 and for the u.s . multicenter study aimed at identifying prevalent and incident cases of diabetes among individuals < 20 years of age at diagnosis . for these analyses , we included children diagnosed with type 1 diabetes in 2004 and 2005 . dka was defined in the search protocol using medical abstraction to identify bicarbonate < 15 mmol / l or venous ph < 7.25 ( arterial / capillary ph < 7.30 ) , international classification of diseases , ninth revision , code 250.1 , or physician s diagnosis of dka . our analysis included 275 search subjects diagnosed with type 1 diabetes when < 5 years of age . swediabkids is the swedish pediatric national quality registry for type 1 diabetes , covering 100% of all children diagnosed in sweden . the incidence register is part of this national online follow - up quality register for pediatric patients , where all pediatric diabetes clinic visits are recorded . in this report , we include data from 651 swedish children diagnosed with type 1 diabetes between 2005 and 2009 and < 5 years of age at diagnosis . the finnish pediatric diabetes register was established in 2002 and collects data on all children in finland diagnosed with type 1 diabetes . our analysis includes 737 children aged < 5 years from the finnish diabetes register diagnosed with type 1 diabetes from 2005 to 2009 . the german / austrian diabetes documentation and quality management system ( dpv ) is a continuous diabetes data acquisition system providing data from 53,485 type 1 diabetes patients throughout germany and austria . multicenter study aimed at identifying prevalent and incident cases of diabetes among individuals < 20 years of age at diagnosis . for these analyses , we included children diagnosed with type 1 diabetes in 2004 and 2005 . dka was defined in the search protocol using medical abstraction to identify bicarbonate < 15 mmol / l or venous ph < 7.25 ( arterial / capillary ph < 7.30 ) , international classification of diseases , ninth revision , code 250.1 , or physician s diagnosis of dka . our analysis included 275 search subjects diagnosed with type 1 diabetes when < 5 years of age . swediabkids is the swedish pediatric national quality registry for type 1 diabetes , covering 100% of all children diagnosed in sweden . the incidence register is part of this national online follow - up quality register for pediatric patients , where all pediatric diabetes clinic visits are recorded . in this report , we include data from 651 swedish children diagnosed with type 1 diabetes between 2005 and 2009 and < 5 years of age at diagnosis . the finnish pediatric diabetes register was established in 2002 and collects data on all children in finland diagnosed with type 1 diabetes . our analysis includes 737 children aged < 5 years from the finnish diabetes register diagnosed with type 1 diabetes from 2005 to 2009 . the german / austrian diabetes documentation and quality management system ( dpv ) is a continuous diabetes data acquisition system providing data from 53,485 type 1 diabetes patients throughout germany and austria . because of differences in data collection and management of newly diagnosed patients within teddy , search , and the population - based registries , we used two definitions of dka to ensure appropriate comparisons ( table 2 ) . for comparisons with search , german - dpv , and finnish registers , broad criteria for dka were used to include the strict criteria and , in cases where ph or bicarbonate values were missing , betahydroxybutyrate > 3.0 mmol / l , urine ketones > 40 mg / dl , or physician s diagnosis . prevalence estimates and 95% confidence intervals were calculated as the proportion presenting with dka at the time of diagnosis by age - group ( < 2 and < 5 years ) based on appropriate definition ( strict or broad ) . teddy screened 424,788 children for type 1 diabetes hla risk , identified 21,588 eligible subjects , and enrolled 8,677 children . as of 31 december 2010 , 80 teddy subjects were diagnosed with type 1 diabetes ( supplementary fig . 40 children were < 2 years of age and 79 were < 5 years of age at diagnosis . in children < 2 years of age at diagnosis , 6 of 40 ( 15% ) had dka under the broad definition of dka whereas 5 of 31 ( 16.1% ) had dka under the strict definition of dka . teddy children diagnosed with type 1 diabetes before 2 years of age had lower rates of mild and total dka when compared with population - based studies and registries ( search , p < 0.0001 ; german dpv register , p < 0.0001 ; swediabkids , p = 0.02 ; finnish register , p = 0.0005 ) ( table 3 ) . dka rates in children aged < 5 years and < 2 years at diagnosis na , data not available . * < 5 years of age at diagnosis , 9 of 79 ( 11.3% ) had dka at diagnosis of diabetes under the broad definition of dka . using the strict definition of dka and excluding those with insufficient data , 8 of 61 ( 13.1% ) < 5 years of age had dka at diagnosis of diabetes . total dka ( mild and severe dka ) at diagnosis was significantly less common in teddy participants ( 11.3% ) when compared with search ( 36.4% ) ( p < 0.0001 ) and german dpv ( 25.3% ) ( p 0.0001 ) children , respectively , but were not statistically different from dka at diagnosis for patients from the swedish ( 16.9% ) ( p = 0.45 ) or finnish ( 18.7% ) ( p = 0.11 ) registers . although numbers were small , the rate of severe dka ( arterial ph < 7.10 ) did not differ significantly between children diagnosed within the teddy study and those reported from national registries in any age - group . notably , all three teddy children diagnosed with severe dka were < 2 years of age ( 8 , 10 , and 14 months , respectively ) and had developed islet autoantibodies before clinical presentation of disease . in children < 2 years of age at diagnosis , 6 of 40 ( 15% ) had dka under the broad definition of dka whereas 5 of 31 ( 16.1% ) had dka under the strict definition of dka . teddy children diagnosed with type 1 diabetes before 2 years of age had lower rates of mild and total dka when compared with population - based studies and registries ( search , p < 0.0001 ; german dpv register , p < 0.0001 ; swediabkids , p = 0.02 ; finnish register , p = 0.0005 ) ( table 3 ) . dka rates in children aged < 5 years and < 2 years at diagnosis na , data not available . in children < 5 years of age at diagnosis , 9 of 79 ( 11.3% ) had dka at diagnosis of diabetes under the broad definition of dka . using the strict definition of dka and excluding those with insufficient data , 8 of 61 ( 13.1% ) < 5 years of age had dka at diagnosis of diabetes . total dka ( mild and severe dka ) at diagnosis was significantly less common in teddy participants ( 11.3% ) when compared with search ( 36.4% ) ( p < 0.0001 ) and german dpv ( 25.3% ) ( p 0.0001 ) children , respectively , but were not statistically different from dka at diagnosis for patients from the swedish ( 16.9% ) ( p = 0.45 ) or finnish ( 18.7% ) ( p = 0.11 ) registers . although numbers were small , the rate of severe dka ( arterial ph < 7.10 ) did not differ significantly between children diagnosed within the teddy study and those reported from national registries in any age - group . notably , all three teddy children diagnosed with severe dka were < 2 years of age ( 8 , 10 , and 14 months , respectively ) and had developed islet autoantibodies before clinical presentation of disease . in this study we compared the prevalence of dka in children participating in teddy and diagnosed with type 1 diabetes before the age of 5 years with national population - based registers . our analyses demonstrate that teddy subjects diagnosed with type 1 diabetes before the age of 2 years experienced significantly less dka at diagnosis ( 15% ) when compared with new - onset patients reported in national diabetes registers from countries participating in teddy and the search study ( 3550% ) . similarly , teddy children diagnosed with type 1 diabetes before the age of 5 years experienced less dka at onset when compared with children in the search study and the german dpv registry , but not compared with children from the finnish or swedish registries . although the relatively high proportion of teddy subjects who were first - degree relatives of type 1 diabetes patients may have confounded our analysis , dka rates within the teddy cohort were similar among children recruited from the general population and those with a first - degree relative with type 1 diabetes , indicating that knowledge of diabetes risk and close longitudinal follow - up may be associated with reduced dka risk regardless of family history . although our analysis is strengthened by the inclusion of data from a national incidence study and registries performed in each of the countries participating in teddy , we should note that children with incident diabetes from colorado and washington could have simultaneously been included in both the search and teddy databases , and the teddy participants in sweden , finland , and germany could have been included in the national registries . while such an occurrence could have biased our data , the effect ( given lower rates of dka noted in teddy ) would have been to mask the differences observed . unfortunately , we were unable to perform an analysis where participants in both teddy and a population - based study or registry were excluded . the finnish and dpv registries used standard biochemical data , but excluded dka in children with negative urine ketones , negative blood ketones , or lack of symptoms . for the purposes of this analysis , teddy defined dka using both strict criteria ( standard cutoffs for ph and bicarbonate ) and broad criteria ( allowing us to document dka status on the basis of urine / blood ketones and clinician documentation ) . when comparing overall dka rates in teddy participants with children reported in the swedish and finnish registry , no significant differences were seen in children diagnosed before 5 years of age . however , rates of dka in teddy participants aged < 5 years were significantly lower when compared with children from the u.s . these observations are concordant with reports that countries with a high incidence of type 1 diabetes ( finland and sweden ) report reduced frequencies of dka when compared with countries with lower incidence of type 1 diabetes . decreased frequencies of dka at onset of disease have been reported from finland between 1982 and 2001 , with a decrease from 22.4 to 15.2% in children 015 years of age and a decrease from 32.1 to 17.7% in children with onset before the age of 5 years ( 6 ) . that said , the rate of dka in children < 2 years of age at onset remained unacceptably high ( 39.1% ) ( 6 ) . on the contrary , german data from the dpv register did not reveal reduced frequency of dka in any age - group between 1995 and 2007 ( 8) . in a swedish study , the incidence of dka at onset of type 1 diabetes in children diagnosed from 2000 to 2004 was 16% , with no difference reported between age - groups . however , children < 2 years of age were not specifically analyzed ( 15 ) . the youngest children with new onset type 1 diabetes ( < 2 years of age ) remain at high risk of dka ( table 1 ) ( 6 ) . more specifically , these young children are prone to severe dka at onset . of the children in teddy diagnosed with severe dka ( ph < 7.10 ) , all three were < 2 years of age ( 8 , 10 , and 14 months , respectively ) . unfortunately , our analysis lacked the power to determine if participation in teddy reduced the risk of severe dka in children < 2 years of age . that said , the observation of reduction in overall dka in the youngest patients with new - onset diabetes suggests that participation in a longitudinal natural history study with provision of updated autoantibody risk information , frequent follow - up , and scheduled laboratory evaluations ( autoantibodies , hba1c , glucose , and ogtt ) to diagnose asymptomatic cases is associated with reduced dka risk . although potentially cost prohibitive outside the constraints of a research protocol , combined approaches to educate communities on signs and symptoms of dka ( as in the parma , italy experiments ) and to provide genetic or antibody screening to further identify high - risk subjects may eventually be required to achieve the goal of entirely preventing dka in children . an early diagnosis of diabetes would therefore be directly beneficial to the child and indirectly beneficial to society through reduction of morbidity , mortality , and cost at onset of disease . in conclusion , intensive longitudinal follow - up and continuous education regarding diabetes risk , as provided in diabetes prediction studies such as teddy , may yield direct benefit to young children diagnosed with type 1 diabetes through early diagnosis and reduction of dka risk . ongoing efforts to reduce and eliminate dka at diagnosis in young children are urgently needed .

the citr is the comprehensive islet transplant registry for 27 national institute of diabetes and digestive and kidney diseases ( niddk)funded north american and jdrf - funded european and australian centers since 1999 , comprising 81% of all allogeneic islet transplants conducted as single - arm phase i / ii trials or standard of care . patients and methods are fully described in previous and current citr annual reports ( 3 ) , which are publicly available . in brief , recipients of allogeneic islet transplants typically are aged between 18 and 65 years . all have had type 1 diabetes for > 5 years , and > 95% had documented negative fasting c - peptide ( < 0.3 ng / ml ) and very problematic diabetes control , including hypoglycemia unawareness complicated by episodes of severe hypoglycemia and/or marked glycemic lability characterized by wide swings of blood glucose levels , often with consistently elevated hba1c levels ( > 8% ) . the registry collects information on the pancreas donor(s ) , islet processing and testing , immunosuppression and concomitant medications , severe hypoglycemic episodes , hba1c , fasting blood glucose and c - peptide levels , daily insulin doses , vital status , islet graft dysfunction and loss , reportable adverse events graded 3 , 4 , and 5 according to the terminology criteria for adverse events of the clinical islet transplantation consortium ( cit ) ( 5 ) , and serious adverse events ( 6 ) . islet recipients enrolled in cit protocols consenting to have their data shared with the citr are registered in the citr and included in the citr reports . cit protocols comprise a series of phase ii and phase iii clinical trials designed to test current immunosuppressive strategies and management practices and pursue licensure for clinical islet transplantation in the u.s . the cit data are coordinated by the university of iowa clinical trials and data management center , william clarke , phd , director , and are made available to the citr through collaborative agreements via the common sponsor , the u.s . citr data are rigorously monitored by the data coordinating center , the emmes corporation , rockville , maryland , to comply with u.s . site participation in the registry requires local research ethics board approval , strict assurance of patient - identifier confidentiality , and written informed consent by the islet recipients . the citr publications and presentations committee approved the manuscript . at preinfusion and at each scheduled follow - up visit , five coprimary end points were assessed by laboratory measurements or clinical evaluations : basal c - peptide ( further divided as 0.3 vs. < 0.3 ng / ml ) , including reported complete graft loss ( defined as fasting c - peptide consistently undetectable with stimulated c - peptide < 0.3 ng / ml by local assay without subsequent recovery to 0.3 ng / ml or reinfusion , also denoted as no function ) ; independence from exogenous insulin for 14 consecutive days ; hba1c ( further divided as < 6.5% and/or a drop by two percentage points or more ) ; fasting blood glucose ( further divided as 60140 vs. < 60 or > 140 mg / dl ) ; and absence of severe hypoglycemia episodes ( requiring assistance of another person ) . the scheduled times for each infusion were immediately before transplant , 7 days , 1 month , 6 months , and annually thereafter , which was reset at each subsequent infusion . annual time points from the last of one or more infusions per recipient were used in this analysis . except for complete graft failure , each of these outcomes can occur , relapse , and then reoccur during follow - up , although with relatively long periods of stable status ; hence , they are analyzed as prevalence ( percentage ) at each follow - up after the last infusion . complete graft failure can not remit by definition ; therefore , this outcome was analyzed by failure - time techniques . when direct data were missing but graft function was known to have been previously lost and not restored , insulin independence was set as dependent and c - peptide was set at 0 . otherwise , missing data were omitted ( i.e. , treated as missing at random ) in the computations and modeling . for infusions given the same day from two to three different donors , the donor , procurement , processing , and isolation characteristics were summarized over the multiple donors ( e.g. , donor ages were averaged , total islet equivalents infused were summed , etc . ) the information was summarized again over two to six infusion events per recipient . trapped ( embedded ) islets are expressed as a percentage of total islet count in the preparation . immunosuppression agents were noted as each given or not at each infusion and during the follow - up . each recipient was classified into induction and maintenance combination categories as indicated in table 1 . all available recipient , donor , islet , and immunosuppression variables were used in the various analyses as possible predictors of the primary outcomes . recipient , donor , islet , and immunosuppression characteristics , based on numbers with data generalized estimating equations with repeated measures per recipient were used to assess the effect of era ( 19992003 , 20032006 , 20072010 ) , follow - up time after the first infusion , and other covariates on the rate ( prevalence ) of the desirable outcome for each primary end point . a multivariate analysis of all available recipient , donor , islet , and medical management factors on the outcomes was also conducted to see if changes in patient selection and management practices accounted for the observed differences in outcomes over the eras . the occurrence and outcomes of clinically reportable adverse events ( craes ) , classified as unlikely , probably , or definitely related to the infusion procedure or to the immunosuppression regimen , were analyzed according to era . each recipient was classified and tabulated according to his or her worst outcome of all infusion - related craes and immunosuppression - related craes during the entire period of infusions and follow - up for the recipient . comparisons were made with mantel - haenszel . comparisons across eras clearly were not randomized , and sample sizes were not experimentally determined . in this registry data , nominal p values are reported without prespecified type i error rates . the citr is the comprehensive islet transplant registry for 27 national institute of diabetes and digestive and kidney diseases ( niddk)funded north american and jdrf - funded european and australian centers since 1999 , comprising 81% of all allogeneic islet transplants conducted as single - arm phase i / ii trials or standard of care . patients and methods are fully described in previous and current citr annual reports ( 3 ) , which are publicly available . in brief , recipients of allogeneic islet transplants typically are aged between 18 and 65 years . all have had type 1 diabetes for > 5 years , and > 95% had documented negative fasting c - peptide ( < 0.3 ng / ml ) and very problematic diabetes control , including hypoglycemia unawareness complicated by episodes of severe hypoglycemia and/or marked glycemic lability characterized by wide swings of blood glucose levels , often with consistently elevated hba1c levels ( > 8% ) . the registry collects information on the pancreas donor(s ) , islet processing and testing , immunosuppression and concomitant medications , severe hypoglycemic episodes , hba1c , fasting blood glucose and c - peptide levels , daily insulin doses , vital status , islet graft dysfunction and loss , reportable adverse events graded 3 , 4 , and 5 according to the terminology criteria for adverse events of the clinical islet transplantation consortium ( cit ) ( 5 ) , and serious adverse events ( 6 ) . islet recipients enrolled in cit protocols consenting to have their data shared with the citr are registered in the citr and included in the citr reports . cit protocols comprise a series of phase ii and phase iii clinical trials designed to test current immunosuppressive strategies and management practices and pursue licensure for clinical islet transplantation in the u.s . the cit data are coordinated by the university of iowa clinical trials and data management center , william clarke , phd , director , and are made available to the citr through collaborative agreements via the common sponsor , the u.s . citr data are rigorously monitored by the data coordinating center , the emmes corporation , rockville , maryland , to comply with u.s . site participation in the registry requires local research ethics board approval , strict assurance of patient - identifier confidentiality , and written informed consent by the islet recipients . at preinfusion and at each scheduled follow - up visit , five coprimary end points were assessed by laboratory measurements or clinical evaluations : basal c - peptide ( further divided as 0.3 vs. < 0.3 ng / ml ) , including reported complete graft loss ( defined as fasting c - peptide consistently undetectable with stimulated c - peptide < 0.3 ng / ml by local assay without subsequent recovery to 0.3 ng / ml or reinfusion , also denoted as no function ) ; independence from exogenous insulin for 14 consecutive days ; hba1c ( further divided as < 6.5% and/or a drop by two percentage points or more ) ; fasting blood glucose ( further divided as 60140 vs. < 60 or > 140 mg / dl ) ; and absence of severe hypoglycemia episodes ( requiring assistance of another person ) . the scheduled times for each infusion were immediately before transplant , 7 days , 1 month , 6 months , and annually thereafter , which was reset at each subsequent infusion . annual time points from the last of one or more infusions per recipient were used in this analysis . except for complete graft failure , each of these outcomes can occur , relapse , and then reoccur during follow - up , although with relatively long periods of stable status ; hence , they are analyzed as prevalence ( percentage ) at each follow - up after the last infusion . complete graft failure can not remit by definition ; therefore , this outcome was analyzed by failure - time techniques . when direct data were missing but graft function was known to have been previously lost and not restored , insulin independence was set as dependent and c - peptide was set at 0 . otherwise , missing data were omitted ( i.e. , treated as missing at random ) in the computations and modeling . for infusions given the same day from two to three different donors , the donor , procurement , processing , and isolation characteristics were summarized over the multiple donors ( e.g. , donor ages were averaged , total islet equivalents infused were summed , etc . ) the information was summarized again over two to six infusion events per recipient . trapped ( embedded ) islets are expressed as a percentage of total islet count in the preparation . immunosuppression agents were noted as each given or not at each infusion and during the follow - up . each recipient was classified into induction and maintenance combination categories as indicated in table 1 . all available recipient , donor , islet , and immunosuppression variables were used in the various analyses as possible predictors of the primary outcomes . recipient , donor , islet , and immunosuppression characteristics , based on numbers with data generalized estimating equations with repeated measures per recipient were used to assess the effect of era ( 19992003 , 20032006 , 20072010 ) , follow - up time after the first infusion , and other covariates on the rate ( prevalence ) of the desirable outcome for each primary end point . a multivariate analysis of all available recipient , donor , islet , and medical management factors on the outcomes was also conducted to see if changes in patient selection and management practices accounted for the observed differences in outcomes over the eras . the occurrence and outcomes of clinically reportable adverse events ( craes ) , classified as unlikely , probably , or definitely related to the infusion procedure or to the immunosuppression regimen , were analyzed according to era . each recipient was classified and tabulated according to his or her worst outcome of all infusion - related craes and immunosuppression - related craes during the entire period of infusions and follow - up for the recipient . comparisons were made with mantel - haenszel . comparisons across eras clearly were not randomized , and sample sizes were not experimentally determined . in this registry data , nominal p values are reported without prespecified type i error rates . this analysis was based on 677 recipients of allogeneic islet transplantation who consented to the reporting of their data to the citr , with 214 recipients in 19992002 ( early ) , 255 in mid-20032006 , and 208 in 20072010 ( recent ) ; 423 ( 62% ) came from north america , and 254 ( 38% ) were reported from the european and australian jdrf sites . transplants comprised islet alone in 575 ( 85% ) and iak or simultaneous islet kidney ( iak / sik ) transplant in 102 ( 15% ) . they received 1,375 islet infusions from 1,502 donors , of which 10% were islets from 2 to 3 donors infused on the same day , considered here as multiple donor infusion . approximately 36% of the recipients received only one infusion , 44% received two , 18% received three , 1.3% received four , and one person received six infusions . the citr data represent 81% of all islet transplants performed in the north american and jdrf european and australian centers between 1999 and 2010 . the number of new islet allograft recipients doubled yearly between 1999 and 2002 ( fig . 1 ) . a marked decline in activity from 2002 to 2003 reflected a saturation of then - existing protocol enrollments , combined with tempered enthusiasm for the procedure after some centers reported waning insulin independence at 23 years ( 7,8 ) . the number of north american centers performing islet transplants continued to rise through 2005 , although the annual number of islet allografts remained less than the 2002 levels . in 2007 , there were fewer than half as many north american centers performing islet transplants and one - third of the total number of islet allografts performed compared with 2005 at a time when the commonly used collagenase enzyme liberase became unavailable . a distinct resurgence in islet transplant activity occurred in 2008 with the available collagenase products and the start - up of the cit trials . islet allograft recipients ( n = 677 ) registered in citr according to type of transplant ( n per year ; top ) , induction immunosuppression at first infusion ( % by year ; center ) , and maintenance immunosuppression at first infusion ( % by year ; bottom ) . the early and mideras were dominated by the edmonton protocol , which used an interleukin 2 receptor antagonist ( e.g. , daclizumab ) for induction and a mammalian target of rapamycin ( mtor ) inhibitor ( e.g. , sirolimus ) , together with a calcineurin inhibitor ( cni , e.g. , tacrolimus ) for maintenance immunosuppression . in the most recent era , there has been a shift to induction with a t - cell depleting ( tcd ) antibody , with or without an inhibitor of tumor necrosis factor- ( tnf- ; e.g. , etanercept ) and maintenance with an mtor inhibitor or an inosine monophosphate dehydrogenase inhibitor ( e.g. , mycophenolic acid ) combined with a cni . table 1 summarizes the preinfusion recipient characteristics according to era . over time , recipients with c - peptide 0.3 ng / ml have been excluded . increasingly , recipients have been selected at older age and with longer type 1 diabetes duration , requiring slightly less insulin and having better kidney function , as indicated by lower serum creatinine , suggesting more appropriate patient selection . consistent with trends in clinical practice , more were using insulin pumps for insulin delivery , which may explain the slightly lower daily insulin requirement , and more were taking lipid - lowering medications . following national trends , donor weight increased , and consistent with trends in critical care medicine , more donors received insulin with a consequent decrease in donor glucose . there were also definite shifts in preservation method and collagenase type , and more islet preparations were cultured . the clinical effects of procurement , processing , and final islet characteristics are the focus of a separate analysis . recent years have seen a substantial decline in the use of daclizumab , with a substantial rise in polyclonal t - cell depleting antibodies and/or etanercept , as well as notable declines in sirolimus use , with increased use of mycophenolic acid . there were increasing levels of missing data with longer follow - up , which is a mixture of data unavailable from the medical record and data still pending entry into the registry . the percentages of missing data for insulin independence were 3% at 1 year , 5% at 3 years , and 7% at 5 years and for other primary end points were 10 to 20% over years 13 . of those who received transplants in the 19992002 era , 51% were insulin - independent at 1-year after the first infusion , regardless of reinfusion , and this declined to 36% at 2 years and to 27% at 3 years . by contrast in the 20072010 era , 66% were insulin - independent at 1 year , 55% at 2 years , and 44% at 3 years ( p = 0.01 , fig . the decline in the rate of insulin independence during 5 years of follow - up in all eras is significant ( p < 0.001 ) . the difference in this decline among the three eras ( p = 0.006 for years - by - era ) indicates that the rate of decline is less steep , showing notable improvement in durability in the most recent era . durability of islet graft function , as measured by fasting c - peptide 0.3 ng / ml , improved significantly over the eras ( p < 0.001 , fig . the rate of graft function loss was significantly reduced if insulin independence was previously achieved , an effect seen in all eras ( fig . nearly all islet recipients had significant improvements in hba1c and fasting blood glucose after islet transplantation . the composite end point of hba1c < 6.5% or a drop by two or more percentage points shows improvement from the early era to the mid era ( p = 0.03 ) , although no further improvement in the most recent era , with 25-year success rates of 5060% in the recent era ( fig . fasting blood glucose showed a marked improvement from the early to mid eras ( p < 0.01 , not shown ) . a : rates of insulin independence after allogeneic islet infusion ( islet transplant alone and iak ) , annually after last infusion . left : by era ( p = 0.02 ) . b : durability of graft function ( basal c - peptide 0.3 ng / ml ) after the last infusion , by era ( p < 0.001 ; left ) . the immediate drop at time 0 is occurrences of primary nonfunction ( i.e. , c - peptide never 0.3 ng / ml ) . in the most recent era , 95% of those who ever achieved insulin independence ( ii ) retained graft function through 3 years after last infusion compared with 70% for those who never achieved ii ( p < 0.001 ; right ) . c : percentage of patients with hba1c < 6.5% or drop by two percentage points ( p = 0.03 ; left ) ; and absence of severe hypoglycemic events regardless of complete graft failure ( p = ns by era ; there were insufficient data from 20072010 ; right ) . d : the percentage with hba1c < 6.5% or drop by 2% increases with increasing c - peptide level ( p < 0.001 ; left ) , as does absence of severe hypoglycemic events ( p < 0.001 ; right ) , annually after the last infusion . ( a high - quality color representation of this figure is available in the online issue . ) severe hypoglycemia was prevalent at first infusion in > 90% of all subjects in all eras . available data on severe hypoglycemic events , regardless of previous graft loss ( c - peptide < 0.3 ng / ml without recovery ) , shows > 90% remained free of severe hypoglycemic events in all eras , and this relationship persisted through 5 years of follow - up ( fig . any differences by era on resolution of severe hypoglycemic events were neither detectable nor important relative to this sustained , high level of benefit . if data on severe hypoglycemic events were missing and previous complete graft loss was counted as return to severe hypoglycemic events an extreme assumption there was still improvement in 20032006 compared with 19992002 at years 24 ( p = 0.03 , not shown ) . concurrent c - peptide is a strong correlate of all the other primary outcomes : the higher the c - peptide , the greater the likelihood of hba1c < 6.5% or a drop by two percentage points ( p < 0.001 ; fig . 2d ) , the greater the likelihood of absence of severe hypoglycemic events ( p < 0.001 ; fig . 2d ) , the greater the likelihood of fasting blood glucose in the 60140 mg / dl range ( p < 0.001 , not shown ) , and the greater the likelihood of insulin independence ( p < 0.001 , not shown ) . a comprehensive model of all predictive factors noting the shifts in patient age and immunosuppression strategies over the eras ( table 1)largely accounted for the differences by era in insulin independence ( table 2 ) . the effect of t - cell depleting agents in conjunction with tnf- inhibitors shows on enduring insulin independence ( 9 ) is confirmed : 5062% of recipients receiving this induction regimen were insulin - independent at years 35 after the last infusion ( fig . factors predictive of insulin independence after last infusion reinfusion is performed when the first graft loses function completely or declining function is proven by declining c - peptide levels . islet reinfusion has decreased substantially during the 12-year period : 48% of recipients were reinfused by 1 year in 20072010 vs. 6065% in 19992006 ( p < 0.01 ) . mortality is low in this group of type 1 diabetic individuals with substantial disease burden , with stable event rates during the 12-year period ( fig . the incidence of life - threatening events has declined ( p = 0.002 ; fig . 3b ) . the incidence of any crae in year 1 declined from 50 to 53% in 19992006 and to 38% in 20072010 ( p = 0.02 ; fig . 3c ) . peritoneal hemorrhage or gallbladder perforation declined from 5.4% in 19992003 to 3.1% in 20072010 . the chronic kidney disease epidemiology collaboration ( ckd - epi ) calculated glomerular filtration rate ( gfr ) declined after islet transplantation ( fig . 3d ) ; however , there are no published comparable follow - up data in similar groups of type 1 diabetes . no primary efficacy or safety end points were associated with recipient or donor sex or ethnicity . a : mortality by era ( p = 0.49 ) . c : incidence of any adverse event ( ae ) in year 1 of first infusion ( p = 0.02 by era ) . in north america , the number of centers performing clinical islet transplants and the total number of islet transplants declined in 20062007 , with a distinct resurgence in 2008 . the reasons for the decline are not directly captured by the registry but likely reflect changes in the production and availability of the collagenase enzymes used for islet digestion , tempered enthusiasm with respect to long - term clinical outcomes of insulin independence ( 7,8,10 ) , concern for effect of immunosuppression on kidney function in islet - alone recipients ( 11,12 ) , concern for risk of sensitization to donor hla ( 1315 ) , and saturation of the referral base for patients with the severest forms of unstable type 1 diabetes . however , with the start of the new cit protocols in 2008 , coupled with more encouraging recent trends in longer - term outcomes with novel protocols using t - cell depletion for induction ( 16,17 ) , the number of new islet cell recipients has increased annually in the most recent era . direct evidence is presented of the importance of durable islet graft function to achieve multiple clinical benefits as a consequent effect . positive c - peptide is strongly associated with all of the other primary clinical outcomes ; hence , the factors that drive positive c - peptide necessarily lead to the other clinical benefits , although additional factors may also contribute to the other benefits . a comprehensive analysis of the effect of all available factors on these primary co - outcomes indicates that older recipient age , lower initial insulin requirement , and the use of t - cell depletion , particularly when given in conjunction with tnf- inhibitors , are significantly associated with improved clinical outcomes . the numbers are too low to definitively assess the impact of a shift in maintenance immunosuppression , with mycophenolic acid replacing mtor inhibitors , and both agents are still usually administered in combination with a cni . it must be noted that the citr data have not been accruing in real time ; rather , as sites have joined over the 12-year life of the registry , large portions of the data , including some of the historical data , have been reported during the last 13 years . hence , the current results may vary somewhat from previously published reports , including the citr annual reports . the present data are the most comprehensive and up - to - date information available for the 12-year period 19992010 . in the present analysis , the increasing levels of missing data with increasing strengths of the analysis are the most complete available set of data and ability to track trends during this 12-year period of steroid - free immunosuppression . stratifying the citr data by era of transplant shows a compelling trend toward better outcomes in the recent era ( p 0.01 ) , despite the still relatively low total number of islet transplant recipients worldwide . there is an indication of moving toward selection of older recipients with longer - standing diabetes and absence of c - peptide to tip the risk - to - benefit ratio in their favor . the trend toward heavier donors is likely due to donor availability in the midst of a global obesity epidemic and possibly to the known association between higher donor weights and the higher number of islet equivalents isolated ( 18 ) . this must be balanced against the detrimental effects of transplanting islets derived from donors with unsuspected type 2 diabetes ( 19 ) , and for this reason , it is important to confirm that the hba1c of an obese donor is within the normal range before transplantation . in the past , transplanting islets rapidly after isolation was believed to be optimal . in recent years , the preference toward transplanting islets after a short culture period emphasizes the current supposition that culturing removes the nonviable islets and decreases tissue factor expression that can lead to nonspecific inflammation and islet loss after transplant ( 20 ) . the percutaneous infusion technique occasionally resulted in intraperitoneal hemorrhage and portal branch vein thrombosis early on ; however , these complications have occurred less often in the present era . whole pancreas transplantation is an approved option for -cell replacement in type 1 diabetes , although it is mostly limited to patients simultaneously receiving a kidney transplant for diabetic nephropathy and often excludes older patients and those with coronary artery disease due to the potential for significant surgical morbidity . thus , islet transplantation may offer a complementary alternative to whole pancreas transplantation in patients who are not candidates for or are unwilling to accept the risks of major surgery , and so some estimation of comparative efficacy is required . in the 20072010 era , islet graft survival ( c - peptide 0.3 ng / ml ) of 92% at 1 year and 83% at 3 years ( fig . 2b ) compares very favorably with whole pancreas graft survival of 80% at 1 year and 61% at 3 years ( 21 ) . in the recent era , these graft survival rates translate to an unconditional 44% insulin independence at 3 years ( fig . 2a ) , the highest long - term islet transplant success rate observed to date . although this is still short of the 61% insulin independence reported in the most successful cohort of type 1 diabetes pancreas - alone transplant recipients ( 22 ) , this difference may be explained by the transplantation of 100% of a normal islet -cell mass with a whole pancreas compared with a variable islet -cell mass surviving the engraftment of isolated islets and resulting in a reduced -cell secretory capacity ( 23 ) . in addition , throughout the 12-year period , these data show an enduring benefit in hba1c reduction and stabilization of fasting blood glucose . importantly , the presence of insulin - dependent islet graft survival defined by c - peptide > 0.3 ng / ml confers protection from severe hypoglycemia , and this effect persists even after the islet graft is lost . this declining rate of islet graft loss by era suggests that more recent strategies of immunosuppression , as identified in the multivariate analysis , may better protect islets from alloimmune rejection and recurrent autoimmunity . the successful strategies have all included cnis that are known to exhibit -cell toxicity at high doses ; however , one study showed modern use of lower - dose , cni - based immunosuppression resulted in a 100% normal -cell secretory capacity in whole pancreas transplant recipients ( 23 ) , supporting that these agents can be used and may even be necessary for successful islet transplantation . finally , the finding that the rate of graft function loss was significantly reduced when insulin independence was previously achieved suggests that the engraftment of a sufficient islet -cell mass to eliminate the need for exogenous insulin may mitigate nonimmunologic islet graft loss believed to occur in the setting of increased -cell demand . present strategies to improve the proportion of islets surviving engraftment are expected to lead to improved functional outcomes for islet recipients ( 24 ) . the citr shows consistent trends toward improved primary outcomes of islet transplantation in the cohort who received transplants in 20072010 compared with those in 19992006 . islet transplantation currently offers substantial protection from severe hypoglycemic episodes and high rates of freedom from exogenous insulin requirements in a minimally invasive setting . emerging innovations in islet production , processing , delivery , and immunosuppressive protection islet transplantation has already moved from phase i / ii to phase iii evaluation , with the results from the cit eagerly awaited to provide efficacy and safety information for a standardized approach to islet isolation and immunosuppression management .

objectiveto describe trends of primary efficacy and safety outcomes of islet transplantation in type 1 diabetes recipients with severe hypoglycemia from the collaborative islet transplant registry ( citr ) from 1999 to 2010.research design and methodsa total of 677 islet transplant - alone or islet - after - kidney recipients with type 1 diabetes in the citr were analyzed for five primary efficacy outcomes and overall safety to identify any differences by early ( 19992002 ) , mid ( 20032006 ) , or recent ( 20072010 ) transplant era based on annual follow - up to 5 years.resultsinsulin independence at 3 years after transplant improved from 27% in the early era ( 19992002 , n = 214 ) to 37% in the mid ( 20032006 , n = 255 ) and to 44% in the most recent era ( 20072010 , n = 208 ; p = 0.006 for years - by - era ; p = 0.01 for era alone ) . c - peptide 0.3 ng / ml , indicative of islet graft function , was retained longer in the most recent era ( p < 0.001 ) . reduction of hba1c and resolution of severe hypoglycemia exhibited enduring long - term effects . fasting blood glucose stabilization also showed improvements in the most recent era . there were also modest reductions in the occurrence of adverse events . the islet reinfusion rate was lower : 48% by 1 year in 20072010 vs. 6065% in 19992006 ( p < 0.01 ) . recipients that ever achieved insulin - independence experienced longer duration of islet graft function ( p < 0.001).conclusionsthe citr shows improvement in primary efficacy and safety outcomes of islet transplantation in recipients who received transplants in 20072010 compared with those in 19992006 , with fewer islet infusions and adverse events per recipient .

RESEARCH DESIGN AND METHODS Patients Statistics RESULTS CONCLUSIONS

the citr is the comprehensive islet transplant registry for 27 national institute of diabetes and digestive and kidney diseases ( niddk)funded north american and jdrf - funded european and australian centers since 1999 , comprising 81% of all allogeneic islet transplants conducted as single - arm phase i / ii trials or standard of care . all have had type 1 diabetes for > 5 years , and > 95% had documented negative fasting c - peptide ( < 0.3 ng / ml ) and very problematic diabetes control , including hypoglycemia unawareness complicated by episodes of severe hypoglycemia and/or marked glycemic lability characterized by wide swings of blood glucose levels , often with consistently elevated hba1c levels ( > 8% ) . the registry collects information on the pancreas donor(s ) , islet processing and testing , immunosuppression and concomitant medications , severe hypoglycemic episodes , hba1c , fasting blood glucose and c - peptide levels , daily insulin doses , vital status , islet graft dysfunction and loss , reportable adverse events graded 3 , 4 , and 5 according to the terminology criteria for adverse events of the clinical islet transplantation consortium ( cit ) ( 5 ) , and serious adverse events ( 6 ) . islet recipients enrolled in cit protocols consenting to have their data shared with the citr are registered in the citr and included in the citr reports . cit protocols comprise a series of phase ii and phase iii clinical trials designed to test current immunosuppressive strategies and management practices and pursue licensure for clinical islet transplantation in the u.s . site participation in the registry requires local research ethics board approval , strict assurance of patient - identifier confidentiality , and written informed consent by the islet recipients . at preinfusion and at each scheduled follow - up visit , five coprimary end points were assessed by laboratory measurements or clinical evaluations : basal c - peptide ( further divided as 0.3 vs. < 0.3 ng / ml ) , including reported complete graft loss ( defined as fasting c - peptide consistently undetectable with stimulated c - peptide < 0.3 ng / ml by local assay without subsequent recovery to 0.3 ng / ml or reinfusion , also denoted as no function ) ; independence from exogenous insulin for 14 consecutive days ; hba1c ( further divided as < 6.5% and/or a drop by two percentage points or more ) ; fasting blood glucose ( further divided as 60140 vs. < 60 or > 140 mg / dl ) ; and absence of severe hypoglycemia episodes ( requiring assistance of another person ) . when direct data were missing but graft function was known to have been previously lost and not restored , insulin independence was set as dependent and c - peptide was set at 0 . immunosuppression agents were noted as each given or not at each infusion and during the follow - up . recipient , donor , islet , and immunosuppression characteristics , based on numbers with data generalized estimating equations with repeated measures per recipient were used to assess the effect of era ( 19992003 , 20032006 , 20072010 ) , follow - up time after the first infusion , and other covariates on the rate ( prevalence ) of the desirable outcome for each primary end point . the occurrence and outcomes of clinically reportable adverse events ( craes ) , classified as unlikely , probably , or definitely related to the infusion procedure or to the immunosuppression regimen , were analyzed according to era . each recipient was classified and tabulated according to his or her worst outcome of all infusion - related craes and immunosuppression - related craes during the entire period of infusions and follow - up for the recipient . the citr is the comprehensive islet transplant registry for 27 national institute of diabetes and digestive and kidney diseases ( niddk)funded north american and jdrf - funded european and australian centers since 1999 , comprising 81% of all allogeneic islet transplants conducted as single - arm phase i / ii trials or standard of care . all have had type 1 diabetes for > 5 years , and > 95% had documented negative fasting c - peptide ( < 0.3 ng / ml ) and very problematic diabetes control , including hypoglycemia unawareness complicated by episodes of severe hypoglycemia and/or marked glycemic lability characterized by wide swings of blood glucose levels , often with consistently elevated hba1c levels ( > 8% ) . the registry collects information on the pancreas donor(s ) , islet processing and testing , immunosuppression and concomitant medications , severe hypoglycemic episodes , hba1c , fasting blood glucose and c - peptide levels , daily insulin doses , vital status , islet graft dysfunction and loss , reportable adverse events graded 3 , 4 , and 5 according to the terminology criteria for adverse events of the clinical islet transplantation consortium ( cit ) ( 5 ) , and serious adverse events ( 6 ) . islet recipients enrolled in cit protocols consenting to have their data shared with the citr are registered in the citr and included in the citr reports . cit protocols comprise a series of phase ii and phase iii clinical trials designed to test current immunosuppressive strategies and management practices and pursue licensure for clinical islet transplantation in the u.s . site participation in the registry requires local research ethics board approval , strict assurance of patient - identifier confidentiality , and written informed consent by the islet recipients . at preinfusion and at each scheduled follow - up visit , five coprimary end points were assessed by laboratory measurements or clinical evaluations : basal c - peptide ( further divided as 0.3 vs. < 0.3 ng / ml ) , including reported complete graft loss ( defined as fasting c - peptide consistently undetectable with stimulated c - peptide < 0.3 ng / ml by local assay without subsequent recovery to 0.3 ng / ml or reinfusion , also denoted as no function ) ; independence from exogenous insulin for 14 consecutive days ; hba1c ( further divided as < 6.5% and/or a drop by two percentage points or more ) ; fasting blood glucose ( further divided as 60140 vs. < 60 or > 140 mg / dl ) ; and absence of severe hypoglycemia episodes ( requiring assistance of another person ) . annual time points from the last of one or more infusions per recipient were used in this analysis . when direct data were missing but graft function was known to have been previously lost and not restored , insulin independence was set as dependent and c - peptide was set at 0 . the information was summarized again over two to six infusion events per recipient . immunosuppression agents were noted as each given or not at each infusion and during the follow - up . recipient , donor , islet , and immunosuppression characteristics , based on numbers with data generalized estimating equations with repeated measures per recipient were used to assess the effect of era ( 19992003 , 20032006 , 20072010 ) , follow - up time after the first infusion , and other covariates on the rate ( prevalence ) of the desirable outcome for each primary end point . the occurrence and outcomes of clinically reportable adverse events ( craes ) , classified as unlikely , probably , or definitely related to the infusion procedure or to the immunosuppression regimen , were analyzed according to era . each recipient was classified and tabulated according to his or her worst outcome of all infusion - related craes and immunosuppression - related craes during the entire period of infusions and follow - up for the recipient . this analysis was based on 677 recipients of allogeneic islet transplantation who consented to the reporting of their data to the citr , with 214 recipients in 19992002 ( early ) , 255 in mid-20032006 , and 208 in 20072010 ( recent ) ; 423 ( 62% ) came from north america , and 254 ( 38% ) were reported from the european and australian jdrf sites . transplants comprised islet alone in 575 ( 85% ) and iak or simultaneous islet kidney ( iak / sik ) transplant in 102 ( 15% ) . the citr data represent 81% of all islet transplants performed in the north american and jdrf european and australian centers between 1999 and 2010 . in 2007 , there were fewer than half as many north american centers performing islet transplants and one - third of the total number of islet allografts performed compared with 2005 at a time when the commonly used collagenase enzyme liberase became unavailable . a distinct resurgence in islet transplant activity occurred in 2008 with the available collagenase products and the start - up of the cit trials . in the most recent era , there has been a shift to induction with a t - cell depleting ( tcd ) antibody , with or without an inhibitor of tumor necrosis factor- ( tnf- ; e.g. over time , recipients with c - peptide 0.3 ng / ml have been excluded . increasingly , recipients have been selected at older age and with longer type 1 diabetes duration , requiring slightly less insulin and having better kidney function , as indicated by lower serum creatinine , suggesting more appropriate patient selection . there were also definite shifts in preservation method and collagenase type , and more islet preparations were cultured . recent years have seen a substantial decline in the use of daclizumab , with a substantial rise in polyclonal t - cell depleting antibodies and/or etanercept , as well as notable declines in sirolimus use , with increased use of mycophenolic acid . there were increasing levels of missing data with longer follow - up , which is a mixture of data unavailable from the medical record and data still pending entry into the registry . the percentages of missing data for insulin independence were 3% at 1 year , 5% at 3 years , and 7% at 5 years and for other primary end points were 10 to 20% over years 13 . of those who received transplants in the 19992002 era , 51% were insulin - independent at 1-year after the first infusion , regardless of reinfusion , and this declined to 36% at 2 years and to 27% at 3 years . by contrast in the 20072010 era , 66% were insulin - independent at 1 year , 55% at 2 years , and 44% at 3 years ( p = 0.01 , fig . the decline in the rate of insulin independence during 5 years of follow - up in all eras is significant ( p < 0.001 ) . the difference in this decline among the three eras ( p = 0.006 for years - by - era ) indicates that the rate of decline is less steep , showing notable improvement in durability in the most recent era . durability of islet graft function , as measured by fasting c - peptide 0.3 ng / ml , improved significantly over the eras ( p < 0.001 , fig . nearly all islet recipients had significant improvements in hba1c and fasting blood glucose after islet transplantation . the composite end point of hba1c < 6.5% or a drop by two or more percentage points shows improvement from the early era to the mid era ( p = 0.03 ) , although no further improvement in the most recent era , with 25-year success rates of 5060% in the recent era ( fig . fasting blood glucose showed a marked improvement from the early to mid eras ( p < 0.01 , not shown ) . a : rates of insulin independence after allogeneic islet infusion ( islet transplant alone and iak ) , annually after last infusion . left : by era ( p = 0.02 ) . b : durability of graft function ( basal c - peptide 0.3 ng / ml ) after the last infusion , by era ( p < 0.001 ; left ) . , c - peptide never 0.3 ng / ml ) . in the most recent era , 95% of those who ever achieved insulin independence ( ii ) retained graft function through 3 years after last infusion compared with 70% for those who never achieved ii ( p < 0.001 ; right ) . c : percentage of patients with hba1c < 6.5% or drop by two percentage points ( p = 0.03 ; left ) ; and absence of severe hypoglycemic events regardless of complete graft failure ( p = ns by era ; there were insufficient data from 20072010 ; right ) . d : the percentage with hba1c < 6.5% or drop by 2% increases with increasing c - peptide level ( p < 0.001 ; left ) , as does absence of severe hypoglycemic events ( p < 0.001 ; right ) , annually after the last infusion . available data on severe hypoglycemic events , regardless of previous graft loss ( c - peptide < 0.3 ng / ml without recovery ) , shows > 90% remained free of severe hypoglycemic events in all eras , and this relationship persisted through 5 years of follow - up ( fig . any differences by era on resolution of severe hypoglycemic events were neither detectable nor important relative to this sustained , high level of benefit . if data on severe hypoglycemic events were missing and previous complete graft loss was counted as return to severe hypoglycemic events an extreme assumption there was still improvement in 20032006 compared with 19992002 at years 24 ( p = 0.03 , not shown ) . concurrent c - peptide is a strong correlate of all the other primary outcomes : the higher the c - peptide , the greater the likelihood of hba1c < 6.5% or a drop by two percentage points ( p < 0.001 ; fig . 2d ) , the greater the likelihood of absence of severe hypoglycemic events ( p < 0.001 ; fig . 2d ) , the greater the likelihood of fasting blood glucose in the 60140 mg / dl range ( p < 0.001 , not shown ) , and the greater the likelihood of insulin independence ( p < 0.001 , not shown ) . islet reinfusion has decreased substantially during the 12-year period : 48% of recipients were reinfused by 1 year in 20072010 vs. 6065% in 19992006 ( p < 0.01 ) . mortality is low in this group of type 1 diabetic individuals with substantial disease burden , with stable event rates during the 12-year period ( fig . the incidence of life - threatening events has declined ( p = 0.002 ; fig . the incidence of any crae in year 1 declined from 50 to 53% in 19992006 and to 38% in 20072010 ( p = 0.02 ; fig . 3d ) ; however , there are no published comparable follow - up data in similar groups of type 1 diabetes . a : mortality by era ( p = 0.49 ) . c : incidence of any adverse event ( ae ) in year 1 of first infusion ( p = 0.02 by era ) . in north america , the number of centers performing clinical islet transplants and the total number of islet transplants declined in 20062007 , with a distinct resurgence in 2008 . the reasons for the decline are not directly captured by the registry but likely reflect changes in the production and availability of the collagenase enzymes used for islet digestion , tempered enthusiasm with respect to long - term clinical outcomes of insulin independence ( 7,8,10 ) , concern for effect of immunosuppression on kidney function in islet - alone recipients ( 11,12 ) , concern for risk of sensitization to donor hla ( 1315 ) , and saturation of the referral base for patients with the severest forms of unstable type 1 diabetes . however , with the start of the new cit protocols in 2008 , coupled with more encouraging recent trends in longer - term outcomes with novel protocols using t - cell depletion for induction ( 16,17 ) , the number of new islet cell recipients has increased annually in the most recent era . direct evidence is presented of the importance of durable islet graft function to achieve multiple clinical benefits as a consequent effect . stratifying the citr data by era of transplant shows a compelling trend toward better outcomes in the recent era ( p 0.01 ) , despite the still relatively low total number of islet transplant recipients worldwide . there is an indication of moving toward selection of older recipients with longer - standing diabetes and absence of c - peptide to tip the risk - to - benefit ratio in their favor . whole pancreas transplantation is an approved option for -cell replacement in type 1 diabetes , although it is mostly limited to patients simultaneously receiving a kidney transplant for diabetic nephropathy and often excludes older patients and those with coronary artery disease due to the potential for significant surgical morbidity . thus , islet transplantation may offer a complementary alternative to whole pancreas transplantation in patients who are not candidates for or are unwilling to accept the risks of major surgery , and so some estimation of comparative efficacy is required . in the 20072010 era , islet graft survival ( c - peptide 0.3 ng / ml ) of 92% at 1 year and 83% at 3 years ( fig . 2b ) compares very favorably with whole pancreas graft survival of 80% at 1 year and 61% at 3 years ( 21 ) . in the recent era , these graft survival rates translate to an unconditional 44% insulin independence at 3 years ( fig . 2a ) , the highest long - term islet transplant success rate observed to date . although this is still short of the 61% insulin independence reported in the most successful cohort of type 1 diabetes pancreas - alone transplant recipients ( 22 ) , this difference may be explained by the transplantation of 100% of a normal islet -cell mass with a whole pancreas compared with a variable islet -cell mass surviving the engraftment of isolated islets and resulting in a reduced -cell secretory capacity ( 23 ) . in addition , throughout the 12-year period , these data show an enduring benefit in hba1c reduction and stabilization of fasting blood glucose . importantly , the presence of insulin - dependent islet graft survival defined by c - peptide > 0.3 ng / ml confers protection from severe hypoglycemia , and this effect persists even after the islet graft is lost . this declining rate of islet graft loss by era suggests that more recent strategies of immunosuppression , as identified in the multivariate analysis , may better protect islets from alloimmune rejection and recurrent autoimmunity . the successful strategies have all included cnis that are known to exhibit -cell toxicity at high doses ; however , one study showed modern use of lower - dose , cni - based immunosuppression resulted in a 100% normal -cell secretory capacity in whole pancreas transplant recipients ( 23 ) , supporting that these agents can be used and may even be necessary for successful islet transplantation . finally , the finding that the rate of graft function loss was significantly reduced when insulin independence was previously achieved suggests that the engraftment of a sufficient islet -cell mass to eliminate the need for exogenous insulin may mitigate nonimmunologic islet graft loss believed to occur in the setting of increased -cell demand . the citr shows consistent trends toward improved primary outcomes of islet transplantation in the cohort who received transplants in 20072010 compared with those in 19992006 . emerging innovations in islet production , processing , delivery , and immunosuppressive protection islet transplantation has already moved from phase i / ii to phase iii evaluation , with the results from the cit eagerly awaited to provide efficacy and safety information for a standardized approach to islet isolation and immunosuppression management .

there are different types of tissues and other structures that can be distinguished within the bone . important constituents of the bone are adipose tissue and the other hematopoietic tissues , along with other structures such as cartilage , articulate surfaces of the joints , periosteum , nerves and vessels . macroscopically , the bone tissue can be divided into compact bone and spongy bone [ 13 ] . the compact bone is more resistant to mechanical forces than spongy bone and it is composed of osteons ( osseous laminae arranged in a concentric pattern around haversian canals ) . the spongy bone is composed of a network of trabecula of different shape and thickness arranged along the directions of action of mechanical pressure forces . therefore , the main protective function of spongy bone is its elasticity in preventing workload . epiphysis and metaphysis of long bones are mainly composed of spongy bone . the bone is a unique type of connective tissue composed of certain types of cells differentiated from mesenchymal line such as pluripotential osteogenic cells , osteoblasts , osteocytes ; and cells differentiated from hematopoietic line such as pro - osteoclasts and osteoclasts and an extracellular matrix which consists of an organic matrix and mineral components ( apatite crystals , calcium salts and water ) [ 46 ] . therefore , the bone structure strength is determined by the mineral components , whereas the elasticity and toughness of the tissue by the organic components . complementary relations between the mineral and the organic components ensure the optimal mechanical properties of bone . changes in tissue organization and composition cause differences in strength and biomechanical properties of the bone . the amount and quality of mineral and organic components , orientation and cross - linking of the collagen fibers , contribute to the quality of both types of bone tissues . the arrangement of collagen fibers and apatite crystals and their composition in the trabecula can be determined at the microstructural level . each trabecula is characterized by different arrangement of collagen fibers , which is understood in this work as different orientation of collagen fibers . in spongy bone tissue the process of organization and composition is more intense than in compact bone tissue because spongy bone is metabolically more active . the frequency of bone fractures is higher in elderly population , the fractures happen unexpectedly during common daily activities [ 13 , 14 ] . a frequent site of typical osteoporotic fracture is below the head of the femur . an array of analytical tools including infrared and raman spectroscopy have been used to study biological material . firstly , these physical techniques give information about the structure and composition of material studied on the microstructural level . secondly , spectroscopic data are obtained in a non - invasive manner , so the same sample can be examined by various analytical methods . thirdly , there is no limit to the size , allowing a study of large area of a sample . raman spectroscopy offers superior spatial resolution 0.61 m compared to infrared spectroscopy 510 m . in this way raman techniques enable analysis of biologically important sites such as individual cement lines , individual lamellae , regions around micro cracks , and human dentin tubules , which would be impossible by infrared techniques [ 18 , 19 ] . in contrast to infrared spectroscopy , raman spectroscopy is relatively insensitive to water , allowing analysis of fully hydrated samples , with minimal sample preparation . nowadays , the newest raman instruments are able to investigate most of the biological samples , especially bone tissue , in the visible range without fluorescence effect . because of heterogeneous nature of bone , a single point in raman microspectroscopy can not sufficiently describe chemical composition and structure of the sample . for this reason , raman spectral imaging is becoming increasingly popular for the study of complex organized systems , because it gives spatial information on samples [ 21 , 22 ] . in earlier studies raman microspectroscopy has been used to determine carbonate to phosphate ratios and phosphate to amide ratios in several tissue types [ 18 , 19 , 23 ] . raman spectral mapping has been used to demonstrate the orientation of mineral and collagen components in osteonal lamellae of the cortical bone and has been shown to permit imaging of two adjacent orthogonal planes in cortical in order to obtain 3d information . raghavan et al . have shown the conditions under which polarized raman spectroscopy can be used to quantitatively measure mineral and matrix orientation in highly turbid bone tissue . systematic errors in the orientation distribution calculations have been minimized by employing a high numerical aperture objective . raman microspectroscopy has been applied to compare cortical and spongy bone tissue from the same femur and tibia of standard laboratory mice . other authors have presented the potential of raman microspectroscopy for the intravital study of bone with using bone chamber . the use of bone chamber in raman studies allows monitoring changes in composition of bone and biomaterials in living animals over time . the study materials were five femur heads from patients who underwent osteoporotic fractures below the head of femur . the material from a group of five patients between 60 and 74 years of age was studied . the treatment of choice in the above mentioned patients was hemialloplastic procedure ( bipolar prosthesis was applied ) . the cross - sections of 5 mm in thickness were obtained from heads of femurs of these patients . in this work , the results of a study on one representative femur head from a 67 year old woman are presented . since the head of femur consists mainly of spongy bone , this part of bone tissue attracted a great deal of attention . the study has got the approval of the local bioethical commission at the wielkopolska medical chamber in pozna ( no 14/2008 from august 27 , 2008 ) . in the present study , raman microspectroscopy was employed to analyze the mineral and organic constituents and orientation of collagen fibers in spongy bone tissue of the human head of the femur . changes in composition and structure of the spongy bone tissue were illustrated using maps of polarized raman spectra . the results demonstrate the versatility of the raman method as the analytical spectroscopic technique and provide insights into the organization of spongy bone tissue at the microstructural level . determination of the composition and collagen fibers arrangement permits better understanding of bone physiology and evaluation of the biomechanical properties of bone . this investigation contributes to development of a method allowing identification of persons at risk of bone fractures . all the measurements were carried out on renishaw invia microscope with diode pumped laser characterized by 500 mw power and emitting 785 nm infrared wavelength . laser beam was focused on the sample through the long working distance of the 50/0.5 objective . multiple scattering in turbid bone tissue cause light depolarization and introduces errors in polarized raman measurements . the use of the smallest depth of field allows minimization of depolarization effects , which is needed for determination of collagen orientation in bone tissue . confocal system improved axial resolution and the depth of field with this objective was 2.2 m . the air - cooled ccd camera detector ( rencam ) and 1200 mm diffraction grating were used . at the beginning of each experimental session the laser was checked for alignment with the optical axis of the microscope and the spectral data ( wavenumber and intensity ) were verified with the use of 521 cm band of a silicon internal sample . thanks to the above procedure , the raman spectra were obtained in the same spectral conditions . for bone studies , the overall spectral resolution was better than 1 cm , while the microscope objective ensured a spatial resolution of 2 m . during measurements , the raman maps of spongy bone tissue were obtained using the motorized in the three axes stage of the microscope allowing to monitor the sample through an optical camera . the raman images were acquired in rectangular areas of 90 90 m and at 10 m step size at five different sites of the spongy bone tissue of particular femur heads . the raman scattered light could be detected in the linear polarization ( vertical and horizontal direction ) using polarisers . the time of exposure to get individual raman spectra was 10 s , the spectra were recoded without accumulation . cosmic ray artefacts were removed and analyses of the spectra were performed in the same wire 3.0 ( renishaw ) software . rayleigh scattering background was subtracted manually from each raw spectrum by using the polynomial curve . the images which display changes in composition and orientation of collagen fibers in spongy bone tissue were generated by the originpro 8.0 software . figure 1a shows a typical raman spectrum of spongy bone tissue . the major bands in raman spectra of bone tissue corresponding to mineral and organic constituents are labeled . information about mineral and organic composition is obtained simultaneously , giving a complete arrangement of the bone constituents in the area surveyed . a very important fact is that the bands corresponding to these two phases are clearly separated . 1b depicts the range of wavenumbers in which the bands in the raman spectrum of bone can occur and specifies the bone structure components corresponding to these bands . the mineral part of the spectrum is dominated by the 1 phosphate ( po4 ) internal mode at 961 cm . the other markers of the mineral component connected with the 2 and 4 phosphate vibrations are detected at 430 and 587 cm , respectively [ 16 , 25 , 27 ] . the 3 phosphate vibration is not well visible in the region 10351048 cm and is overlapped with that of carbonate , which is substituted in the apatite structure , appearing in the region 10701075 cm , so it can not be used to detect changes in the spongy bone tissue . both b - type and a - type co3 bands are observed at 1073 and 1103 cm , respectively . however , the weakness of the a - type carbonate band does not permit getting information about the composition of spongy bone tissue , therefore b - type mode only is used . the major bands assigned to the organic components are found in region : ~12001320 cm ( amide iii ) , ~15951700 cm ( amide i ) , ~14001470 cm and ~28003100 cm ( bending and stretching modes of c h groups , respectively ) [ 16 , 24 ] . in this study the maxima of the bands assigned to amide i and amide iii are at 1655 and 1300 cm respectively . the bands assigned to c h groups present in both collagenous and noncollagenous moieties , are visible at 1443 cm and 2852 , 2877 , 2935 cm.fig . 1typical raman spectrum of spongy bone tissue showing the major bands and the corresponding compounds . the wavenumber ranges in which the bands can occur in the raman spectrum of bone tissue ( b ) typical raman spectrum of spongy bone tissue showing the major bands and the corresponding compounds . the wavenumber ranges in which the bands can occur in the raman spectrum of bone tissue ( b ) the raman signal depends not only on the composition but also on the local orientation of collagen fibers or apatite crystals and changes according to the polarization of incident and scattered light , so interpretation of the spectra of bone tissues is more complicated than that of those of isotropic materials [ 24 , 27 , 28 ] . the collagen triple helix structure determines the positions of the amide bonds with respect to the backbone . the amide i band is predominantly associated with the c = o stretching vibration , whereas the amide iii mode is mostly connected with c therefore , the behavior of polarized bands of amide i and iii is completely different . the intensity of amide i band is higher for the polarization perpendicular to the collagen fibers , while the amide iii band is characterized by two different c n vibration modes corresponding to perpendicular and parallel conformations . when both conformations are combined , no orientation effect is detectable . the crystallographic c - axis of apatite crystals is along the collagen fibers and is associated with 1 phosphate vibrations . thus the 1po4 band intensity has a maximum value when the polarization of incident light is parallel to the orientation of collagen fibers . reported that raman band intensities depend strongly on both chemical composition and collagen fibers orientation of cortical bone tissue with respect to the linear polarization of the incident light . this work has also shown that the bands , such as 1po4 and amide i are quite sensitive to the orientation and the polarization of the incident light , so these bands give information about the orientation of collagen fibers . in contrast , the bands such as those assigned to amide iii , 2po4 and 4po4 are less sensitive to the orientation effects and indicate changes in mineral and organic composition of bone tissue . we are concerned with a similar but not the same problem . in our work raman microspectroscopy was employed to determine chemical composition and orientation of collagen fibers in spongy bone tissue . in this study the linearly polarized incident and also scattered light was used to obtain raman maps to show changes in the structure of trabecula surface . the intensity of individual raman bands can not be used as an empirical measure of the content of mineral and organic components in bone tissue , because the irregularity of biological material surface strongly influences the bands intensity . during measurements the distance from the objective to the sample is changed , therefore the focus of the laser beam is also changed . in order to remove the impact of this factor , the ratios of intensities of the appropriate bands in the raman spectra were employed . figure 2 shows the line spectra of the ( a ) 2po4/amide iii ( b ) 4po4/amide iii , ( c ) 1po4/amide i and ( d ) 1co3/2po4 ratios . this result is obtained from raman maps of spongy bone tissue for different polarizations of the incident light . 2e illustrates the area of the bone tissue studied by raman spectra with 10 measurement lines . the band intensities of the raman spectra obtained from these lines are presented on ratio plots . the investigation was carried out over rectangular areas of 90 90 m and the raman spectra were acquired at 10 m step size.fig . 2plots of 2po4/amide iii ( a ) , 4po4/amide iii ( b ) , 1po4/amide i ( c ) and 1co3/2po4 ( d ) ratios obtained from raman maps of spongy bone tissue with horizontal ( thin gray line ) and vertical ( thick black line ) polarization of laser light . the lines show directions of successive measurements plots of 2po4/amide iii ( a ) , 4po4/amide iii ( b ) , 1po4/amide i ( c ) and 1co3/2po4 ( d ) ratios obtained from raman maps of spongy bone tissue with horizontal ( thin gray line ) and vertical ( thick black line ) polarization of laser light . the lines show directions of successive measurements as markers of chemical composition the ratios 2po4/amide iii , 4po4/amide iii and 1co3/2po4 were used . the bands used in the above ratios are less sensitive to orientation effect as shown in fig . 2a , b and d. the ratios of 2po4/amide iii and 4po4/amide iii ( fig . 2a and b ) do not change significantly for the horizontal and vertical polarization of incident light . as a result , the maps of these ratios could be used to give the information on the relation of the mineral and organic components content in the spongy bone tissue . similarly , no considerable differences were noted for the ratio of 1co3/2po4 ratio ( fig . however , the 1co3/2po4 ratio reveals more pronounced differences for the above two polarizations , but is not sensitive to fibers orientation . thus the maps of 1co3/2po4 ratio inform about the relative content of two mineral components , carbonates to phosphates , in the same area of the spongy bone tissue . the collagen orientation in trabecula is shown on the maps of 1po4/amide i ratio because these bands are quite sensitive to the orientation effect . 2c ) displays pronounced orientational effect , which is not observed in the other ratio plots . changes in the fibers orientation in the trabecula surface studied appear in the seventh line . then the 1po4/amide i ratio decreases for the vertical polarization of incident light and increases for the horizontal polarization . in the ratio plot , the orientation effect connected with collagen fibers arrangement is visible due to the phase differences between 1po4 and amide i components appearing because their vibration directions are perpendicular to each other . the highest value of 1po4/amide i ratio is obtained when the polarization of incident light is parallel to the collagen fiber orientation , in contrast , the lowest value of 1po4/amide i ratio is obtained when the polarization of incident light is perpendicular to the collagen fiber orientation . consequently , the maximum ratio of 1po4/amide i in the vertical polarization corresponds to the minimum value of this ratio in the horizontal polarization ( fig . 2c ) raman spectroscopy with a micro - level spatial resolution allows generation of images mapping the raman spectra and in consequence permits identification of local variations in composition and structure of bone tissue . figures 3 and 4 present images of the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . the maps of raman spectra generated for the vertical and horizontal polarization of incident light are presented in fig . 3 and those generated for different polarizations of the incident and scattered light the maximum values of the ratios are specified and the minimum value is 0 for all images . the bright contrast corresponds to the maximum ratio , while dark to the lowest one.fig . 3the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . arrows indicate polarization of laser light , and the color bar displays the maximum ratio for each imagefig . 4the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . arrows indicate polarisation of laser incident and scattered beams , and the color bar displays the maximum ratio for each image the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . arrows indicate polarization of laser light , and the color bar displays the maximum ratio for each image the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . arrows indicate polarisation of laser incident and scattered beams , and the color bar displays the maximum ratio for each image conclusions on the chemical composition can be drawn from fig . 3 showing the ratios 2po4/amide iii ( a ) , 4po4/amide iii ( b ) and 1co3/2po4 ( c ) obtained for the vertical polarization of laser light . 3a , b indicate the degree of mineralization and give information about changes in the content of hydroxyapatite crystals with respect to that of collagen in the trabecula . the images present very similar contrast changes ; i.e. , brighter and darker areas are in the same regions and correspond to higher and lower ratios of mineral to organic content . in fig . . the higher ratio of phosphate to carbonate crystals occurs in the same area as the higher ratio of hydroxyapatite to collagen content in fig . 3a , b. figure 3d displays 1po4/amide i ratio contrast image of the bands quite sensitive to the orientation effect . this image should allow determination of the collagen fiber orientation in trabecula , but it does not show a pronounced structural effect . figure 3d reveals slight contrast changes in comparison with those in the other ratio images referring to the chemical composition ( fig . the similarity in the character of the maps referring to the collagen orientation and chemical composition is probably related to the distribution of bone tissue constituents . this means that differences in contrast in the maps referring to chemical composition are too large with respect to those referring to collagen fibers orientation so the orientation effect is undetectable . figure 3 displays images for 2po4/amide iii ( e ) , 4po4/amide iii ( f ) , 1co3/2po4 ( g ) and 1po4/amide i ( h ) ratios obtained for the horizontal polarization of laser light . 3e g ) are similar and the contrasts in them change a similar way . the small differences in contrast are justified because the bands used in these ratios are less sensitive to orientation effect . the images obtained in the orthogonal polarization vertical and horizontal are quite similar and do not show any significant differences . the slight changes in the images based on the ratios 2po4/amide iii , 4po4/amide iii , 1co3/2po4 are a result of different behavior of the amide iii band in light of different polarizations . however , the changes in the amide iii band are much smaller than in that of amide i , so only the first band gives information about organic composition . 3h based on the bands sensitive to collagen fiber orientation shows contrast changes similar to those in the maps referring to chemical composition in trabecula ( fig . therefore , it is difficult to conclude if the highest contrast corresponds to the orientation of collagen fibers or to the changes in chemical composition . 3d and h are almost identical and their contrasts do not change much for different light polarizations . perhaps , such small changes in contrast result from a particular orientation of collagen fibers on this surface . if the orientation is not parallel to the horizontal or vertical polarization of laser light , so if the fibers are skewed , then the changes in polarizability of collagen molecule are detected for both light polarization . therefore , the ratio images obtained for vertical and horizontal polarizations are very similar . detailed comparative analysis of fig . 3h indicate the area with bright color contrast that corresponds to the dark contrast areas in fig . it could mean that collagen orientation is parallel to horizontal direction in this part of trabecula surface . in the other sites of the images , no distinct differences in contrast appear , so it is impossible to say anything more about collagen orientation . images in fig . 4 were obtained for appropriate different polarizations of the incident and scattered light . 4a d were obtained for the vertical polarization of the incident and scattered beams , while fig . the maps based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) do not show considerable changes , so these images were taken as an indicator of mineral content to organic content in spongy bone tissue . the images based on the ratios of 1co3/2po4 ( c , g ) do not show distinct changes for different polarizations of scattered light , similarly as those of 2po4/amide iii and 4po4/amide iii , thus they also refer to the spongy bone composition , i.e. , give the information about relative amount of carbonate apatite to hydroxyapatite . g prove that 2po4 , 4po4 , 1co3 and amide iii bands are less susceptible to polarization effects . unlike these bands , those of 1po4 and amide i are characterized by higher sensitivity to polarization effects , therefore the ratio of 1po4/amide i could show the fiber orientation . 3d , h obtained for the incident light polarized in two different directions do not reveal considerable differences , whereas the contrast images in fig . 4d , h obtained for the polarized incident and scattered laser beams display pronounced changes . when the polarization of laser incident and scattered light is the same , the contrast in ( fig . 4d ) is similar to that in the images which give information about the bone composition ( fig . 4a , b ) . on the other hand , when the polarizations of incident and scattered light are mutually perpendicular , then the contrast ( fig . such different behavior for the polarized scattered light is a result of the presence of collagen fibers in the structure of spongy bone tissue . a certain area on the trabecula surface corresponds to high intensity values ( bright contrast ) in fig . changes in intensity ratio induced by changes in the polarization of scattered light allow determination of the orientation of collagen fibers . for example , we can conclude that the collagen fibers are arranged in parallel to the incident beam polarization ( or scattered beam polarization ) only when the polarizations of these two beams are the same , which corresponds to bright color contrast . however , to verify this conclusion , the area of bright contrast must correspond to the area of dark contrast on the map for the orthogonal polarizations of the incident and scattered light ( fig . the analysis of collagen orientation in trabecula is performed on the basis of fig . 4d , h. the arrows in fig . 4d indicate collagen fibers arrangement , which is close to vertical direction . the arrows in fig . 4h indicate the region of dark contrast , so taking into account the above , it is possible to conclude that the collagen fibers are arranged in perpendicular to these arrows . the results allow concluding that 1po4/amide i ratios permit determination of the orientations of collagen fibers in spongy bone tissue . this work presents possibilities of raman spectroscopy application for determination of chemical composition and orientation of collagen fibers in human spongy bone tissue . raman microspectroscopy had been employed to study organization of collagen fibers and material composition in cortical bone by kazanci et al . . this study shows that the raman spectral maps allow determination of local variations in mineral and organic content in the human spongy bone and changes in orientation of collagen fibers on the bone surface . analysis of the ratios of 2po4/amide iii , 4po4/amide iii and 1co3/2po4 could be used to conclude about the relative amount of bone tissue components . in this study , it is not important which polarization of laser beam is used , because these ratios are less sensitive to the orientation effect . changes in the ratio of 1po4/amide i for the polarized incident and scattered light allow determination of the arrangement of collagen fibers in trabecula . the ratio maps obtained for the same polarization of incident and scattered light give information about the orientation of collagen fibers , but also on the distribution of the material components . the ratio maps obtained in the orthogonal polarization of incident and scattered light reveal the sites at which the ratios change in comparison with the ratio maps obtained in the same polarization . this process separates the orientational and compositional changes and permits identification of the sites of collagen fibers occurrence in trabecula . the versatility of the raman method as the analytical spectroscopic technique offers a possibility to get insights into the organization of bone . raman spectroscopy with micro - level spatial resolution permits detection of local variations in composition and structure of the spongy bone tissue . the above information in combination with evaluation of bone mineral density could allow earlier detection of fracture risk . understanding the bone tissue organization at the microstructural level can help finding the origins of bone diseases such as osteoporosis or osteoarthrosis .

biomechanical properties of bone depend on the composition and organization of collagen fibers . in this study , raman microspectroscopy was employed to determine the content of mineral and organic constituents and orientation of collagen fibers in spongy bone in the human head of femur at the microstructural level . changes in composition and structure of trabecula were illustrated using raman spectral mapping . the polarized raman spectra permit separate analysis of local variations in orientation and composition . the ratios of 2po43/amide iii , 4po43/amide iii and 1co32/2po43 are used to describe relative amounts of spongy bone components . the 1po43/amide i ratio is quite susceptible to orientation effect and brings information on collagen fibers orientation . the results presented illustrate the versatility of the raman method in the study of bone tissue . the study permits better understanding of bone physiology and evaluation of the biomechanical properties of bone .

Introduction Materials and methods Results and discussion Conclusion

macroscopically , the bone tissue can be divided into compact bone and spongy bone [ 13 ] . the compact bone is more resistant to mechanical forces than spongy bone and it is composed of osteons ( osseous laminae arranged in a concentric pattern around haversian canals ) . the spongy bone is composed of a network of trabecula of different shape and thickness arranged along the directions of action of mechanical pressure forces . therefore , the main protective function of spongy bone is its elasticity in preventing workload . epiphysis and metaphysis of long bones are mainly composed of spongy bone . complementary relations between the mineral and the organic components ensure the optimal mechanical properties of bone . changes in tissue organization and composition cause differences in strength and biomechanical properties of the bone . the amount and quality of mineral and organic components , orientation and cross - linking of the collagen fibers , contribute to the quality of both types of bone tissues . the arrangement of collagen fibers and apatite crystals and their composition in the trabecula can be determined at the microstructural level . each trabecula is characterized by different arrangement of collagen fibers , which is understood in this work as different orientation of collagen fibers . in spongy bone tissue the process of organization and composition is more intense than in compact bone tissue because spongy bone is metabolically more active . a frequent site of typical osteoporotic fracture is below the head of the femur . firstly , these physical techniques give information about the structure and composition of material studied on the microstructural level . in this way raman techniques enable analysis of biologically important sites such as individual cement lines , individual lamellae , regions around micro cracks , and human dentin tubules , which would be impossible by infrared techniques [ 18 , 19 ] . in contrast to infrared spectroscopy , raman spectroscopy is relatively insensitive to water , allowing analysis of fully hydrated samples , with minimal sample preparation . nowadays , the newest raman instruments are able to investigate most of the biological samples , especially bone tissue , in the visible range without fluorescence effect . because of heterogeneous nature of bone , a single point in raman microspectroscopy can not sufficiently describe chemical composition and structure of the sample . for this reason , raman spectral imaging is becoming increasingly popular for the study of complex organized systems , because it gives spatial information on samples [ 21 , 22 ] . in earlier studies raman microspectroscopy has been used to determine carbonate to phosphate ratios and phosphate to amide ratios in several tissue types [ 18 , 19 , 23 ] . raman spectral mapping has been used to demonstrate the orientation of mineral and collagen components in osteonal lamellae of the cortical bone and has been shown to permit imaging of two adjacent orthogonal planes in cortical in order to obtain 3d information . have shown the conditions under which polarized raman spectroscopy can be used to quantitatively measure mineral and matrix orientation in highly turbid bone tissue . raman microspectroscopy has been applied to compare cortical and spongy bone tissue from the same femur and tibia of standard laboratory mice . other authors have presented the potential of raman microspectroscopy for the intravital study of bone with using bone chamber . the use of bone chamber in raman studies allows monitoring changes in composition of bone and biomaterials in living animals over time . the study materials were five femur heads from patients who underwent osteoporotic fractures below the head of femur . in this work , the results of a study on one representative femur head from a 67 year old woman are presented . since the head of femur consists mainly of spongy bone , this part of bone tissue attracted a great deal of attention . the study has got the approval of the local bioethical commission at the wielkopolska medical chamber in pozna ( no 14/2008 from august 27 , 2008 ) . in the present study , raman microspectroscopy was employed to analyze the mineral and organic constituents and orientation of collagen fibers in spongy bone tissue of the human head of the femur . changes in composition and structure of the spongy bone tissue were illustrated using maps of polarized raman spectra . the results demonstrate the versatility of the raman method as the analytical spectroscopic technique and provide insights into the organization of spongy bone tissue at the microstructural level . determination of the composition and collagen fibers arrangement permits better understanding of bone physiology and evaluation of the biomechanical properties of bone . laser beam was focused on the sample through the long working distance of the 50/0.5 objective . multiple scattering in turbid bone tissue cause light depolarization and introduces errors in polarized raman measurements . the use of the smallest depth of field allows minimization of depolarization effects , which is needed for determination of collagen orientation in bone tissue . at the beginning of each experimental session the laser was checked for alignment with the optical axis of the microscope and the spectral data ( wavenumber and intensity ) were verified with the use of 521 cm band of a silicon internal sample . thanks to the above procedure , the raman spectra were obtained in the same spectral conditions . during measurements , the raman maps of spongy bone tissue were obtained using the motorized in the three axes stage of the microscope allowing to monitor the sample through an optical camera . the raman images were acquired in rectangular areas of 90 90 m and at 10 m step size at five different sites of the spongy bone tissue of particular femur heads . the raman scattered light could be detected in the linear polarization ( vertical and horizontal direction ) using polarisers . cosmic ray artefacts were removed and analyses of the spectra were performed in the same wire 3.0 ( renishaw ) software . the images which display changes in composition and orientation of collagen fibers in spongy bone tissue were generated by the originpro 8.0 software . figure 1a shows a typical raman spectrum of spongy bone tissue . the major bands in raman spectra of bone tissue corresponding to mineral and organic constituents are labeled . information about mineral and organic composition is obtained simultaneously , giving a complete arrangement of the bone constituents in the area surveyed . 1b depicts the range of wavenumbers in which the bands in the raman spectrum of bone can occur and specifies the bone structure components corresponding to these bands . the 3 phosphate vibration is not well visible in the region 10351048 cm and is overlapped with that of carbonate , which is substituted in the apatite structure , appearing in the region 10701075 cm , so it can not be used to detect changes in the spongy bone tissue . however , the weakness of the a - type carbonate band does not permit getting information about the composition of spongy bone tissue , therefore b - type mode only is used . in this study the maxima of the bands assigned to amide i and amide iii are at 1655 and 1300 cm respectively . 1typical raman spectrum of spongy bone tissue showing the major bands and the corresponding compounds . the wavenumber ranges in which the bands can occur in the raman spectrum of bone tissue ( b ) typical raman spectrum of spongy bone tissue showing the major bands and the corresponding compounds . the wavenumber ranges in which the bands can occur in the raman spectrum of bone tissue ( b ) the raman signal depends not only on the composition but also on the local orientation of collagen fibers or apatite crystals and changes according to the polarization of incident and scattered light , so interpretation of the spectra of bone tissues is more complicated than that of those of isotropic materials [ 24 , 27 , 28 ] . the collagen triple helix structure determines the positions of the amide bonds with respect to the backbone . the intensity of amide i band is higher for the polarization perpendicular to the collagen fibers , while the amide iii band is characterized by two different c n vibration modes corresponding to perpendicular and parallel conformations . thus the 1po4 band intensity has a maximum value when the polarization of incident light is parallel to the orientation of collagen fibers . reported that raman band intensities depend strongly on both chemical composition and collagen fibers orientation of cortical bone tissue with respect to the linear polarization of the incident light . this work has also shown that the bands , such as 1po4 and amide i are quite sensitive to the orientation and the polarization of the incident light , so these bands give information about the orientation of collagen fibers . in contrast , the bands such as those assigned to amide iii , 2po4 and 4po4 are less sensitive to the orientation effects and indicate changes in mineral and organic composition of bone tissue . in our work raman microspectroscopy was employed to determine chemical composition and orientation of collagen fibers in spongy bone tissue . in this study the linearly polarized incident and also scattered light was used to obtain raman maps to show changes in the structure of trabecula surface . the intensity of individual raman bands can not be used as an empirical measure of the content of mineral and organic components in bone tissue , because the irregularity of biological material surface strongly influences the bands intensity . during measurements the distance from the objective to the sample is changed , therefore the focus of the laser beam is also changed . in order to remove the impact of this factor , the ratios of intensities of the appropriate bands in the raman spectra were employed . figure 2 shows the line spectra of the ( a ) 2po4/amide iii ( b ) 4po4/amide iii , ( c ) 1po4/amide i and ( d ) 1co3/2po4 ratios . this result is obtained from raman maps of spongy bone tissue for different polarizations of the incident light . 2e illustrates the area of the bone tissue studied by raman spectra with 10 measurement lines . the band intensities of the raman spectra obtained from these lines are presented on ratio plots . the investigation was carried out over rectangular areas of 90 90 m and the raman spectra were acquired at 10 m step size.fig . 2plots of 2po4/amide iii ( a ) , 4po4/amide iii ( b ) , 1po4/amide i ( c ) and 1co3/2po4 ( d ) ratios obtained from raman maps of spongy bone tissue with horizontal ( thin gray line ) and vertical ( thick black line ) polarization of laser light . the lines show directions of successive measurements plots of 2po4/amide iii ( a ) , 4po4/amide iii ( b ) , 1po4/amide i ( c ) and 1co3/2po4 ( d ) ratios obtained from raman maps of spongy bone tissue with horizontal ( thin gray line ) and vertical ( thick black line ) polarization of laser light . the lines show directions of successive measurements as markers of chemical composition the ratios 2po4/amide iii , 4po4/amide iii and 1co3/2po4 were used . the bands used in the above ratios are less sensitive to orientation effect as shown in fig . 2a , b and d. the ratios of 2po4/amide iii and 4po4/amide iii ( fig . as a result , the maps of these ratios could be used to give the information on the relation of the mineral and organic components content in the spongy bone tissue . thus the maps of 1co3/2po4 ratio inform about the relative content of two mineral components , carbonates to phosphates , in the same area of the spongy bone tissue . the collagen orientation in trabecula is shown on the maps of 1po4/amide i ratio because these bands are quite sensitive to the orientation effect . changes in the fibers orientation in the trabecula surface studied appear in the seventh line . in the ratio plot , the orientation effect connected with collagen fibers arrangement is visible due to the phase differences between 1po4 and amide i components appearing because their vibration directions are perpendicular to each other . the highest value of 1po4/amide i ratio is obtained when the polarization of incident light is parallel to the collagen fiber orientation , in contrast , the lowest value of 1po4/amide i ratio is obtained when the polarization of incident light is perpendicular to the collagen fiber orientation . 2c ) raman spectroscopy with a micro - level spatial resolution allows generation of images mapping the raman spectra and in consequence permits identification of local variations in composition and structure of bone tissue . figures 3 and 4 present images of the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . 3 and those generated for different polarizations of the incident and scattered light the maximum values of the ratios are specified and the minimum value is 0 for all images . 3the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . 4the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . arrows indicate polarisation of laser incident and scattered beams , and the color bar displays the maximum ratio for each image the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . arrows indicate polarization of laser light , and the color bar displays the maximum ratio for each image the contrast images based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) , 1co3/2po4 ( c , g ) and 1po4/amide i ( d , h ) . 3a , b indicate the degree of mineralization and give information about changes in the content of hydroxyapatite crystals with respect to that of collagen in the trabecula . , brighter and darker areas are in the same regions and correspond to higher and lower ratios of mineral to organic content . 3a , b. figure 3d displays 1po4/amide i ratio contrast image of the bands quite sensitive to the orientation effect . figure 3d reveals slight contrast changes in comparison with those in the other ratio images referring to the chemical composition ( fig . the similarity in the character of the maps referring to the collagen orientation and chemical composition is probably related to the distribution of bone tissue constituents . this means that differences in contrast in the maps referring to chemical composition are too large with respect to those referring to collagen fibers orientation so the orientation effect is undetectable . the small differences in contrast are justified because the bands used in these ratios are less sensitive to orientation effect . the slight changes in the images based on the ratios 2po4/amide iii , 4po4/amide iii , 1co3/2po4 are a result of different behavior of the amide iii band in light of different polarizations . however , the changes in the amide iii band are much smaller than in that of amide i , so only the first band gives information about organic composition . 3h based on the bands sensitive to collagen fiber orientation shows contrast changes similar to those in the maps referring to chemical composition in trabecula ( fig . therefore , it is difficult to conclude if the highest contrast corresponds to the orientation of collagen fibers or to the changes in chemical composition . perhaps , such small changes in contrast result from a particular orientation of collagen fibers on this surface . if the orientation is not parallel to the horizontal or vertical polarization of laser light , so if the fibers are skewed , then the changes in polarizability of collagen molecule are detected for both light polarization . it could mean that collagen orientation is parallel to horizontal direction in this part of trabecula surface . in the other sites of the images , no distinct differences in contrast appear , so it is impossible to say anything more about collagen orientation . the maps based on the ratios of 2po4/amide iii ( a , e ) , 4po4/amide iii ( b , f ) do not show considerable changes , so these images were taken as an indicator of mineral content to organic content in spongy bone tissue . the images based on the ratios of 1co3/2po4 ( c , g ) do not show distinct changes for different polarizations of scattered light , similarly as those of 2po4/amide iii and 4po4/amide iii , thus they also refer to the spongy bone composition , i.e. such different behavior for the polarized scattered light is a result of the presence of collagen fibers in the structure of spongy bone tissue . changes in intensity ratio induced by changes in the polarization of scattered light allow determination of the orientation of collagen fibers . however , to verify this conclusion , the area of bright contrast must correspond to the area of dark contrast on the map for the orthogonal polarizations of the incident and scattered light ( fig . the analysis of collagen orientation in trabecula is performed on the basis of fig . the results allow concluding that 1po4/amide i ratios permit determination of the orientations of collagen fibers in spongy bone tissue . this work presents possibilities of raman spectroscopy application for determination of chemical composition and orientation of collagen fibers in human spongy bone tissue . raman microspectroscopy had been employed to study organization of collagen fibers and material composition in cortical bone by kazanci et al . this study shows that the raman spectral maps allow determination of local variations in mineral and organic content in the human spongy bone and changes in orientation of collagen fibers on the bone surface . analysis of the ratios of 2po4/amide iii , 4po4/amide iii and 1co3/2po4 could be used to conclude about the relative amount of bone tissue components . in this study , it is not important which polarization of laser beam is used , because these ratios are less sensitive to the orientation effect . changes in the ratio of 1po4/amide i for the polarized incident and scattered light allow determination of the arrangement of collagen fibers in trabecula . the ratio maps obtained for the same polarization of incident and scattered light give information about the orientation of collagen fibers , but also on the distribution of the material components . the ratio maps obtained in the orthogonal polarization of incident and scattered light reveal the sites at which the ratios change in comparison with the ratio maps obtained in the same polarization . this process separates the orientational and compositional changes and permits identification of the sites of collagen fibers occurrence in trabecula . the versatility of the raman method as the analytical spectroscopic technique offers a possibility to get insights into the organization of bone . raman spectroscopy with micro - level spatial resolution permits detection of local variations in composition and structure of the spongy bone tissue . the above information in combination with evaluation of bone mineral density could allow earlier detection of fracture risk . understanding the bone tissue organization at the microstructural level can help finding the origins of bone diseases such as osteoporosis or osteoarthrosis .

cancer of the prostate is the most frequently diagnosed cancer in men , accounting for 27% of new cancer cases in the united states ( usa ) in 2014 . in the same year , 10% of the total cancer deaths in men were due to prostate cancer , the second leading cause of cancer death in this population . although the cause of prostate cancer remains unknown , its incidence has been associated with age , ethnicity , family history , physical activity , body mass index , diet , region , and sexually transmitted infections . the seriousness of this disease warrants a more definitive investigation of its association with a history of sexually transmitted diseases . caused by the bacterium neisseria gonorrhoeae , in the year 2008 the world health organization estimated that there were 106 million infected adults globally , making it the most prevalent sexually transmitted bacterial infection . several epidemiologic studies have investigated an association between gonorrhea infection and incidence of prostate cancer , but results have been inconsistent . according to a meta - analysis published by dennis et al . in 2002 , men with a history of gonorrhea were at elevated risk of prostate cancer ( pooled relative risk ratio [ rr ] 1.36 , 95% confidence interval [ ci ] 1.151.61 ) . a meta - analysis reported by taylor et al . in 2005 also indicated that gonorrhea was associated with increased prostate cancer risk ( odds ratio [ or ] 1.39 , 95% ci 1.051.83 ) . however , both of these studies were based predominantly on case - control data for white men . studies that include african american subjects , serologic measures , and prospective data are lacking . more recently , 2 large , prospective , cohort studies and 2 case - control studies failed to confirm an association between a history of gonorrhea and prostate cancer . however , some factors were not considered in their analyses that might limit the evaluation of prostate cancer risk . these include adjustments for confounders , study design , study region , ethnicity , the method of gonorrhea exposure assessment , study quality , and the introduction of psa screening . herein we provide an updated review and meta - analysis of the association between a history of gonorrhea and incidence of prostate cancer , conducting subgroup analyses based on the factors aforementioned . we performed a systematic search of pubmed , embase , and cochrane library databases , for all papers published up to june 2014 . the following search terms were used : ( gonorrhea or neisseria gonorrhoeae or sexually transmitted diseases or sexually transmitted infections or venereal disease ) and ( prostate cancer or prostatic neoplasms or prostatic cancer or prostate neoplasms ) . we also searched the reference lists of all the retrieved articles to identify any other potentially relevant articles . to be included in this meta - analysis , the papers had to report a case - control or cohort study ; evaluate the association between gonorrhea and the incidence of prostate cancer ; provide relative risk ratios , ors , and 95% cis , or sufficient information to calculate these ; and be published in english . if multiple publications from the same study population were available , only the one with the largest sample size was included . studies were excluded if they did not conform to the inclusion criteria above , or contained duplicate data , or were based on incomplete raw data or irrelevant data . no case reports , letters , reviews , editorials , or correspondence articles were considered . two investigators ( wen - qing lian and fei luo ) independently reviewed and extracted information from all eligible publications , in accordance with the inclusion and exclusion criteria listed above . disagreement was resolved by discussion between the 2 authors until a consensus was reached . if a consensus could not be reached , a third author ( xian - lu song ) was consulted and a final decision was determined by majority opinion . data extracted from the publications included the first author , year of publication , country in which the study was performed , study design , study period , sample size , ages and ethnicities of the subjects , exposure assessment , and the confounders adjusted for . the subjects ethnicities were categorized as white , african american , asian , other , or mixed ( a population with individuals of different ethnicities ) . for studies conducted in the usa , when the paper did not specify the ethnicity of the study population , the most probable ethnicity was recorded , based on the predominant ethnic group in the study country . the methodological quality of each eligible study was independently assessed by 2 reviewers ( wen - qing lian and fei luo ) based on the newcastle - ottawa scale , in which the total score ( in stars ) can range from 0 to 9 . a third party ( xian - lu song ) was involved if a consensus could not be reached . a study was considered to be of high quality if the newcastle - ottawa scale score was 7 stars ; studies given 56 stars were judged to be of moderate quality . due to the low incidence of prostate cancer , the relative risk ratio can be mathematically approximated by the or . in the present study , the or and its 95% ci was used to assess the association between gonorrhea and the risk of prostate cancer . in some studies , risk estimates were stratified according to ethnic categories , and risk to the total group was not reported . for these studies , the study - specific effect size in the overall analysis was recalculated using the inverse - variance method , by pooling the risk estimates of the various ethnic categories . the statistical heterogeneity among the studies was evaluated using cochrane s q test and the i statistic . regarding the former ( q ) , the included studies were identified as having acceptable heterogeneity , and the fixed - effects model was used . otherwise , the random - effects model was used . to explore the sources of heterogeneity across studies , subgroup analyses were conducted according to study design ( e.g. , case - control vs. cohort study and population - based vs. hospital - based case - control study ) , geographic region ( e.g. , north america vs. europe vs. asia ) , adjustment of confounders ( e.g. , crude vs. adjusted ) , ethnicity ( e.g. , white vs. african american ) , exposure assessment ( e.g. , self - reported vs. medical record vs. serum antibody ) , and study quality ( e.g. , high vs. moderate ) . to determine whether estimates were influenced by the introduction of prostate - specific antigen ( psa ) screening , we performed another subgroup analysis ( i.e. , pre - psa vs. psa - era screening ) using 1994 as the cutoff . for studies that spanned numerous years , we considered the middle year as the determining date . the influence of individual studies was evaluated by estimating the pooled ors after omission of each study in turn . potential publication bias was assessed by visual inspection of begg s funnel plots , in which the log relative risk ratios were plotted against their standard errors . we also performed begg s and egger s tests to assess the presence of publication bias . if the p - value for the egger s test was < 0.05 , we assumed that there was publication bias . all of the statistical analyses were performed with stata statistical software ( version 12.0 ; college station , tx ) , using 2-sided p - values . we performed a systematic search of pubmed , embase , and cochrane library databases , for all papers published up to june 2014 . the following search terms were used : ( gonorrhea or neisseria gonorrhoeae or sexually transmitted diseases or sexually transmitted infections or venereal disease ) and ( prostate cancer or prostatic neoplasms or prostatic cancer or prostate neoplasms ) . we also searched the reference lists of all the retrieved articles to identify any other potentially relevant articles . to be included in this meta - analysis , the papers had to report a case - control or cohort study ; evaluate the association between gonorrhea and the incidence of prostate cancer ; provide relative risk ratios , ors , and 95% cis , or sufficient information to calculate these ; and be published in english . if multiple publications from the same study population were available , only the one with the largest sample size was included . studies were excluded if they did not conform to the inclusion criteria above , or contained duplicate data , or were based on incomplete raw data or irrelevant data . no case reports , letters , reviews , editorials , or correspondence articles were considered . two investigators ( wen - qing lian and fei luo ) independently reviewed and extracted information from all eligible publications , in accordance with the inclusion and exclusion criteria listed above . disagreement was resolved by discussion between the 2 authors until a consensus was reached . if a consensus could not be reached , a third author ( xian - lu song ) was consulted and a final decision was determined by majority opinion . data extracted from the publications included the first author , year of publication , country in which the study was performed , study design , study period , sample size , ages and ethnicities of the subjects , exposure assessment , and the confounders adjusted for . the subjects ethnicities were categorized as white , african american , asian , other , or mixed ( a population with individuals of different ethnicities ) . for studies conducted in the usa , when the paper did not specify the ethnicity of the study population , the most probable ethnicity was recorded , based on the predominant ethnic group in the study country . the methodological quality of each eligible study was independently assessed by 2 reviewers ( wen - qing lian and fei luo ) based on the newcastle - ottawa scale , in which the total score ( in stars ) can range from 0 to 9 . a third party ( xian - lu song ) a study was considered to be of high quality if the newcastle - ottawa scale score was 7 stars ; studies given 56 stars were judged to be of moderate quality . due to the low incidence of prostate cancer , the relative risk ratio can be mathematically approximated by the or . in the present study , the or and its 95% ci was used to assess the association between gonorrhea and the risk of prostate cancer . in some studies , risk estimates were stratified according to ethnic categories , and risk to the total group was not reported . for these studies , the study - specific effect size in the overall analysis was recalculated using the inverse - variance method , by pooling the risk estimates of the various ethnic categories . the statistical heterogeneity among the studies was evaluated using cochrane s q test and the i statistic . regarding the former ( q ) , the included studies were identified as having acceptable heterogeneity , and the fixed - effects model was used . otherwise , the random - effects model was used . to explore the sources of heterogeneity across studies , subgroup analyses were conducted according to study design ( e.g. , case - control vs. cohort study and population - based vs. hospital - based case - control study ) , geographic region ( e.g. , north america vs. europe vs. asia ) , adjustment of confounders ( e.g. , crude vs. adjusted ) , ethnicity ( e.g. , white vs. african american ) , exposure assessment ( e.g. , self - reported vs. medical record vs. serum antibody ) , and study quality ( e.g. , high vs. moderate ) . to determine whether estimates were influenced by the introduction of prostate - specific antigen ( psa ) screening , we performed another subgroup analysis ( i.e. , pre - psa vs. psa - era screening ) using 1994 as the cutoff . for studies that spanned numerous years , we considered the middle year as the determining date . the influence of individual studies was evaluated by estimating the pooled ors after omission of each study in turn . potential publication bias was assessed by visual inspection of begg s funnel plots , in which the log relative risk ratios were plotted against their standard errors . we also performed begg s and egger s tests to assess the presence of publication bias . if the p - value for the egger s test was < 0.05 , we assumed that there was publication bias . all of the statistical analyses were performed with stata statistical software ( version 12.0 ; college station , tx ) , using 2-sided p - values . initially , we retrieved 605 articles from the pubmed , embase , and cochrane library databases that were relevant to the search terms ( figure 1 ) . of these , 110 were removed as duplicates , which left 495 . by screening the titles and abstracts , 457 articles were excluded because they were reviews , editorials , or otherwise not relevant to our meta - analysis . through full - text review of the remaining 38 articles , 5 more were found by reviewing the reference lists , while 22 were excluded because the data were incomplete or irrelevant . finally , 21 studies [ 710,1935 ] were included for meta - analysis . of the 22 included studies , 19 were case - control studies [ 9,10,1935 ] and 2 were cohort studies ; all were published between 1975 and 2011 ( table 1 ) . fourteen were conducted in north america [ 79,1921,23,24,27,3033,35 ] , 5 in europe , and 2 in asia . the sample size per study ranged from 104 to 68 675 , with a total of 118 765 participants and 9965 incident cases . most of these studies adjudged exposure or history of gonorrhea through self - report by the participants , while 2 used medical records and 1 used serology for neisseria gonorrhoeae antibodies . the quality score of studies ranged from 5 stars to 9 stars according to the 9-star newcastle - ottawa scale ( supplementary table 1 ) . based on the combined results of the 21 studies , gonorrhea was significantly associated with increased risk of prostate cancer ( or 1.31 , 95% ci 1.141.52 ) under the random - effects model ( heterogeneity i=38.2% , p=0.039 ; figure 2 ) . the pooled or did not substantially change even after adjustments for confounders , study quality , or the introduction of psa screening ( table 2 ) . we also performed subgroup analyses based on study design , study region , ethnicity , and the method of gonorrhea exposure assessment ( table 2 ) . in the subgroup analysis based on study design , we found a significantly increased risk of prostate cancer in the case - control studies ( or 1.41 , 95% ci 1.241.61 ) , especially in those that were population - based ( or 1.38 , 95% ci 1.191.61 ) . however , the results from the cohort studies were nil ( or 1.07 , 95% ci 0.951.21 ) . regarding geographic area , there was a significant association between gonorrhea and prostate cancer risk in studies conducted in north america ( or 1.33 , 95% ci 1.131.57 ) , but no significant association was found in studies conducted in europe ( or 1.18 , 95% ci 0.781.78 ) or asia ( or 1.44 , 95% ci 0.842.48 ) . the association between gonorrhea and prostate cancer was higher for african american men ( or 1.32 , 95% ci 1.061.65 ) than whites ( or 1.05 , 95% ci 0.901.21 ) . the association was more significant in studies relying on self - reports of gonorrhea exposure ( or 1.34 , 95% ci 1.151.57 ) than those that used medical records ( or 1.01 , 95% ci 0.561.82 ) or serum antibodies ( or 1.07 , 95% ci 0.422.73 ) to determine exposure . to assess the influence of the individual data sets on the pooled ors , repeated meta - analyses that excluded each single study in turn were performed . begg s funnel plot and egger s test were conducted to assess the publication bias of the studies . the shape of the funnel plots did not reveal any evidence of obvious asymmetry ( figure 3 ) , and the results indicated no publication bias ( pbeggs=0.695 , peggers=0.054 ) . initially , we retrieved 605 articles from the pubmed , embase , and cochrane library databases that were relevant to the search terms ( figure 1 ) . of these , 110 were removed as duplicates , which left 495 . by screening the titles and abstracts , 457 articles were excluded because they were reviews , editorials , or otherwise not relevant to our meta - analysis . through full - text review of the remaining 38 articles , 5 more were found by reviewing the reference lists , while 22 were excluded because the data were incomplete or irrelevant . finally , 21 studies [ 710,1935 ] were included for meta - analysis . of the 22 included studies , 19 were case - control studies [ 9,10,1935 ] and 2 were cohort studies ; all were published between 1975 and 2011 ( table 1 ) . fourteen were conducted in north america [ 79,1921,23,24,27,3033,35 ] , 5 in europe , and 2 in asia . the sample size per study ranged from 104 to 68 675 , with a total of 118 765 participants and 9965 incident cases . most of these studies adjudged exposure or history of gonorrhea through self - report by the participants , while 2 used medical records and 1 used serology for neisseria gonorrhoeae antibodies . the quality score of studies ranged from 5 stars to 9 stars according to the 9-star newcastle - ottawa scale ( supplementary table 1 ) . based on the combined results of the 21 studies , gonorrhea was significantly associated with increased risk of prostate cancer ( or 1.31 , 95% ci 1.141.52 ) under the random - effects model ( heterogeneity i=38.2% , p=0.039 ; figure 2 ) . the pooled or did not substantially change even after adjustments for confounders , study quality , or the introduction of psa screening ( table 2 ) . we also performed subgroup analyses based on study design , study region , ethnicity , and the method of gonorrhea exposure assessment ( table 2 ) . in the subgroup analysis based on study design , we found a significantly increased risk of prostate cancer in the case - control studies ( or 1.41 , 95% ci 1.241.61 ) , especially in those that were population - based ( or 1.38 , 95% ci 1.191.61 ) . however , the results from the cohort studies were nil ( or 1.07 , 95% ci 0.951.21 ) . regarding geographic area , there was a significant association between gonorrhea and prostate cancer risk in studies conducted in north america ( or 1.33 , 95% ci 1.131.57 ) , but no significant association was found in studies conducted in europe ( or 1.18 , 95% ci 0.781.78 ) or asia ( or 1.44 , 95% ci 0.842.48 ) . the association between gonorrhea and prostate cancer was higher for african american men ( or 1.32 , 95% ci 1.061.65 ) than whites ( or 1.05 , 95% ci 0.901.21 ) . the association was more significant in studies relying on self - reports of gonorrhea exposure ( or 1.34 , 95% ci 1.151.57 ) than those that used medical records ( or 1.01 , 95% ci 0.561.82 ) or serum antibodies ( or 1.07 , 95% ci 0.422.73 ) to determine exposure . to assess the influence of the individual data sets on the pooled ors , repeated meta - analyses that excluded each single study in turn were performed . begg s funnel plot and egger s test were conducted to assess the publication bias of the studies . the shape of the funnel plots did not reveal any evidence of obvious asymmetry ( figure 3 ) , and the results indicated no publication bias ( pbeggs=0.695 , peggers=0.054 ) . we performed a meta - analysis of 21 relevant studies published up to june 2014 to determine the association between a history of gonorrhea and prostate cancer , and found a significantly increased risk of prostate cancer among men with prior gonorrhea . our results were consistent with the meta - analyses of case - control studies conducted by dennis et al . in 2002 and however , the present meta - analysis involved 19 case - control studies and 2 cohort studies , and while a significant increased risk of prostate cancer was found from the case - control studies , the association according to the cohort studies was nil . the discrepancy between the case - control and cohort studies might be due to the potential bias of case - control studies , including selection bias or recall bias . we also found that there was a stronger association shown in population - based case - control studies than in hospital - based case - control studies . although both population- and hospital - based case - control studies contain biases , we consider the former more reliable because the cases and controls are more representative . in the present study , subgroup analyses by ethnicity revealed a stronger association between a history of gonorrhea and prostate cancer among african americans than among whites . these results suggest that ethnic or cultural differences exist in susceptibility to prostate cancer after gonorrhea exposure . data from the centers for disease control and prevention ( cdc ) in the united states showed that in 2012 the gonorrhea rate among african american males ( 467.7 cases per 100 000 population ) was 16 times the gonorrhea rate among white males ( 28.8 cases per 100 000 population ) , and the disparities were striking across all age groups and regions . kwame owusu - edusei jr . et al . also found that racial disparities in income were associated with racial disparities in gonorrhea rates . in addition , the higher gonorrhea rates might be due to the average lower rate of insurance , later diagnosis , less effective treatment , and greater incidence of relevant genetic polymorphisms in african americans . because rates of gonorrhea infection and rates of prostate cancer are each higher among african american men than among white men , we consider that many factors influencing these rates likely exist that are beyond the scope of this analysis , and any conclusions require further verification . in the present meta - analysis , subgroup analysis showed that gonorrhea was significantly associated with increased incidence of prostate cancer in north america , but not in europe or asia . one possible explanation for this finding is that there was not sufficient published evidence representing european ( 5 studies ) and asian ( 2 studies ) countries . this is possibly due to the exclusion criteria of the study , since we only included articles published in english . other potential factors are the relatively low incidence of prostate cancer in asians and the greater incidence in african americans . in this study , we found a significant association between gonorrhea and prostate cancer risk in the studies in which the history of gonorrhea was based on self - reports of the subjects , while the association was insignificant in studies that used medical records or serum antibodies . this finding may be due to the small number of studies that used medical records ( 2 studies ) or serum antibodies ( 1 study ) . a general association between infections , infection - induced chronic inflammation , and the development of cancer is well - known , and increasing evidence indicates that chronic inflammatory states contribute to prostate carcinogenesis . gonorrhea infection by neisseria gonorrhoeae has been shown to induce a chronic inflammatory environment within the prostate . inflammatory cells are recruited after damage or an infection , and they can secrete a large number of cytokines ( e.g. , interleukin 6 ) and chemokines ( e.g. , interleukin 8) that promote the growth of neoplastic cells and ultimately lead to carcinogenesis within the prostate . evidence from pathology also shows that proliferative inflammatory atrophy , which is often associated with chronic and , at times , acute inflammation , may be the direct precursor lesion to prostatic intraepithelial neoplasia , prostate cancer , or both . in addition , several genes have been studied for their role in prostate cancer development , and in some cases , ( e.g. , ribonuclease l[rnasel]/ hereditary prostate cancer 1 [ hpc1 ] , toll - like receptor 4 [ tlr4 ] , macrophage scavenger receptor 1 [ msr1 ] ) mutations or variants in these genes also increase an individual s susceptibility to infection . the present meta - analysis has some limitations . first , most of the included studies were case - control studies , which are susceptible to recall and selection biases . the statistical effect of these kinds of biases might be reduced somewhat by cohort studies , but we found only 2 suitable cohort studies . gonorrhea history was mostly based on the self - report of the subjects and only 1 study conducted serological tests . third , substantial heterogeneity was observed among the studies , although we were able to find the major sources of heterogeneity through subgroup analyses . fourth , our results may also have been biased by restricting studies to those published in english , but there was no evidence of publication bias , based on either egger s or begg s test . fifth , the effect of gonorrhea on prostate cancer outcomes may be different for cases of prostate cancer detected in the early stages through psa screening than for patients who had aggressive fatalities before psa screening was widely available . however , the pooled ors in the pre - psa and psa - era subgroups were similar . moreover , data stratified by prostate cancer aggressiveness were not available from the included studies . we also note that , although we performed a careful search for papers published up to june 2014 , the 21 included studies all appeared between 1975 and 2011 . our analysis found a potential association between gonorrhea and increased risk of prostate cancer , especially among african american males . because of the limited number of studies , more prospective cohort or intervention studies such findings also warrant investigations of the underlying mechanisms that may be responsible for this association .

backgroundthe association between gonorrhea and prostate cancer risk has been investigated widely , but the results remain inconsistent and contradictory . we conducted an updated meta - analysis to obtain a more precise estimate of this association.material/methodspubmed , embase , and the cochrane library were searched for papers up to june 2014 to identify eligible studies . pooled odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated to assess the influence of gonorrhea on prostate cancer risk.resultstwenty-one observational studies ( 19 case - control and 2 cohort ) were eligible , comprising 9965 prostate cancer patients and 118 765 participants . pooled results indicated that gonorrhea was significantly associated with increased incidence of prostate cancer ( or 1.31 , 95% ci 1.141.52 ) . the association between gonorrhea and prostate cancer was stronger in african american males ( or 1.32 , 95% ci 1.061.65 ) than in whites ( or 1.05 , 95% ci 0.901.21).conclusionsour findings suggest that gonorrhea is associated with an increased risk of prostate cancer , especially among african american males . these results warrant further well - designed , large - scale cohort studies to draw definitive conclusions .

Background Material and Methods Literature search Study selection Data extraction and quality score assessment Statistical analysis Results Literature search Study characteristics Meta-analysis results Sensitivity analysis Publication bias Discussion Conclusions Supplementary materials

in the same year , 10% of the total cancer deaths in men were due to prostate cancer , the second leading cause of cancer death in this population . although the cause of prostate cancer remains unknown , its incidence has been associated with age , ethnicity , family history , physical activity , body mass index , diet , region , and sexually transmitted infections . the seriousness of this disease warrants a more definitive investigation of its association with a history of sexually transmitted diseases . several epidemiologic studies have investigated an association between gonorrhea infection and incidence of prostate cancer , but results have been inconsistent . according to a meta - analysis published by dennis et al . in 2002 , men with a history of gonorrhea were at elevated risk of prostate cancer ( pooled relative risk ratio [ rr ] 1.36 , 95% confidence interval [ ci ] 1.151.61 ) . a meta - analysis reported by taylor et al . in 2005 also indicated that gonorrhea was associated with increased prostate cancer risk ( odds ratio [ or ] 1.39 , 95% ci 1.051.83 ) . however , both of these studies were based predominantly on case - control data for white men . studies that include african american subjects , serologic measures , and prospective data are lacking . more recently , 2 large , prospective , cohort studies and 2 case - control studies failed to confirm an association between a history of gonorrhea and prostate cancer . however , some factors were not considered in their analyses that might limit the evaluation of prostate cancer risk . these include adjustments for confounders , study design , study region , ethnicity , the method of gonorrhea exposure assessment , study quality , and the introduction of psa screening . herein we provide an updated review and meta - analysis of the association between a history of gonorrhea and incidence of prostate cancer , conducting subgroup analyses based on the factors aforementioned . we performed a systematic search of pubmed , embase , and cochrane library databases , for all papers published up to june 2014 . the following search terms were used : ( gonorrhea or neisseria gonorrhoeae or sexually transmitted diseases or sexually transmitted infections or venereal disease ) and ( prostate cancer or prostatic neoplasms or prostatic cancer or prostate neoplasms ) . we also searched the reference lists of all the retrieved articles to identify any other potentially relevant articles . to be included in this meta - analysis , the papers had to report a case - control or cohort study ; evaluate the association between gonorrhea and the incidence of prostate cancer ; provide relative risk ratios , ors , and 95% cis , or sufficient information to calculate these ; and be published in english . data extracted from the publications included the first author , year of publication , country in which the study was performed , study design , study period , sample size , ages and ethnicities of the subjects , exposure assessment , and the confounders adjusted for . the subjects ethnicities were categorized as white , african american , asian , other , or mixed ( a population with individuals of different ethnicities ) . due to the low incidence of prostate cancer , the relative risk ratio can be mathematically approximated by the or . in the present study , the or and its 95% ci was used to assess the association between gonorrhea and the risk of prostate cancer . in some studies , risk estimates were stratified according to ethnic categories , and risk to the total group was not reported . the statistical heterogeneity among the studies was evaluated using cochrane s q test and the i statistic . regarding the former ( q ) , the included studies were identified as having acceptable heterogeneity , and the fixed - effects model was used . , case - control vs. cohort study and population - based vs. hospital - based case - control study ) , geographic region ( e.g. , white vs. african american ) , exposure assessment ( e.g. , self - reported vs. medical record vs. serum antibody ) , and study quality ( e.g. to determine whether estimates were influenced by the introduction of prostate - specific antigen ( psa ) screening , we performed another subgroup analysis ( i.e. the influence of individual studies was evaluated by estimating the pooled ors after omission of each study in turn . we also performed begg s and egger s tests to assess the presence of publication bias . if the p - value for the egger s test was < 0.05 , we assumed that there was publication bias . we performed a systematic search of pubmed , embase , and cochrane library databases , for all papers published up to june 2014 . the following search terms were used : ( gonorrhea or neisseria gonorrhoeae or sexually transmitted diseases or sexually transmitted infections or venereal disease ) and ( prostate cancer or prostatic neoplasms or prostatic cancer or prostate neoplasms ) . we also searched the reference lists of all the retrieved articles to identify any other potentially relevant articles . to be included in this meta - analysis , the papers had to report a case - control or cohort study ; evaluate the association between gonorrhea and the incidence of prostate cancer ; provide relative risk ratios , ors , and 95% cis , or sufficient information to calculate these ; and be published in english . data extracted from the publications included the first author , year of publication , country in which the study was performed , study design , study period , sample size , ages and ethnicities of the subjects , exposure assessment , and the confounders adjusted for . the subjects ethnicities were categorized as white , african american , asian , other , or mixed ( a population with individuals of different ethnicities ) . due to the low incidence of prostate cancer , the relative risk ratio can be mathematically approximated by the or . in the present study , the or and its 95% ci was used to assess the association between gonorrhea and the risk of prostate cancer . in some studies , risk estimates were stratified according to ethnic categories , and risk to the total group was not reported . the statistical heterogeneity among the studies was evaluated using cochrane s q test and the i statistic . regarding the former ( q ) , the included studies were identified as having acceptable heterogeneity , and the fixed - effects model was used . , case - control vs. cohort study and population - based vs. hospital - based case - control study ) , geographic region ( e.g. , white vs. african american ) , exposure assessment ( e.g. , self - reported vs. medical record vs. serum antibody ) , and study quality ( e.g. to determine whether estimates were influenced by the introduction of prostate - specific antigen ( psa ) screening , we performed another subgroup analysis ( i.e. , pre - psa vs. psa - era screening ) using 1994 as the cutoff . the influence of individual studies was evaluated by estimating the pooled ors after omission of each study in turn . we also performed begg s and egger s tests to assess the presence of publication bias . if the p - value for the egger s test was < 0.05 , we assumed that there was publication bias . initially , we retrieved 605 articles from the pubmed , embase , and cochrane library databases that were relevant to the search terms ( figure 1 ) . by screening the titles and abstracts , 457 articles were excluded because they were reviews , editorials , or otherwise not relevant to our meta - analysis . finally , 21 studies [ 710,1935 ] were included for meta - analysis . of the 22 included studies , 19 were case - control studies [ 9,10,1935 ] and 2 were cohort studies ; all were published between 1975 and 2011 ( table 1 ) . fourteen were conducted in north america [ 79,1921,23,24,27,3033,35 ] , 5 in europe , and 2 in asia . the sample size per study ranged from 104 to 68 675 , with a total of 118 765 participants and 9965 incident cases . most of these studies adjudged exposure or history of gonorrhea through self - report by the participants , while 2 used medical records and 1 used serology for neisseria gonorrhoeae antibodies . based on the combined results of the 21 studies , gonorrhea was significantly associated with increased risk of prostate cancer ( or 1.31 , 95% ci 1.141.52 ) under the random - effects model ( heterogeneity i=38.2% , p=0.039 ; figure 2 ) . we also performed subgroup analyses based on study design , study region , ethnicity , and the method of gonorrhea exposure assessment ( table 2 ) . in the subgroup analysis based on study design , we found a significantly increased risk of prostate cancer in the case - control studies ( or 1.41 , 95% ci 1.241.61 ) , especially in those that were population - based ( or 1.38 , 95% ci 1.191.61 ) . however , the results from the cohort studies were nil ( or 1.07 , 95% ci 0.951.21 ) . regarding geographic area , there was a significant association between gonorrhea and prostate cancer risk in studies conducted in north america ( or 1.33 , 95% ci 1.131.57 ) , but no significant association was found in studies conducted in europe ( or 1.18 , 95% ci 0.781.78 ) or asia ( or 1.44 , 95% ci 0.842.48 ) . the association between gonorrhea and prostate cancer was higher for african american men ( or 1.32 , 95% ci 1.061.65 ) than whites ( or 1.05 , 95% ci 0.901.21 ) . the association was more significant in studies relying on self - reports of gonorrhea exposure ( or 1.34 , 95% ci 1.151.57 ) than those that used medical records ( or 1.01 , 95% ci 0.561.82 ) or serum antibodies ( or 1.07 , 95% ci 0.422.73 ) to determine exposure . to assess the influence of the individual data sets on the pooled ors , repeated meta - analyses that excluded each single study in turn were performed . begg s funnel plot and egger s test were conducted to assess the publication bias of the studies . the shape of the funnel plots did not reveal any evidence of obvious asymmetry ( figure 3 ) , and the results indicated no publication bias ( pbeggs=0.695 , peggers=0.054 ) . initially , we retrieved 605 articles from the pubmed , embase , and cochrane library databases that were relevant to the search terms ( figure 1 ) . by screening the titles and abstracts , 457 articles were excluded because they were reviews , editorials , or otherwise not relevant to our meta - analysis . through full - text review of the remaining 38 articles , 5 more were found by reviewing the reference lists , while 22 were excluded because the data were incomplete or irrelevant . finally , 21 studies [ 710,1935 ] were included for meta - analysis . of the 22 included studies , 19 were case - control studies [ 9,10,1935 ] and 2 were cohort studies ; all were published between 1975 and 2011 ( table 1 ) . fourteen were conducted in north america [ 79,1921,23,24,27,3033,35 ] , 5 in europe , and 2 in asia . the sample size per study ranged from 104 to 68 675 , with a total of 118 765 participants and 9965 incident cases . most of these studies adjudged exposure or history of gonorrhea through self - report by the participants , while 2 used medical records and 1 used serology for neisseria gonorrhoeae antibodies . based on the combined results of the 21 studies , gonorrhea was significantly associated with increased risk of prostate cancer ( or 1.31 , 95% ci 1.141.52 ) under the random - effects model ( heterogeneity i=38.2% , p=0.039 ; figure 2 ) . we also performed subgroup analyses based on study design , study region , ethnicity , and the method of gonorrhea exposure assessment ( table 2 ) . in the subgroup analysis based on study design , we found a significantly increased risk of prostate cancer in the case - control studies ( or 1.41 , 95% ci 1.241.61 ) , especially in those that were population - based ( or 1.38 , 95% ci 1.191.61 ) . however , the results from the cohort studies were nil ( or 1.07 , 95% ci 0.951.21 ) . regarding geographic area , there was a significant association between gonorrhea and prostate cancer risk in studies conducted in north america ( or 1.33 , 95% ci 1.131.57 ) , but no significant association was found in studies conducted in europe ( or 1.18 , 95% ci 0.781.78 ) or asia ( or 1.44 , 95% ci 0.842.48 ) . the association between gonorrhea and prostate cancer was higher for african american men ( or 1.32 , 95% ci 1.061.65 ) than whites ( or 1.05 , 95% ci 0.901.21 ) . the association was more significant in studies relying on self - reports of gonorrhea exposure ( or 1.34 , 95% ci 1.151.57 ) than those that used medical records ( or 1.01 , 95% ci 0.561.82 ) or serum antibodies ( or 1.07 , 95% ci 0.422.73 ) to determine exposure . to assess the influence of the individual data sets on the pooled ors , repeated meta - analyses that excluded each single study in turn were performed . begg s funnel plot and egger s test were conducted to assess the publication bias of the studies . the shape of the funnel plots did not reveal any evidence of obvious asymmetry ( figure 3 ) , and the results indicated no publication bias ( pbeggs=0.695 , peggers=0.054 ) . we performed a meta - analysis of 21 relevant studies published up to june 2014 to determine the association between a history of gonorrhea and prostate cancer , and found a significantly increased risk of prostate cancer among men with prior gonorrhea . our results were consistent with the meta - analyses of case - control studies conducted by dennis et al . in 2002 and however , the present meta - analysis involved 19 case - control studies and 2 cohort studies , and while a significant increased risk of prostate cancer was found from the case - control studies , the association according to the cohort studies was nil . the discrepancy between the case - control and cohort studies might be due to the potential bias of case - control studies , including selection bias or recall bias . we also found that there was a stronger association shown in population - based case - control studies than in hospital - based case - control studies . although both population- and hospital - based case - control studies contain biases , we consider the former more reliable because the cases and controls are more representative . in the present study , subgroup analyses by ethnicity revealed a stronger association between a history of gonorrhea and prostate cancer among african americans than among whites . these results suggest that ethnic or cultural differences exist in susceptibility to prostate cancer after gonorrhea exposure . data from the centers for disease control and prevention ( cdc ) in the united states showed that in 2012 the gonorrhea rate among african american males ( 467.7 cases per 100 000 population ) was 16 times the gonorrhea rate among white males ( 28.8 cases per 100 000 population ) , and the disparities were striking across all age groups and regions . et al . also found that racial disparities in income were associated with racial disparities in gonorrhea rates . in addition , the higher gonorrhea rates might be due to the average lower rate of insurance , later diagnosis , less effective treatment , and greater incidence of relevant genetic polymorphisms in african americans . because rates of gonorrhea infection and rates of prostate cancer are each higher among african american men than among white men , we consider that many factors influencing these rates likely exist that are beyond the scope of this analysis , and any conclusions require further verification . in the present meta - analysis , subgroup analysis showed that gonorrhea was significantly associated with increased incidence of prostate cancer in north america , but not in europe or asia . one possible explanation for this finding is that there was not sufficient published evidence representing european ( 5 studies ) and asian ( 2 studies ) countries . other potential factors are the relatively low incidence of prostate cancer in asians and the greater incidence in african americans . in this study , we found a significant association between gonorrhea and prostate cancer risk in the studies in which the history of gonorrhea was based on self - reports of the subjects , while the association was insignificant in studies that used medical records or serum antibodies . a general association between infections , infection - induced chronic inflammation , and the development of cancer is well - known , and increasing evidence indicates that chronic inflammatory states contribute to prostate carcinogenesis . gonorrhea infection by neisseria gonorrhoeae has been shown to induce a chronic inflammatory environment within the prostate . inflammatory cells are recruited after damage or an infection , and they can secrete a large number of cytokines ( e.g. , interleukin 6 ) and chemokines ( e.g. evidence from pathology also shows that proliferative inflammatory atrophy , which is often associated with chronic and , at times , acute inflammation , may be the direct precursor lesion to prostatic intraepithelial neoplasia , prostate cancer , or both . in addition , several genes have been studied for their role in prostate cancer development , and in some cases , ( e.g. , ribonuclease l[rnasel]/ hereditary prostate cancer 1 [ hpc1 ] , toll - like receptor 4 [ tlr4 ] , macrophage scavenger receptor 1 [ msr1 ] ) mutations or variants in these genes also increase an individual s susceptibility to infection . the present meta - analysis has some limitations . first , most of the included studies were case - control studies , which are susceptible to recall and selection biases . the statistical effect of these kinds of biases might be reduced somewhat by cohort studies , but we found only 2 suitable cohort studies . gonorrhea history was mostly based on the self - report of the subjects and only 1 study conducted serological tests . fourth , our results may also have been biased by restricting studies to those published in english , but there was no evidence of publication bias , based on either egger s or begg s test . fifth , the effect of gonorrhea on prostate cancer outcomes may be different for cases of prostate cancer detected in the early stages through psa screening than for patients who had aggressive fatalities before psa screening was widely available . moreover , data stratified by prostate cancer aggressiveness were not available from the included studies . we also note that , although we performed a careful search for papers published up to june 2014 , the 21 included studies all appeared between 1975 and 2011 . our analysis found a potential association between gonorrhea and increased risk of prostate cancer , especially among african american males . because of the limited number of studies , more prospective cohort or intervention studies such findings also warrant investigations of the underlying mechanisms that may be responsible for this association .

extracellular vesicles ( evs ) are particles of 20 nm up to 5 m in diameter composed of proteins , nucleic acid , and lipids that are found in body fluids such as plasma , serum , saliva , urine , breast milk , ascites , and cerebrospinal fluids . these particles are involved in intercellular communication , modulating a wide range of signaling events during innate and acquired immune responses ( figure 1 and table 1 ) [ 24 ] . evs are secreted during health conditions or upon inflammation during the course of diseases by all mammalian cells types [ 2 , 3 , 5 ] . evs include different types of particles and may be named or classified depending on the cell type or function . they can be derived from dendritic cells ( dexosomes ) , prostate tissue ( prostasomes ) , bone , cartilage and atherosclerotic plaques ( matrix vesicles ) , neurons ( synaptic vesicles ) , apoptotic blebs or apoptotic bodies ( microparticles , exosomes , and apoptotic vesicles ) , shed vesicles , shedding microvesicles or microparticles ( ectosomes or microvesicles ) , and membrane fragments of virus infected cells , protozoa , fungi , and bacteria outer membrane vesicles [ 1 , 2 , 4 , 610 ] . the vesicles derived from mammalian cells contain a family of integral membrane proteins that cross four times the lipid bilayer and are called tetraspanins , including the surface markers of lymphocytes and antigen - presenting cells such as cd37 , cd9 , cd53 , cd63 , cd81 , and cd82 . evs also contain molecules of the major histocompatibility complex ( mhc classes i and ii ) ( http://www.exocarta.org/ ) [ 11 , 12 ] . evs derived from normal cells cause either suppression or activation of the immune response by modulating the production of inflammatory mediators . for example , t - cells and monocytes secrete vesicles that contain fasl on the surface that modulate apoptosis of the other cells ( figure 2 ) . vesicles isolated from monocytes deliver proinflammatory mediators that activate endothelial cells [ 14 , 15 ] . tumor cells secrete evs that are able to downregulate the immune system , allowing the escape from the immune system . furthermore , these vesicles can control tumor development and growth , by decreasing the expression and release of il-2 reducing the proliferation of natural killer ( nk ) cells [ 14 , 15 ] . therefore , evs are potential biomarkers and antigens for vaccination , with potential uses for early diagnostic , and therapeutic applications in several diseases . the purpose of this review is to provide an updated overview of the vesicles released by distinct pathogens and mammalian tissues , highlighting their potential use in vaccination and diagnostic of cancer and infectious diseases . evs derived from tumors may be involved in tumor growth control and in the communication events between tumor and normal cells by delivering oncogenic proteins and growth factors [ 16 , 17 ] . in some cases , evs suppress tumor growth by exposing dendritic cells mhc classes i or ii molecules , peptides , and costimulatory molecules for the immune system . moreover , evs contain proteins and genetic material from the originating tumor cells that can be used as diagnostic biomarkers . in this regard , recent efforts to elucidate different roles and signaling pathways of evs have been conducted . a pivotal role of evs during cancer cell migration and invasion has been reported in different cell types . for instance , evs derived from 786 - 0 renal tumor cells enhance their migration and invasion properties . this occurs through induction of type 4-chemokine receptor ( cxcr4 ) and matrix metalloproteinase-9 ( mmp-9 ) expression by evs . in addition , adhesion and invasion of the gastrointestinal interstitial stroma are enhanced by the oncogenic protein tyrosine kinase ( kit ) present in tumor cell evs . tamoxifen - resistant breast tumor cells release exosomes that contain micrornas ( mir221/222 ) and promote drug resistance in naive cells . similarly , resistance to docetaxel in breast tumors and prostate cancer , as well as cisplatin in human lung cancer line ( a549 cells ) , was associated with the content of vesicular micrornas transferred to susceptible cells [ 4749 ] . moreover , evs from a549 cells containing trkb , egfr , and sortilin receptors ( tes complex ) were related to angiogenesis induction through endothelial cells . in hepatocellular carcinoma ( hcc ) , one of the most lethal cancers , the tumor becomes more resistant to tgf-dependent chemotherapy through long noncoding rnas ( lncrnas ) obtained from evs . therefore , the extracellular communication through evs is an important mechanism to activate / deactivate certain crucial events in tumor cell biology . for instance , mir-105 , detected in evs from breast tumor , is associated with metastasis formation via destruction of endothelial monolayers . interestingly , it is possible to detect mir-105 in the blood circulation before the metastasis establishment reinforcing its potential role as a diagnostic biomarker . likewise , expression of mir-214 , mir-221 , and mir-222 present in these evs is related to lymph node metastasis , venous invasion , and tumor development . in some cases , mir - containing evs repress proangiogenic events and impair tumor development on a bone cancer model . the study of biogenesis of stress - induced vesicles also becomes crucial to understand the development of metastasis . for example , the elevated expression of rab22a gene in breast tumor cells induced by hypoxia , common in advanced tumors , increases the shedding of vesicles , and the rab protein colocalizes with the sites of budding evs . moreover , the knockdown of rab22a prevents metastasis , supporting the idea that rab is involved in the generation of evs . evs released from heat - stressed tumors in a mouse model can induce antitumor immunity . these vesicles showed chemotactic effects on cd4 + and cd8 + t - cells , efficiently activating dendritic cells ( dc ) . another study showed that evs derived from breast cancers can alter the tumor microenvironment and promote tumorigenesis of normal cells via induction of autophagy , response to dna damage repair ( ddr ) , and induction of reactive oxygen species ( ros ) in normal breast epithelial cells . this includes the polyadenylate - binding protein 1 ( pabp1 ) , predominant in evs from metastatic duodenal tumor cell lines , prostate - specific membrane antigen ( psa ) related to prostate cancer progression , angiogenesis , and metastasis , mir-21 and mir-146a in cervical cancer , and finally lncrnas in skin cancer ( secreted into the blood or urine through evs ) . all the above - mentioned micrornas are proposed as potential biomarkers for cancer noninvasive diagnosis . it was also shown that evs from pancreatic tumor cells contain fragments of double - stranded genomic dna ( dsdna ) , suggesting that mutations may be identified in this dsdna as predictors of cancer and streamline therapeutics . based on these findings , it is clear that new biomarkers , once optimized , could be used in therapeutic conducts , offering great advantage over other established methods . in cancer therapy , evs from tumor cells are able to associate better with their recipient cells than liposomes ( > 10-fold ) , due to their lipid and protein composition . in addition , micrornas can be delivered to tumor cells and interfere with cancer progression and metastasis . in this logic , synthetic mir-143 was introduced into mesenchymal stem cells , and the secreted exosomes containing mir-143 was transferred to osteosarcoma cells to reduce the migration of the latter cell . interestingly , a feedback regulatory mechanism for controlling exosome release was suggested , in which exosomes derived from normal human mammary epithelial cells could impair the release of exosomes from breast tumor cells . these authors suggest that this may be used as a novel therapeutic approach , attenuating carcinogenic effects of tumor exosomes . another interesting strategy is to use a synthetic structure based on tumor - derived exosomes and staphylococcal enterotoxin b to induce apoptosis in breast tumor cells [ 59 , 60 ] . the vesicles could be used as a diagnostic , because tumor cells release vesicles in biologic fluids like urine , blood , ascites , and pleural fluids . for example , patients with ovarian cancer shed vesicles derived from tumor cells in the circulation . these vesicles are enriched up to 4-fold more in patients with cancer than healthy controls . therefore , they can be used as biomarkers to identify early cancers in asymptomatic patients that will potentially develop malignancy . in addition , specific mirnas are found in extracellular vesicles from patients with lung cancers . for example , dc were primed with interferon - gamma ( ifn- ) to induce the expression cd40 , cd80 , cd86 , and cd54 in exosomes , endowing a potent cd8 + t - cell - triggering potential in vitro and in vivo . yao et al . compared the antitumor immunities between eg7 tumor cell - derived exosomes [ exo ( eg7 ) ] and exo- ( eg7- ) targeted dendritic cells [ dc ( exo ) ] . they showed that the latter dc ( exo ) was more effective in inducing antitumor immunity , and this was independent from the host dc , emphasizing the role of the host dc in tumor cell - derived exosomes ( tex ) vaccines . in contrast , cd8 + t - cell responses could be induced in vivo when mice were immunized with protein - loaded instead of peptide - loaded dexosomes . recently , protein - loaded dexosomes were used to protect against tumor growth , whereby cd8 + t - cell responses occurred in vivo [ 63 , 64 ] . purified mhc classes i and ii inserted in exosomes and delivered to melanoma were recognized by specific t - cells . this was used to transfer functional mhc / peptide complexes to antigen - presenting cells . in this way , antitumor response could be elicited as these complexes may stimulate cd8 + and cd4 + t - cell responses in an exosomes from rab27a overexpressing cells increased significantly cd4 + t - cell proliferation in vitro because these exosomes upregulated mhc class ii , cd80 , and cd86 molecules in dc . moreover , exosomes containing a small gtpase protein involved in secretion of exosomes also were capable of retaining tumor growth in vivo . plasmid dna vaccines encoding ev - associated antigens were recently used as vaccines in mice in order to produce ovalbumin containing - ev antigens in vivo , either exposed on the surface of vesicles or incorporated inside membrane - enclosed virus - like particles . in both cases , these vaccines were able to induce specific t - cell responses and efficiently prevent the growth of ovalbumin - expressing tumors in vivo , showing that immunotherapy based on evs may be a valuable method to promote tumor control and other diseases . bacteria release vesicles sizing from 20 to 250 nm [ 33 , 67 ] are named outer membrane vesicles ( omvs ) for gram - negative and membrane vesicles , or blebs , for gram - positive bacteria [ 68 , 69 ] . evs are required for the exchange of genetic information between bacteria such as bacillus anthracis , staphylococcus aureus , mycobacterium ulcerans , bacillus spp . , escherichia coli , pseudomonas aeruginosa , and helicobacter pylori . additionally , evs contain toxins and deliver virulence factors to host cells [ 32 , 7080 ] . bacterial evs are composed of cytosolic and membrane proteins , lipoproteins , phospholipids , glycolipids , and nucleic acids [ 31 , 32 , 8184 ] . detailed composition analysis and biogenesis of omvs from different gram - negative bacteria are available . for example , omvs from bordetella parapertussis contain surface immunogenic molecules , porin , outer membrane protein ompq , and pertactin that were used in a murine model to assess the protection against infection . in the same way , omvs of pseudomonas putida kt2440 have outer membrane proteins such as oprc , oprd , opre , oprf , oprh , oprg , and oprw which can serve as adjuvants or vaccine . vibrio cholerae omvs contain several proteins that contribute for the virulence and are essential for cell growth and colonization in vivo . another interesting aspect of omvs is their role in delivering endotoxins to host cells as demonstrated for enterogenic and uropathogenic escherichia coli ( ( etec ) and ( upec ) ) , the causative agents of traveler 's diarrhea and human urinary tract infections . both etec and upec strains are able to produce many virulence factors including the heat - labile enterotoxin ( lt ) , homologous to cholera toxin , and cytotoxic necrotizing factor type 1 ( cnf1 ) . these toxins are released from bacteria in omvs and delivered to host cells through vesicle internalization [ 74 , 87 ] . in particular , lt also acts as a ligand for vesicle binding , which is internalized via lipid rafts . once inside the cell , the toxin is trafficked via retrograde transport through the golgi and the endoplasmic reticulum . an outstanding role of omvs in biotechnology is their use as general vehicles to deliver human , heterologous , or viral antigens [ 33 , 88 ] . neisseria meningitidis serogroup b omvs showed remarkable adjuvant properties for anti - hiv-1 antigens and induced a production of ifn- and il-4 . vesicles isolated from dc infected with mycobacterium tuberculosis were able to induce a protective host immunity response [ 90 , 91 ] . omvs from different species of gram - negative bacteria contain lipopolysaccharide ( lps ) , proteins , and nucleic acids , which are strong agonists in the modulation of inflammatory reactions through the activation of toll - like receptors ( tlrs ) . these activations require the action of lps , which is sensed by toll - like receptor 4 ( tlr4 ) on host cells , and induce an innate immune response to gram - negative bacteria leading to inflammatory cytokine production [ 9396 ] . in the case of pseudomonas aeruginosa , omvs appear to deliver virulence factors to distant locations by fusing with lipid rafts of several host cell membranes . proteins present in secreted vesicles released from p. aeruginosa also seem to play important roles in pathogenesis . this is the case of the inhibitory factor of the cystic fibrosis transmembrane conductance regulator , which promotes changes in the epithelium , allowing reduced clearance of p. aeruginosa toxin a that hijacks the host ubiquitin proteolytic system . therefore , p. aeruginosa evs have the potential to protect the immunized host against subsequent infection and for this reason they have been proposed as vaccines candidates against infection . another interesting example is omvs isolated from haemophilus influenzae , which increases the expression of cd69 and cd86 and activating of the humoral response . in addition , they induce tlr9 signaling through bacterial dna , which causes a significant proliferative response of inflammatory cells . vesicles from gram - negative bacteria are released naturally as blebs of the outer membrane through bulging and pinching off . alternatively , vesicles can be prepared from the detergent - treated bacteria either from normal or from bacteria carrying genetic modifications such as the generalized modules for membrane antigens ( gmma ) to induce a strong immune response . all these vesicles are called omvs , but it is important to note that they have different composition and properties . naturally shed blebs are almost free of cytoplasmic and inner membrane components and maintain lipophilic proteins , unlike detergent extracted omvs derived from bacteria . these differences are relevant when considering the use of vesicles for immunization or diagnostic purposes . one example is the case of neisseria meningitidis - omvs vaccine , named bexsero ( novartis ) . these particles activate the immune response and protection against a challenge with bacteria in murine models [ 82 , 101107 ] . there are , however , several cases that vaccination with omvs requires further developments to improve better antigenicity , manufacturability , and reduction of pyrogenicity , detergent extract , and toxicity via lps detoxification . the mechanism of how gram - negative bacteria - derived omvs elicit a vaccination effect , for example e. coli used as a model to study the effect of the adaptive immune response decrease against bacteria - induced lethality . however , with high doses these omvs induced systemic inflammatory , characterized by hypothermia , tachypnea , and leukopenia ( sepsis ) . because of the thick cell wall of gram - positive bacteria , extracellular vesicle secretion has been less studied in these bacteria . nevertheless , it has been reported that s. aureus and bacillus subtilis secrete membrane vesicles to the extracellular milieu . furthermore , s. aureus extracellular vesicles induce atopic dermatitis - like skin inflammation in mice . these observations recently , a study on the immune activating role of gram - positive bacteria - derived ev has been published . the gram - positive bacillus anthracis , the agent of the anthrax disease , also shed membrane - derived vesicles . these evs are formed by a double membrane and have a spherical shape sizing from 50 to 150 nm . they are enriched by molecular chaperons and molecules of the cell wall involved in the cellular architecture and include the lethal toxin ( letx ) and the antholysin ( alo ) . balb / c mice immunized with these evs were able to produce more protective igm to the toxin in comparison with the isolated toxin , prompting to further use these preparations to elaborate vaccines . the protection induced by vesicles obtained from gram - positive bacteria was not as effective when compared to gram - negative bacteria omvs indicating that further work might be necessary to improve their potential . in summary , cultured protozoan parasites release evs that contain several molecules that might affect the host ( figure 1 ) . they are composed of membrane fragments and cytosolic components , including proteins , lipids , and nucleic acids that accumulate in the supernatant of the protozoan cultivated in the presence or absence of host cells [ 40 , 41 ] . when injected in animal models or added to in vitro systems , these evs were found to affect the course of infection and alter the disease progression caused by the parasite , through the modulation of the host innate and acquired immune response . [ 37 , 38 , 90 , 111 , 112 ] , trypanosoma cruzi [ 40 , 41 , 113115 ] , trypanosoma brucei , plasmodium spp . [ 117 , 118 ] , trichomonas vaginalis , toxoplasma gondii [ 120122 ] , and eimeria parasites . it is acquired by humans either by the insect vector , blood transfusion , or through maternal transmission during new born delivery . when t. cruzi enters the host , the first line of defense is the innate immune response , which initiates when receptors that recognize microbial products are activated . this occurs through toll - like receptors ( tlr ) signaling and macrophage activation by mucin - like glycoproteins , which corresponds to 6080% of the parasite surface molecules [ 35 , 127 ] , resulting in the increased production of il-12 , ifn- , and nitric oxide ( no ) . the production of these proinflammatory cytokines leads to the activation of several kinds of cells such as natural killer ( nk ) typical of the acute phase of chagas disease . very little is known about how mucin - like glycoproteins and other surface components are presented to the host . infective parasites obtained from cultured mammalian cells shed large amounts of evs that are rich in these surface molecules . evs isolated from infective t. cruzi forms promote macrophage activation with an increase in parasitemia levels and amastigotes nests in the heart tissue . these effects are caused by parasite surface glycoproteins present in the vesicles that attenuate the host immune system . t. cruzi evs are enriched in -gal containing glycoconjugates , found preferentially in the mucin - like molecules , and several surface glycoproteins , known as members of a trans - sialidase ( ts ) family that participate in adhesion and invasion of host cells [ 40 , 41 , 114 , 130 ] . mucins containing -gal residues elicit high titers of igg antibodies decreasing parasitemia during the chronic phase . therefore , the production and release of evs might have a key role in the establishment of infection and may be considered a platform to develop preventive or prophylactic vaccines for chagas disease . leishmania genus encloses protozoan species that cause visceral , cutaneous , and mucocutaneous leishmaniasis in humans . the disease is transmitted by sandfly vectors ( lutzomyia and phlebotomus ) , which inject parasites into the host during the insect blood meal . in culture , the insect stages of several leishmania species release evs containing parasite antigens , such as the surface glycoprotein of 63 kda ( gp63 ) that has a strong suppressive effect on host macrophages [ 37 , 38 ] . however , a missing step in leishmania evs biogenesis is whether those structures also contain the major surface lipophosphoglycan ( lpg ) , a multivirulence factor involved in the interaction with the vertebrate and invertebrate host . evs derived from leishmania donovani are involved in immune response evasion mechanisms , enabling parasite survival in the host [ 37 , 133 , 134 ] . in contrast , evs derived from macrophages infected with leishmania amazonensis induce proinflammatory response in vitro by stimulating the production of proinflammatory cytokines tnf- , il-12 , and il-1 . these host - derived vesicles have been characterized and contain both parasite and host components [ 37 , 38 ] , which indicates that a cross talk of signaling events occurs during infection . indeed , the immunization of mice with dexosomes derived from dc pulsed with leishmania major antigen was able to provide protection against the parasite . this finding could help to improve the available canine vaccines , used to stop transmission , and eventually develop a preventive prophylactic human therapy for leishmaniasis . trichomonas vaginalis , a flagellated protozoan that colonizes human vaginal and urethral epithelia , also secretes vesicles that act at the host - parasite interface . t. vaginalis evs stimulate the immune response by increasing the production of il-6 and il-8 and promote greater adherence of less adherent strains of the parasite to the epithelium . t. vaginalis evs fuse with and deliver their contents to host cells and are clearly involved in the colonization of the genital host 's tract . it is also possible that evs from this parasite could provide a more suitable environment to other sexually transmitted diseases such as hiv or hpv . , apicomplexa parasites that cause human and animal malaria focusing mainly in the immunization alternatives . for example , evs derived from reticulocytes infected with plasmodium yoelii , a rodent malaria , induce protection to infection in mice . plasmodium berghei , another rodent malaria , secretes microparticles in the plasma of infected mice that induce an intense macrophage activation , which results in inflammatory reaction via tlr4 and myd88 . therefore , these evs are key components in the modulation and communication between the parasite and the host . however , one of the main difficulties in working with human plasmodium , especially plasmodium vivax , is the availability to have enough amounts of evs . the disease is usually transmitted by eating contaminated meat , accidental ingestion of cat feces with oocytes , and congenital contact . evs derived from dc incubated with t. gondii antigens induce an intense immune response , increasing the levels of mhc class ii and the specific production of t - cells and cytokines . studies of immunization with these dc are promising alternatives in promoting protection against t. gondii [ 141 , 142 ] . eimeria tenella , eimeria maxima , and eimeria acervulina are also coccidian parasite of chickens that also release evs , which confer protective immune response against the parasite [ 123 , 143 , 144 ] . in summary , evs isolated from several parasites or from infected cells have major effects on the immune response and are also potential candidates for immunoprevention of parasitic diseases . fungi have the capacity to cause devastating human diseases , some of them with high mortality rates , in both immunocompetent and immunocompromised individuals . pathogenic fungi exhibit a singular genetic flexibility that facilitates rapid adaptation to the host or environment . however , there are several open questions of how these pathogens colonize and cause morbidity . as other eukaryotic organisms , fungi use membrane trafficking to connect intracellular and extracellular compartments allowing sorting of protein and lipids to their final cellular sites . for a variety of proteins , the extracellular milieu is the final destination of the cell wall components , digestive enzymes , and , in the pathogenic species , virulence factors . in fungi , the cell wall represents the final step of secretion , an event that brings additional complexity to the secretory mechanisms used by these cells . the cell wall is a complex and rigid structure basically composed of chitin , chitosan , -1,3-glucan , -1,6-glucan , mixed -1,3-/-1,4-glucan , -1,3-glucan , melanin , and glycoproteins as major constituents . evs are now recognized as important structures for transcell transport of virulence factors that modulate host immune responses [ 147 , 148 , 150 , 151 ] , suggesting the importance of these structures in the pathogenesis of many fungal diseases . currently , evs were identified in several pathogenic fungi such as histoplasma capsulatum , paracoccidioides brasiliensis , sporothrix schenckii , candida albicans , candida parapsilosis , malassezia sympodialis [ 42 , 78 , 152 , 153 ] , and nonpathogenic yeast saccharomyces cerevisiae . different proteins , sterols , phospholipids , polysaccharides , and pigments have been characterized in these fungal evs isolated from culture supernatants [ 42 , 78 , 150158 ] . many of these molecules have been identified as known virulence factors or inducers of host humoral responses . for example , in c. neoformans the most important virulence factor and immunomodulator , the glucuronoxylomannan ( gxm ) , was detected in vesicles released during in vitro macrophage infection . in p. brasiliensis , similar gxm that interacts with 1,3-glucans was detected in evs . gxm acts differently on the host immune response , depending on its specific molecular characteristics [ 44 , 45 ] making it a possible target for antifungal therapy or vaccination . another key molecule in fungal infection is glucosylceramide ( glccer ) , a glycolipid component of the fungal cell wall , which has been detected in evs of c. neoformans [ 152 , 158 ] , p. brasiliensis , and c. albicans . fungal glccer is an antigenic glycosphingolipid that elicits antibody responses in experimental infection models and in patients affected by some mycoses , such as cryptococcosis . glccer is described as a virulence regulator of c. albicans and c. neoformans [ 163 , 164 ] . furthermore , glccer from p. brasiliensis , aspergillus fumigatus , and s. schenckii inhibited t - cell proliferation in vitro . the glccer from a. fumigatus was able to activate in vitro mouse and human nk cells and to induce airway hyperreactivity in mice . these findings indicate that fungal glccer may influence both humoral and cellular responses and that inhibition or blocking the glccer action can be a therapeutic approach . other studies have evidenced that vesicles isolated from c. neoformans culture supernatant were able to melanize after incubation with l - dopa , a substrate for melanization . melanin has been identified in several pathogenic fungi . although it is immunologically active , little is known about its role in the immune response . melanin protects fungal cells from phagocytosis by macrophages , a key step in the host defense against these pathogens . it also reduces proinflammatory cytokines and decreases their susceptibility to antifungal drugs , mainly to amphotericin b and caspofungin , and is less evident or absent in ketoconazole , fluconazole , or itraconazole [ 171 , 172 ] . therefore , it seems that melanization is a distinguished feature observed in evs released during fungal infections and its role should be further explored in the fungal pathogenesis . vesicular components reacted with immune serum from patients with cryptococcosis , histoplasmosis , and paracoccidioidomycosis ( pcm ) [ 42 , 78 , 153 ] or with serum from c. albicans - infected mice . particularly , evs of p. brasiliensis transport components carrying -galactopyranosyl ( -gal ) epitopes , a highly immunogenic molecule , which were efficiently recognized by anti--gal antibodies from patient with pcm . these data showed that the fungal vesicular products might be important serological markers produced during this disease . the immunomodulatory activity of fungal evs is still poorly understood . in vitro studies have demonstrated that mammalian macrophages can incorporate fungal evs , resulting in increased levels of both pro- and anti - inflammatory cytokines [ 150 , 151 ] . specifically , in c. neoformans , the exposure of macrophages to evs resulted in their internalization and production of il-10 , tgf- , and tnf- , while for c. albicans , the production of il-10 , il-12 , and tgf- was observed . in both studies , fungal evs stimulated murine macrophages to produce higher levels of no [ 150 , 151 ] . this effect probably occurred due to the fungal evs preparations , which were composed of heterogeneous populations of different size and probably content [ 148 , 150 ] . m. sympodialis releases evs carrying allergen , which induce high levels of tnf- and il-4 , suggesting that vesicles have multiple immunoregulatory functions in atopic eczema . despite this controversy in host immune response , recently , vargas et al . showed that inoculation of galleria mellonella , a larvae model , with evs followed by challenge with c. albicans reduced the number of recovered viable yeasts in comparison to infected larvae control . moreover , these authors also observed immunomodulation of dc after internalization of evs from c. albicans . the synthesis of il-12 , il-10 , tgf- , and tnf- was also significantly increased in comparison to nonstimulated dc . proteomic - based approaches have been used to characterize c. neoformans , p. brasiliensis , h. capsulatum , and c. albicans and s. cerevisiae evs [ 78 , 150 , 151 , 153 , 156 ] . interestingly , most of the identified proteins in p. brasiliensis and c. neoformans lacked the characteristic signal peptide required for conventional secretion [ 78 , 156 ] , suggesting that fungal vesicles can also be derived from unconventional secretory mechanisms , as observed in mammalian cells . these proteomic analyses also revealed a large complexity of proteins with diverse biological functions in fungi evs . remarkably , we notice the presence of four proteins repeated in all evs analyzed as follows : glyceraldehyde-3-phosphate dehydrogenase ( gadph ) , phosphoglycerate kinase , elongation factor 1-alpha , and 6-phosphogluconate dehydrogenase . thus , it is possible to consider the potential of these molecules as biomarkers of fungal evs . . the major reported functions of evs are highlighted in figure 1 . in normal and tumor cells , they affect the following : antigen presentation , immune suppression , intercellular communication , inflammation , cellular homeostasis , and coagulation . in pathogens , they are considered virulence factors and are involved in the following : cell adhesion and invasion , evasion and modulation of the immune response , and drug resistance . the evs from mammalian cells contain molecules such as mhc classes i and ii , mrna , mirna , caspase 3 , signaling factors , structural proteins , and cytokines . the evs isolated from tumor cells express , for example , fasl , mhc classes i and ii , mrna , mirna , fadd , p - glycoprotein , mmps , ps , and tf . in protozoan , evs are formed by key membrane components involved in host - parasite interaction . omv or evs from bacteria have antigenic material providing gene transference of resistance to antibiotics and adaptation factors . fungal evs are structures for transcell transport of virulence factors , immunomodulatory molecules , and serological markers . therefore , evs extend the cell - to - cell communication between host and pathogens . by preventing this communication , in addition , the presence of evs and the characterization of their composition can provide new diagnostic information on several diseases . furthermore , studies on evs in the different situations can be useful to understand the intimate mechanisms of pathogenesis . in conclusion , evs represent a rich and challenging subject for basic and applied research enabling the understanding of a plethora of different mechanisms and opening new tools to combat diseases ( figure 2 ) .

almost all cells and organisms release membrane structures containing proteins , lipids , and nucleic acids called extracellular vesicles ( evs ) , which have a wide range of functions concerning intercellular communication and signaling events . recently , the characterization and understanding of their biological role have become a main research area due to their potential role in vaccination , as biomarkers antigens , early diagnostic tools , and therapeutic applications . here , we will overview the recent advances and studies of evs shed by tumor cells , bacteria , parasites , and fungi , focusing on their inflammatory role and their potential use in vaccination and diagnostic of cancer and infectious diseases .

1. Introduction 2. Extracellular Vesicles in Cancer 3. Bacterial Vesicles 4. Parasite Vesicles 5. Fungal Vesicles 6. Concluding Remarks

extracellular vesicles ( evs ) are particles of 20 nm up to 5 m in diameter composed of proteins , nucleic acid , and lipids that are found in body fluids such as plasma , serum , saliva , urine , breast milk , ascites , and cerebrospinal fluids . these particles are involved in intercellular communication , modulating a wide range of signaling events during innate and acquired immune responses ( figure 1 and table 1 ) [ 24 ] . they can be derived from dendritic cells ( dexosomes ) , prostate tissue ( prostasomes ) , bone , cartilage and atherosclerotic plaques ( matrix vesicles ) , neurons ( synaptic vesicles ) , apoptotic blebs or apoptotic bodies ( microparticles , exosomes , and apoptotic vesicles ) , shed vesicles , shedding microvesicles or microparticles ( ectosomes or microvesicles ) , and membrane fragments of virus infected cells , protozoa , fungi , and bacteria outer membrane vesicles [ 1 , 2 , 4 , 610 ] . the vesicles derived from mammalian cells contain a family of integral membrane proteins that cross four times the lipid bilayer and are called tetraspanins , including the surface markers of lymphocytes and antigen - presenting cells such as cd37 , cd9 , cd53 , cd63 , cd81 , and cd82 . for example , t - cells and monocytes secrete vesicles that contain fasl on the surface that modulate apoptosis of the other cells ( figure 2 ) . tumor cells secrete evs that are able to downregulate the immune system , allowing the escape from the immune system . therefore , evs are potential biomarkers and antigens for vaccination , with potential uses for early diagnostic , and therapeutic applications in several diseases . the purpose of this review is to provide an updated overview of the vesicles released by distinct pathogens and mammalian tissues , highlighting their potential use in vaccination and diagnostic of cancer and infectious diseases . moreover , evs contain proteins and genetic material from the originating tumor cells that can be used as diagnostic biomarkers . in this regard , recent efforts to elucidate different roles and signaling pathways of evs have been conducted . a pivotal role of evs during cancer cell migration and invasion has been reported in different cell types . for instance , evs derived from 786 - 0 renal tumor cells enhance their migration and invasion properties . tamoxifen - resistant breast tumor cells release exosomes that contain micrornas ( mir221/222 ) and promote drug resistance in naive cells . similarly , resistance to docetaxel in breast tumors and prostate cancer , as well as cisplatin in human lung cancer line ( a549 cells ) , was associated with the content of vesicular micrornas transferred to susceptible cells [ 4749 ] . moreover , evs from a549 cells containing trkb , egfr , and sortilin receptors ( tes complex ) were related to angiogenesis induction through endothelial cells . in hepatocellular carcinoma ( hcc ) , one of the most lethal cancers , the tumor becomes more resistant to tgf-dependent chemotherapy through long noncoding rnas ( lncrnas ) obtained from evs . therefore , the extracellular communication through evs is an important mechanism to activate / deactivate certain crucial events in tumor cell biology . interestingly , it is possible to detect mir-105 in the blood circulation before the metastasis establishment reinforcing its potential role as a diagnostic biomarker . likewise , expression of mir-214 , mir-221 , and mir-222 present in these evs is related to lymph node metastasis , venous invasion , and tumor development . for example , the elevated expression of rab22a gene in breast tumor cells induced by hypoxia , common in advanced tumors , increases the shedding of vesicles , and the rab protein colocalizes with the sites of budding evs . moreover , the knockdown of rab22a prevents metastasis , supporting the idea that rab is involved in the generation of evs . these vesicles showed chemotactic effects on cd4 + and cd8 + t - cells , efficiently activating dendritic cells ( dc ) . another study showed that evs derived from breast cancers can alter the tumor microenvironment and promote tumorigenesis of normal cells via induction of autophagy , response to dna damage repair ( ddr ) , and induction of reactive oxygen species ( ros ) in normal breast epithelial cells . this includes the polyadenylate - binding protein 1 ( pabp1 ) , predominant in evs from metastatic duodenal tumor cell lines , prostate - specific membrane antigen ( psa ) related to prostate cancer progression , angiogenesis , and metastasis , mir-21 and mir-146a in cervical cancer , and finally lncrnas in skin cancer ( secreted into the blood or urine through evs ) . it was also shown that evs from pancreatic tumor cells contain fragments of double - stranded genomic dna ( dsdna ) , suggesting that mutations may be identified in this dsdna as predictors of cancer and streamline therapeutics . in cancer therapy , evs from tumor cells are able to associate better with their recipient cells than liposomes ( > 10-fold ) , due to their lipid and protein composition . in addition , micrornas can be delivered to tumor cells and interfere with cancer progression and metastasis . in this logic , synthetic mir-143 was introduced into mesenchymal stem cells , and the secreted exosomes containing mir-143 was transferred to osteosarcoma cells to reduce the migration of the latter cell . interestingly , a feedback regulatory mechanism for controlling exosome release was suggested , in which exosomes derived from normal human mammary epithelial cells could impair the release of exosomes from breast tumor cells . the vesicles could be used as a diagnostic , because tumor cells release vesicles in biologic fluids like urine , blood , ascites , and pleural fluids . for example , patients with ovarian cancer shed vesicles derived from tumor cells in the circulation . recently , protein - loaded dexosomes were used to protect against tumor growth , whereby cd8 + t - cell responses occurred in vivo [ 63 , 64 ] . in this way , antitumor response could be elicited as these complexes may stimulate cd8 + and cd4 + t - cell responses in an exosomes from rab27a overexpressing cells increased significantly cd4 + t - cell proliferation in vitro because these exosomes upregulated mhc class ii , cd80 , and cd86 molecules in dc . , escherichia coli , pseudomonas aeruginosa , and helicobacter pylori . bacterial evs are composed of cytosolic and membrane proteins , lipoproteins , phospholipids , glycolipids , and nucleic acids [ 31 , 32 , 8184 ] . for example , omvs from bordetella parapertussis contain surface immunogenic molecules , porin , outer membrane protein ompq , and pertactin that were used in a murine model to assess the protection against infection . in the same way , omvs of pseudomonas putida kt2440 have outer membrane proteins such as oprc , oprd , opre , oprf , oprh , oprg , and oprw which can serve as adjuvants or vaccine . another interesting aspect of omvs is their role in delivering endotoxins to host cells as demonstrated for enterogenic and uropathogenic escherichia coli ( ( etec ) and ( upec ) ) , the causative agents of traveler 's diarrhea and human urinary tract infections . both etec and upec strains are able to produce many virulence factors including the heat - labile enterotoxin ( lt ) , homologous to cholera toxin , and cytotoxic necrotizing factor type 1 ( cnf1 ) . in particular , lt also acts as a ligand for vesicle binding , which is internalized via lipid rafts . once inside the cell , the toxin is trafficked via retrograde transport through the golgi and the endoplasmic reticulum . omvs from different species of gram - negative bacteria contain lipopolysaccharide ( lps ) , proteins , and nucleic acids , which are strong agonists in the modulation of inflammatory reactions through the activation of toll - like receptors ( tlrs ) . these activations require the action of lps , which is sensed by toll - like receptor 4 ( tlr4 ) on host cells , and induce an innate immune response to gram - negative bacteria leading to inflammatory cytokine production [ 9396 ] . another interesting example is omvs isolated from haemophilus influenzae , which increases the expression of cd69 and cd86 and activating of the humoral response . in addition , they induce tlr9 signaling through bacterial dna , which causes a significant proliferative response of inflammatory cells . there are , however , several cases that vaccination with omvs requires further developments to improve better antigenicity , manufacturability , and reduction of pyrogenicity , detergent extract , and toxicity via lps detoxification . however , with high doses these omvs induced systemic inflammatory , characterized by hypothermia , tachypnea , and leukopenia ( sepsis ) . because of the thick cell wall of gram - positive bacteria , extracellular vesicle secretion has been less studied in these bacteria . furthermore , s. aureus extracellular vesicles induce atopic dermatitis - like skin inflammation in mice . these observations recently , a study on the immune activating role of gram - positive bacteria - derived ev has been published . the gram - positive bacillus anthracis , the agent of the anthrax disease , also shed membrane - derived vesicles . the protection induced by vesicles obtained from gram - positive bacteria was not as effective when compared to gram - negative bacteria omvs indicating that further work might be necessary to improve their potential . they are composed of membrane fragments and cytosolic components , including proteins , lipids , and nucleic acids that accumulate in the supernatant of the protozoan cultivated in the presence or absence of host cells [ 40 , 41 ] . [ 117 , 118 ] , trichomonas vaginalis , toxoplasma gondii [ 120122 ] , and eimeria parasites . when t. cruzi enters the host , the first line of defense is the innate immune response , which initiates when receptors that recognize microbial products are activated . this occurs through toll - like receptors ( tlr ) signaling and macrophage activation by mucin - like glycoproteins , which corresponds to 6080% of the parasite surface molecules [ 35 , 127 ] , resulting in the increased production of il-12 , ifn- , and nitric oxide ( no ) . infective parasites obtained from cultured mammalian cells shed large amounts of evs that are rich in these surface molecules . t. cruzi evs are enriched in -gal containing glycoconjugates , found preferentially in the mucin - like molecules , and several surface glycoproteins , known as members of a trans - sialidase ( ts ) family that participate in adhesion and invasion of host cells [ 40 , 41 , 114 , 130 ] . therefore , the production and release of evs might have a key role in the establishment of infection and may be considered a platform to develop preventive or prophylactic vaccines for chagas disease . leishmania genus encloses protozoan species that cause visceral , cutaneous , and mucocutaneous leishmaniasis in humans . the disease is transmitted by sandfly vectors ( lutzomyia and phlebotomus ) , which inject parasites into the host during the insect blood meal . in culture , the insect stages of several leishmania species release evs containing parasite antigens , such as the surface glycoprotein of 63 kda ( gp63 ) that has a strong suppressive effect on host macrophages [ 37 , 38 ] . however , a missing step in leishmania evs biogenesis is whether those structures also contain the major surface lipophosphoglycan ( lpg ) , a multivirulence factor involved in the interaction with the vertebrate and invertebrate host . in contrast , evs derived from macrophages infected with leishmania amazonensis induce proinflammatory response in vitro by stimulating the production of proinflammatory cytokines tnf- , il-12 , and il-1 . these host - derived vesicles have been characterized and contain both parasite and host components [ 37 , 38 ] , which indicates that a cross talk of signaling events occurs during infection . indeed , the immunization of mice with dexosomes derived from dc pulsed with leishmania major antigen was able to provide protection against the parasite . plasmodium berghei , another rodent malaria , secretes microparticles in the plasma of infected mice that induce an intense macrophage activation , which results in inflammatory reaction via tlr4 and myd88 . however , one of the main difficulties in working with human plasmodium , especially plasmodium vivax , is the availability to have enough amounts of evs . the disease is usually transmitted by eating contaminated meat , accidental ingestion of cat feces with oocytes , and congenital contact . evs derived from dc incubated with t. gondii antigens induce an intense immune response , increasing the levels of mhc class ii and the specific production of t - cells and cytokines . studies of immunization with these dc are promising alternatives in promoting protection against t. gondii [ 141 , 142 ] . eimeria tenella , eimeria maxima , and eimeria acervulina are also coccidian parasite of chickens that also release evs , which confer protective immune response against the parasite [ 123 , 143 , 144 ] . as other eukaryotic organisms , fungi use membrane trafficking to connect intracellular and extracellular compartments allowing sorting of protein and lipids to their final cellular sites . for a variety of proteins , the extracellular milieu is the final destination of the cell wall components , digestive enzymes , and , in the pathogenic species , virulence factors . in fungi , the cell wall represents the final step of secretion , an event that brings additional complexity to the secretory mechanisms used by these cells . the cell wall is a complex and rigid structure basically composed of chitin , chitosan , -1,3-glucan , -1,6-glucan , mixed -1,3-/-1,4-glucan , -1,3-glucan , melanin , and glycoproteins as major constituents . different proteins , sterols , phospholipids , polysaccharides , and pigments have been characterized in these fungal evs isolated from culture supernatants [ 42 , 78 , 150158 ] . for example , in c. neoformans the most important virulence factor and immunomodulator , the glucuronoxylomannan ( gxm ) , was detected in vesicles released during in vitro macrophage infection . another key molecule in fungal infection is glucosylceramide ( glccer ) , a glycolipid component of the fungal cell wall , which has been detected in evs of c. neoformans [ 152 , 158 ] , p. brasiliensis , and c. albicans . the glccer from a. fumigatus was able to activate in vitro mouse and human nk cells and to induce airway hyperreactivity in mice . although it is immunologically active , little is known about its role in the immune response . it also reduces proinflammatory cytokines and decreases their susceptibility to antifungal drugs , mainly to amphotericin b and caspofungin , and is less evident or absent in ketoconazole , fluconazole , or itraconazole [ 171 , 172 ] . vesicular components reacted with immune serum from patients with cryptococcosis , histoplasmosis , and paracoccidioidomycosis ( pcm ) [ 42 , 78 , 153 ] or with serum from c. albicans - infected mice . particularly , evs of p. brasiliensis transport components carrying -galactopyranosyl ( -gal ) epitopes , a highly immunogenic molecule , which were efficiently recognized by anti--gal antibodies from patient with pcm . specifically , in c. neoformans , the exposure of macrophages to evs resulted in their internalization and production of il-10 , tgf- , and tnf- , while for c. albicans , the production of il-10 , il-12 , and tgf- was observed . this effect probably occurred due to the fungal evs preparations , which were composed of heterogeneous populations of different size and probably content [ 148 , 150 ] . despite this controversy in host immune response , recently , vargas et al . moreover , these authors also observed immunomodulation of dc after internalization of evs from c. albicans . the synthesis of il-12 , il-10 , tgf- , and tnf- was also significantly increased in comparison to nonstimulated dc . proteomic - based approaches have been used to characterize c. neoformans , p. brasiliensis , h. capsulatum , and c. albicans and s. cerevisiae evs [ 78 , 150 , 151 , 153 , 156 ] . interestingly , most of the identified proteins in p. brasiliensis and c. neoformans lacked the characteristic signal peptide required for conventional secretion [ 78 , 156 ] , suggesting that fungal vesicles can also be derived from unconventional secretory mechanisms , as observed in mammalian cells . remarkably , we notice the presence of four proteins repeated in all evs analyzed as follows : glyceraldehyde-3-phosphate dehydrogenase ( gadph ) , phosphoglycerate kinase , elongation factor 1-alpha , and 6-phosphogluconate dehydrogenase . the major reported functions of evs are highlighted in figure 1 . in normal and tumor cells , they affect the following : antigen presentation , immune suppression , intercellular communication , inflammation , cellular homeostasis , and coagulation . the evs from mammalian cells contain molecules such as mhc classes i and ii , mrna , mirna , caspase 3 , signaling factors , structural proteins , and cytokines . the evs isolated from tumor cells express , for example , fasl , mhc classes i and ii , mrna , mirna , fadd , p - glycoprotein , mmps , ps , and tf . fungal evs are structures for transcell transport of virulence factors , immunomodulatory molecules , and serological markers . by preventing this communication , in addition , the presence of evs and the characterization of their composition can provide new diagnostic information on several diseases . in conclusion , evs represent a rich and challenging subject for basic and applied research enabling the understanding of a plethora of different mechanisms and opening new tools to combat diseases ( figure 2 ) .

research into the amino acid glutamate ( glu ) and its role in neurotransmission and disease has a long history spanning over four decades . recognized as the predominant excitatory neurotransmitter in the central nervous system ( cns ) , a byproduct of glutamine ( gln ) deamination , a critical step in nitrogen metabolism and a source of energy for rapidly dividing cells , this non - essential amino acid has been well - studied for its role in cellular homeostasis 1 . in the nervous system , glu is the primary fast excitatory neurotransmitter participating in a wide range of neural functions such as learning and memory , long - term potentiation and synaptic plasticity 2 , 3 . the early observation that increasing amounts of glu can cause excitotoxicity and neural cell death opened the door to a whole field of research implicating glu toxicity in neurodegenerative diseases ( ndds ) . in depth study of glu signaling mechanisms has facilitated the development of treatments for glu - related ndds such as alzheimer 's and parkinson 's disease and multiple schlerosis 4 . most recently , glu signaling has been implicated in cellular transformation and cancer progression in multiple organs such as brain , skin , breast and prostate 5 . beginning with the many and varied glu receptors ( glurs ) , glurs are divided into two families : ionotropic ( iglur ) and metabotropic ( mglur ) ( reviewed in 6 , 7 ) . further , the ionotropic receptor family includes the n - methyl - d - aspartate ( nmda ) , a - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) , and kainate receptor subfamilies - so named for the chemical agonist that selectively binds to the subfamily members . the iglur subfamilies all share a common voltage - gated ion channel function . metabotropic glurs , on the contrary , are g - protein - coupled receptors ( gpcrs ) containing the classic seven transmembrane domain structure and initiate signaling cascades or cation influx upon glu binding . based on sequence homology , pharmacology and second messenger associations , mglurs are further categorized into the group i , ii and iii subfamilies 8 . although iglur signaling plays a critical role in neural function and has been shown in a few cases to play a role in cancer progression , for the purposes of this review , we will provide a cursory discussion of iglurs while focusing more on the detail of glu - mglur signaling mechanisms . in general , all iglurs are quaternary ligand - gated ion channels that allow cation influx upon glu binding 7 . each of the four subunits that comprise the channel share a similar structure of four well - conserved domains including the extracellular amino - terminal ( atd ) , ligand - binding ( lbd ) , transmembrane ( tmd ) and intracellular carboxy - terminal ( ctd ) domains ( figure 1a ) . glu or agonist interaction with the lbd results in a conformation shift which changes the angle of the tmd regions , opening a pore in the membrane where influx of sodium , potassium and/or calcium occurs ( figure 1b ) . interestingly , the nmda family of iglurs can also be activated by voltage gating and are often found paired with mglurs . if enough iglurs are stimulated simultaneously , high concentrations of cation influx will result in an action potential - the fastest type of excitatory synaptic transmission throughout the central and peripheral nervous systems ( cns , pns ) and the retina 7 . after the signal is received by the target cell , excitatory amino acid transporters ( eaats ; also known as glutamate transporters ) expressed on postsynaptic and supporting glial cells are tasked with emptying the synaptic cleft of glu to effectively turn off the signal and reset the system for generation and propagation of new action potentials 7 . prolonged stimulation of the iglurs and other post - synaptic signaling components results in a phenomenon called excitotoxicity - a major contributor to ndds and nervous system injuries 9 . cells associated with trauma or disease release glu , stimulating further glu release by normal signaling mechanisms , and the resulting excessive ca influx leads to aberrant over - activation of proteases and caspases 4 , 10 . since eaats require normal membrane voltage to function , they are not able to overcome the bombardment of cations caused by the overstimulation of iglurs and apoptosis can occur , further exacerbating the injury and/or contributing to disease onset and progression 11 , 12 . in fact , ischemia and hypoxia after cns injury results in such a buildup of glu in the synaptic cleft that depolarization and eaat activity is inhibited leading to paralysis . both nmda and ampa receptor subfamily members have been implicated in excitotoxicity after nervous system trauma 13 . it is clear that reactive oxygen species ( ros ) and overstimulation of calcium - dependent signaling molecules is responsible , at least in part , for neuronal cell death by a similar mechanism as outlined for nervous system trauma 9 . research into exactly which iglurs are involved in disease onset and progression has led to treatments with varying success rates ( reviewed in 4 , 14 ) . the mglur superfamily consists of 12 members encoded by eight genes ( reviewed in 15 ) . this is accomplished by multiple alternative splicing products produced by some mglur genes , while others encode only one ( figure 2 ) . for example , mglur1 encodes five isoforms while there is only one isoform of mglur2 . within the superfamily , the three aforementioned subgroups ( i , ii and iii ) emerge based on amino acid sequence homology , agonist binding pharmacology and the second messenger system that is activated upon glu binding to each receptor ( see 8 for a review ) . activation of group i mglurs ( mglur1 and 5 ) and coupled gq g - proteins initiates signaling cascades involving phospholipase c / inositol 1,4,5-triphosphate / diacylglycerol ( plc / ip3/dag ) . groups ii and iii both reduce camp levels by signaling through the inhibitory g - protein , gi . group ii receptors include mglur2 and 3 while group iii includes mglur4 , 6 , 7 and 8 ( figure 2a ) . within each group , amino acid sequence homologies are more than of 70% , while between groups , homology is only , on average , 45% ( figure 3 ) . the n - terminus exists a large bi - lobed extracellular domain known as the amino terminal domain ( atd ) , which in the protein crystallography literature is also referred to as the venus fly trap domain ( vft ) because of its unique shape 16 . following the atd is a cysteine - rich domain ( crd ) which is critical for dimerization and activation 17 . c - terminal to the crd , the classic seven alpha - helical transmembrane domains ( tmd ) shared by all gpcrs are found and finally , an intracellular cytoplasmic tail domain ( ctd ) ( reviewed in 18 ) . some isoforms of the group i mglurs ( mglur1a and 5a ) contain a very long proline - rich domain which has been studied for its intricate protein - protein interactions and complex formations 19 . this domain , called the homer1 binding motif , facilitates coupling of mglurs with nmda receptors among other proteins and will be described in detail below . aside from the tmd and g - protein coupling , mglurs are highly dissimilar at the structural and functional levels to any other classical gpcr groups studied to date and are therefore referred to as atypical gpcrs . in general , gpcrs are organized into five groups and virtually all of them belong to groups a , b , d and e. mglurs , gaba receptors , two groups of taste receptors , and a ca - sensing receptor ( casrs ) class belong to group c - a clearly divergent group based on amino acid sequence , size and structure 20 , 21 . interestingly , casr receptors are not sensitive to glu or mglur agonists , but share amino acid identity of 28% with group i mglurs 22 . casrs are mainly involved in ca sensing and homeostasis in the parathyroid gland and detection of l - amino acid levels 20 . first , both mglurs and casrs form homodimers in vivo through disulfide bridging between crds and this event is critical for function 17 , 24 , 25 . in addition , unlike other gpcrs which typically have a short extracellular ligand binding domain , the atd is a large two - lobed structure allowing for glu binding between the two lobes ( figure 4 ) . the atd in human mglurs is , on average , 56 kda ( 470 - 510 amino acids ) which is far larger than the extracellular domain of other gpcrs that usually measure about 5 kda , or 50 amino acids and correspond in size with ligand size 26 . for example , gpcrs which are activated by small molecules such as cations generally have a very small extracellular loop , while larger ligands require a larger lbd . this does not hold true for group c gpcrs since ca and glu are small molecules , indeed . the atd is related to bacterial periplasmic amino acid binding proteins , the structure of which has been extensively modeled and provides an excellent basis for models of the mglur atd 27 . interestingly , some allosteric modifiers do not bind to mglurs in glu - binding pocket within the atd . instead , they typically bind to the small loop structure connecting the atd to the tmd or within the tmd itself which is similar to activation of other gpcrs 25 . reports have suggested that ca ( which can activate both mglurs and casrs ) and l - amino acids ( which can activate casr signaling ) may bind to the extracellular loops connecting the seven transmembrane domains , further separating group c gpcrs from the other groups 28 , 29 . finally , despite the canonical role of the third intracellular loop ( i3 ; figure 4 ) in g - protein activation , group c gpcrs appear to use the second loop for this function ( i2 ; 30 - 33 ) . the downstream signaling effects of glu binding and the pharmacology pharmacodynamics of agonist / antagonist binding to mglurs has been studied extensively due to this receptor group 's clear implications in both benign and malignant disease progression . mglurs are localized throughout the cns and pns and have been shown to play a role in homeostasis in many organ systems 34 . groups ii and iii receptors are generally found on presynaptic cells in the nervous system which correlates well with their function in regulating ion channel opening , while group i receptors are generally found on postsynaptic cells ( 35 and reviewed in 4 ) . this is not a rule , however , since all three receptor groups have been found on both pre - and postsynaptic cells . with regards to non - neural tissues , mglurs have been found in all of the major organ systems including epithelium , connective and muscle tissues . more specifically , one or more mglurs are expressed in the gastrointestinal system , immune system , genitourinary tract , bone , and hepatosplenic system ( reviewed in 6 ) . in many cases , the first member of the human mglur gene superfamily , mglur1 ( sometimes referred to as the glutamate receptor metabotropic 1 ; grm1 ) encodes five alternatively spliced isoforms ( figure 5 ) . a complete literature review on this gene is stymied by multiple confusing nomenclatures that have been used interchangeably throughout the decades . for example , the first isoform of mglur1 is synonymously referred to as the following : grm1a , mglur1a , mglur1 , gprc1a , and hmglu1a . surprisingly , a search for any of the synonyms excludes articles using the others , necessitating many searches to ensure completeness in collecting reliable and informative data in a review on this topic . we will use the simplest nomenclature which is the most utilized in recent studies , mglur1a , for this review and will always be referring to the homo sapiens gene unless explicitly noted . adding to this complexity is the fact that many of the studies on the mglur gene family have been performed using orthologues from the norway rat , rattus norvegicus . in fact , a large body of experimental data exists on the pharmacology , expression , mutational analysis and drug targeting of rodent mglur1s . this phenomenon is so pervasive that it is difficult to find a study in the last decade that has used the human gene or protein . the rodent orthologues have certainly facilitated our understanding of mglur signaling , but their translational value when interpreted in the context of non - malignant or malignant human diseases may remain questionable - at least in some cases . many of the initial studies on the human mglur1 gene cloning and isoform identification were published in the mid-1990s , before the human genome sequence became available and was regularly annotated with newly published information 36 - 41 . as a result , despite numerous publications on the human mglur1c and mglur1d isoforms , there are no references to them in the ncbi human genome database at either the mrna or protein levels . a nucleotide blast search with rat mglur1d found similarity with the human mglur1 and mglur5 precursor gene sequences , but since isoforms c and d were not annotated in the human genome databases , and human mglur1c and mglur1d do not exist there as either mrna or protein sequences . this issue is highlighted by the fact that annotation of isoforms a , b , c and d exists for the rattus norvegicus and mus musculus homologues of mglur1 in the ncbi databases while the human gene shows annotation of only isoforms a and b. the rat mglur1a protein sequence is 94% identical to the human mglur1a protein at the amino acid level ( figure 6 ) and is certainly an excellent model for studies on mglur1a function , however , it is a detriment to the field that genes with such a critical role in many organ systems , in ndds and in benign and malignant diseases have been far better characterized with modern bioinformatics in rodents than their human orthologues . figure 6 details the percent amino acid identity when the mouse and rat proteins are compared to the human protein . this analysis was performed using william pearson 's lalign program with huang and miller 's algorithm which can be found at http://www.ch.embnet.org/software/lalign_form.html 42 . the highest homology between the mouse and rat protein and the human protein exists in the tmd , while the lowest percent identity is found in the crd ( figure 6 ) . although all of the cysteine residues in the crd are conserved , it is unclear whether or not the changes in the surrounding amino acids change the three dimensional structure of the domain or influence how it interacts with other mglur1a proteins during homodimerization . human mglur1a was first expressed and characterized in vitro in 1998 by hermans and colleagues using an inducible expression system 43 , 44 . the localization ( diffuse cytoplasmic ) , size ( 145 kda ) and induction of receptor activity were assayed in the common rodent model , chinese hamster ovary ( cho ) cells 43 . subsequent studies have utilized this inducible rodent cell line system , known in the field as cho - lac - hmglu1a . one such study noted that the induction of high levels of mglur1a expression in cho cells leads to a notable morphology change which is modulated by changing amounts of ca in the culture medium , but follow up studies in which the consequences of this change on cell motility or wound healing were investigated were not found 28 . it would be a great service to the field for a study to be undertaken in which full - length rodent and human mglur1a are expressed in a human cell line and downstream signaling and biological assays are shown to yield identical results in side - by - side experiments . we were unable to find such a study , however , recent work by esseltine and colleagues showed the result of human mglur1a expression in human embryonic kidney 293 ( hek293 ) cells 45 . the mglur1a protein that was expressed in in esseltine and colleagues ' study began at amino acid 19 , therefore removing the amino - terminal end of the protein to include their own localization domain 45 . although this study confirmed that the truncated mglur1a localized to the plasma membrane and was able to initiate downstream signaling cascades upon stimulation , no biological effects such as cell growth , migration , morphology , etc . many studies have been aimed at determining how many isoforms were produced by the mglur1 gene and the function and localization of each ( see 46 , 47 for reviews ) . the shortest and least well - characterized , mglur1e , is truncated before the tmd , leading to the predicted expression of only the atd ( figure 5 ) 46 . follow up studies on a possible function for this isoform are not available . excluding mglur1e , the human mglur1 isoforms are virtually identical except for alternative splicing changes near the 3 ' end of the mrna which result in much smaller cytoplasmic domains at their c - termini for the mglur1b , c and d proteins than the mglur1a isoform ( figure 5 ) . this difference in size is dramatic : an amino acid sequence alignment of human mglur1a and mglur1b are 100% identical until amino acid 886 . afterwards , a small exon containing a stop codon is incorporated in mglur1b that is not included in mglur1a , explaining the difference in size between the two isoforms ( figure 5 ) . alternative splicing for human mglur1a results in a significantly longer protein than the other isoforms and includes the proline - rich homer1 binding motif at the c - terminus . the extended length of the mglur1a isoform prompts the question of why such a large cytoplasmic tail is required : studies have shown that extensive protein - protein interactions are facilitated by the proline - rich region which contains the homer - binding motif 19 . this phenomenon is not limited to mglur1a since mglur5a ( a member of group i ) also possesses this domain , however , gpcr group c member , casr does not 21 . the homer - binding motif found in the ctd of mglur1a and mglur5a is a poly - proline region that has been shown to interact with proteins containing a homer ena / vasp homology 1 domain ( homer evh1 ) 19 , 48 - 51 . homer1 , in particular , has been well - characterized for its role in the regulation of mglur1a and mglur5a activity . binding of full - length homer1isoforms to the consensus site found in the homer - binding motif of mglur1a/5a , ppxxf ( where x is any amino acid ) , has been shown to influence not only the trafficking of the receptor between the cytoplasm and the membrane , but also receptor clustering within the membrane through its scaffolding activity ( figure 4 ) . further , homer1 proteins can form homodimers and therefore link mglurs to other homer binding motif - containing proteins . this has been shown to be the case for plc signaling where homer1 homodimers create a scaffold which brings mglur1a/5a in close proximity to the inositol triphosphate receptor ( ip3r ) . activation of mglur1a/5a results in ip3 production and rapid , localized activation of intracellular ca release through ip3r - the speed of which is facilitated by the close physical proximity of ip3r to active mglur1a/5a via homer1 scaffolding 52 . in addition to the previously described domains within the mglur proteins , a cysteine - rich domain referred to as the 9-cystein domain in some studies or the cysteine - rich domain ( crd ) in others is found between the atd and tmd ( figure 4 ) 24 , 34 . this domain , unique to group c gpcrs , has been crystallized and studied in rat group ii and iii mglurs . this short domain ( only 69 amino acids in mglur1 ) consists of three -pleated sheets and nine cysteine residues - all nine of which are very well - conserved in the other mglur proteins , irrespective of subgroup classifications and are also conserved in the human orthologues . although the exact structure and function of this domain is still under investigation , it is clear from mutational analyses that certain cysteine 's are critical for receptor clustering and for facilitating the interaction between the atd and the tmd regions during ligand binding and activation 17 . activation of the mglur dimer has been shown to elicit a conformational change which brings the c - terminal regions of the crd in closer proximity to one another ( represented by a * in figure 4 ) and to involve a cysteine - cysteine interaction in the e2 loop of the tmd . crystal structures have indicated that this conformation change also involves a shift in the tmd , therefore coupling the atd with g - protein activation 25 . the mutational analyses and crystallography that was used to obtain these models were all completed using the rat orthologues of the mglur family members and although all of the cysteine 's in the crd are conserved in the human protein , as shown in figure 6 , studies will need to be performed to confirm the residues of importance in the human orthologues . glu binding and aforementioned conformational changes can precipitate a number of varied signaling responses including : activation of coupled ca , k and na channels , activation of gq and consequent downstream signaling cascades , activation of extracellular - signal - regulated kinase ( erk ) 1/2 ( also known as mitogen activated protein kinase ; mapk ) and retrovirus ak thymoma [ akt ; also known as protein kinase b ( pkb ) ] signaling and even stimulation of sarcoma ( src ) kinase signaling ( figure 7 ) 4 , 15 , 53 - 56 . interestingly , mglur1 differs from fellow group i mglur family member , mglur5 , in that its activation results in a single transient rise in intracellular ca while active mglur5 causes recurrent ca oscillations of unknown function 57 . each of these second messenger systems and their functions in specific tissues will be expanded upon below . glu stimulation of both mglurs and iglurs in neural cells results in increased intracellular ca levels via differing signaling mechanisms . unlike iglur signaling , which directly involves the opening of an ion channel upon glu - binding , mglur signaling can lead to ca influx by indirect ion channel opening and/or by second messenger signaling systems . homer1 binding to the homer binding motif can recruit the scaffold protein , shank , which facilitates the direct interaction of mglurs and nmdars resulting in ca influx 58 . further , activation of plcs by g11 results in the cleavage of phosphatidylinositol 4,5-bisphosphate ( pip2 ) into two products : the phosphate head group ( ip3 ) and the fatty acid chains ( e.g. , dag ) . dag remains in the plasma membrane while ip3 diffuses freely in the cytosol and binds to its cognate receptor , ip3r . each of these two products not only initiates a signaling cascade of its own but can also work cooperatively to achieve further protein activation ( figure 7 ) . protein kinase c ( pkc ) is recruited to the plasma membrane by cooperatively binding dag and ca and is maintained there in its active state by membrane - bound receptor for activated protein kinase c proteins ( e.g. , receptor for activated c kinases ( racks ) ) . for excellent reviews on neural pkc signaling , moreover , active ip3r results in further cytosolic increases in ca through the release of intracellular ca stores from the endoplasmic reticulum ( er ) . pkc activation plays a role in receptor desensitization through phosphorylation of mglur1a itself , a critical mechanism allowing for return to resting membrane potential after action potential propagation 60 . in addition to coupling of mglur1 to ip3 and to nmdars , homer1 proteins also appear to couple active mglur1 to pi3k , initiating signaling which has been shown to have potent effects on cell survival , metabolism , proliferation and tumorigenesis / cancer progression 61 , 62 . activated pi3k leads to the accumulation of phosphoinsitol 3,4,5-triphosphate ( pip3 ) and consequent recruitment of pleckstrin homology ( ph ) domain - containing proteins such as akt1 . native akt1 is recruited to the membrane via its ph domain where it is subsequently phosphorylated first at serine 473 by mammalian target of rapamycin complex 2 ( mtorc2 ) and this event triggers a second phosphorylation at threonine 308 by phosphoinositide dependent kinase 1 ( pdk1 ) 63 . fully active akt1 participates in a number of important downstream signaling events that certainly occur in neurons but are not limited to this cell type ( reviewed in 64 , 65 ) . akt1 and akt2 activation by mglur1 is not only neuroprotective but is also pro - proliferation in many neuronal cell types 61 , 66 , 67 . it should be noted , however , that activation of mglur1 is also considered a pro - apoptosis signal in subsets of neural cells and in certain experimental paradigms 68 ( reviewed in 69 ) . therefore , the link between survival and mglur1 signaling may be complex . as a survival signal , akt1 can phosphorylate bcl2-associated death protein ( bad ) resulting in bad 's dissociation from the bcl-2/bcl - x complex and therefore leading to its anti - apoptotic and cell survival activities 70 , 71 . in addition , nuclear factor kappa - light - chain - enhancer of activated b cells ( nfb ) activation following akt1-mediated phosphorylation of ib kinase ( ikk ) results in active transcription of survival and growth promoting genes 72 , 73 . further , phosphorylation of mtorc1 complex members by akt1 results in increased translation of mrnas required for cell growth 74 ( figure 7 ) . therefore , mglur1 activation is responsible for regulating cell cycle and proliferation in many cell types . glu signaling through nfb and ib kinase ( ikk ) has been shown to play a role in many neuronal processes including learning , memory and other processes where synaptic remodeling and plasticity are critical 75 . in addition , nfb target genes are implicated in the growth of dendritic spines and other neural processes 76 , 77 . interestingly , not only is nfb activated by this mechanism , but it can also be phosphorylated by pkc downstream of gq signaling through plc . active glurs ( both mglur and iglur families ) stimulate release of intracellular calcium stores via multiple previously - discussed mechanisms . in signal transduction , calcium can act as either second messenger ( when influx or release is downstream of g - protein signaling ) or as direct effector of enzyme activity ( where influx occurs as a direct or indirect result of ion - channel opening ) . as outlined above further , ca binds and activates calmodulin ( cam ) , the main second messenger that transduces ca - related signals 79 . the binding of 4 ca molecules in cam 's 4 ef - hand motifs results in the activation of ca / cam - dependent kinases ( camk ) and subsequent signaling has effects on learning and memory and long - term potentiation of action potentials ( figure 7 ) 4 , 78 . g - protein independent signaling mechanisms involving mglurs have been documented in rodents 54 , 56 . indeed , an additional consequence of active mglurs appears to be the activation of src tyrosine kinase and downstream messengers such as erk 56 . the functional role for g - protein independent glutamatergic signaling clearly involves long - term potentiation in the cns , since certain tyrosine kinase inhibitors have negative effects on this process in rats 54 , 56 , 80 . studies have also demonstrated that activation of erk / mapk signaling in the cns is also critical for synaptic plasticity and memory formation - two processes that require long - term potentiation 56 , 81 , 82 . canonical neural pathways involving mglurs have been well - established by a large body of literature ( see 4 for example ) . first , mglur1a expressed in baby hamster kidney ( bhk ) cells inhibits adenylate cyclase ( ac ) activity via signaling through g1/o , suggesting that mglur1a can couple to inhibitory g proteins 83 . in contrast , mglur1a in chinese hamster ovary ( cho ) cells does not affect ac or appear to couple with g1/o 84 . several overexpression models including cho , human embryonic kidney ( hek 293 ) and xenopus oocytes demonstrated that mglur1a can couple with gs to stimulate camp production 30 , 85 , 86 . therefore , there is no established rule for which g proteins mglur1s couple with - only that in the nervous system , they most commonly couple with g protein complex q and more specifically , g11 . a large body of work has linked mglurs to the development and progression of cancer . rodent mglur1 expression is sufficient for cellular transformation of melanocytes in culture , indicating that mglur genes can function as oncogenes 87 , 88 . furthermore , recent work has shown that not only is glu a serum biomarker for prostate cancer aggressiveness , but also that mglur1 is overexpressed in prostate tumors 89 . a detailed review on the involvement of mglurs in malignant disease has been recently published by our group 90 . in addition to the described role for mglur signaling in normal nervous system function , glutamatergic signaling through the mglur family has been implicated in ndds and learning and memory disorders 91 . for example , in severe autism and alzeimer 's disease , secretion of amyloid precursor protein- ( sapp ) is a known contributor to disease severity 92 , 93 . many reports have postulated that targeting mglur signaling may provide symptomatic relief from alzheimer 's and autism 90 , 91 , 94 . the mechanism underlying this phenomenon has not been documented as of this review , but it clearly involves antagonizing or inhibition of the pi3k / akt / mtor pathway which is a known target of active mglur signaling 91 . decades of studies have clearly established that glutamatergic signaling is critical for normal nervous system function . much of the research done to characterize this complex network of receptors and second messenger systems has been performed using rodent models . this has led to a critical lack of information on the human orthologues of the mglur family - a fact which may have far - reaching impacts on drug discovery for ndds and other nervous system disorders as well as for anti - cancer therapies . for example , it is clear from an analysis of the literature on the crystal structure of the mglur proteins that much of this work has been done on the rodent orthologues . in fact , when analyzing publications on this subject , a protein blast search is often necessary to determine the species of origin for the currently discussed peptides or proteins . the exact amino acid sequence of the human mglur that are involved in the disulfide bridging between members of the receptor dimer are not known and are inferred from these studies . although it is true that in the majority of cases , the information obtained from rodent crystallography may be applicable to the human proteins , however , without mutational analysis , the question will remain . furthermore , no studies were found that provide an analysis of the rodent and human mglur proteins expressed in the same cell line to allow a comparison between the orthologues in terms of expression level and function . hopefully , future work will address these issues and the field will gain insight into exactly how identical the rodent and human proteins are in terms of function and therapeutic targeting potential .

glutamate is a nonessential amino acid , a major bioenergetic substrate for proliferating normal and neoplastic cells , and an excitatory neurotransmitter that is actively involved in biosynthetic , bioenergetic , metabolic , and oncogenic signaling pathways . glutamate signaling activates a family of receptors consisting of metabotropic glutamate receptors ( mglurs ) and ionotropic glutamate receptors ( iglurs ) , both of which have been implicated in chronic disabling brain disorders such as schizophrenia and neurodegenerative diseases like alzheimer 's , parkinson 's , and multiple sclerosis . in this review , we discuss the structural and functional relationship of mglurs and iglurs and their downstream signaling pathways . the three groups of mglurs , the associated second messenger systems , and subsequent activation of pi3k / akt , mapk , nfkb , plc , and ca / cam signaling systems will be discussed in detail . the current state of human mglur1a as one of the most important isoforms of group i - mglurs will be highlighted . the lack of studies on the human orthologues of mglurs family will be outlined . we conclude that upon further study , human glutamate - initiated signaling pathways may provide novel therapeutic opportunities for a variety of non - malignant and malignant human diseases .

Introduction The ionotropic GluRs: Basic functions and implications in disease states The metabotropic GluRs are atypical G-protein coupled receptors Structure and function of the mGluR1 gene mGluR1 signaling in the nervous system mGluR1 signaling in malignant diseases Concluding Remarks

research into the amino acid glutamate ( glu ) and its role in neurotransmission and disease has a long history spanning over four decades . recognized as the predominant excitatory neurotransmitter in the central nervous system ( cns ) , a byproduct of glutamine ( gln ) deamination , a critical step in nitrogen metabolism and a source of energy for rapidly dividing cells , this non - essential amino acid has been well - studied for its role in cellular homeostasis 1 . in the nervous system , glu is the primary fast excitatory neurotransmitter participating in a wide range of neural functions such as learning and memory , long - term potentiation and synaptic plasticity 2 , 3 . in depth study of glu signaling mechanisms has facilitated the development of treatments for glu - related ndds such as alzheimer 's and parkinson 's disease and multiple schlerosis 4 . most recently , glu signaling has been implicated in cellular transformation and cancer progression in multiple organs such as brain , skin , breast and prostate 5 . beginning with the many and varied glu receptors ( glurs ) , glurs are divided into two families : ionotropic ( iglur ) and metabotropic ( mglur ) ( reviewed in 6 , 7 ) . further , the ionotropic receptor family includes the n - methyl - d - aspartate ( nmda ) , a - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) , and kainate receptor subfamilies - so named for the chemical agonist that selectively binds to the subfamily members . metabotropic glurs , on the contrary , are g - protein - coupled receptors ( gpcrs ) containing the classic seven transmembrane domain structure and initiate signaling cascades or cation influx upon glu binding . based on sequence homology , pharmacology and second messenger associations , mglurs are further categorized into the group i , ii and iii subfamilies 8 . although iglur signaling plays a critical role in neural function and has been shown in a few cases to play a role in cancer progression , for the purposes of this review , we will provide a cursory discussion of iglurs while focusing more on the detail of glu - mglur signaling mechanisms . each of the four subunits that comprise the channel share a similar structure of four well - conserved domains including the extracellular amino - terminal ( atd ) , ligand - binding ( lbd ) , transmembrane ( tmd ) and intracellular carboxy - terminal ( ctd ) domains ( figure 1a ) . interestingly , the nmda family of iglurs can also be activated by voltage gating and are often found paired with mglurs . prolonged stimulation of the iglurs and other post - synaptic signaling components results in a phenomenon called excitotoxicity - a major contributor to ndds and nervous system injuries 9 . cells associated with trauma or disease release glu , stimulating further glu release by normal signaling mechanisms , and the resulting excessive ca influx leads to aberrant over - activation of proteases and caspases 4 , 10 . both nmda and ampa receptor subfamily members have been implicated in excitotoxicity after nervous system trauma 13 . it is clear that reactive oxygen species ( ros ) and overstimulation of calcium - dependent signaling molecules is responsible , at least in part , for neuronal cell death by a similar mechanism as outlined for nervous system trauma 9 . within the superfamily , the three aforementioned subgroups ( i , ii and iii ) emerge based on amino acid sequence homology , agonist binding pharmacology and the second messenger system that is activated upon glu binding to each receptor ( see 8 for a review ) . activation of group i mglurs ( mglur1 and 5 ) and coupled gq g - proteins initiates signaling cascades involving phospholipase c / inositol 1,4,5-triphosphate / diacylglycerol ( plc / ip3/dag ) . some isoforms of the group i mglurs ( mglur1a and 5a ) contain a very long proline - rich domain which has been studied for its intricate protein - protein interactions and complex formations 19 . this domain , called the homer1 binding motif , facilitates coupling of mglurs with nmda receptors among other proteins and will be described in detail below . aside from the tmd and g - protein coupling , mglurs are highly dissimilar at the structural and functional levels to any other classical gpcr groups studied to date and are therefore referred to as atypical gpcrs . in general , gpcrs are organized into five groups and virtually all of them belong to groups a , b , d and e. mglurs , gaba receptors , two groups of taste receptors , and a ca - sensing receptor ( casrs ) class belong to group c - a clearly divergent group based on amino acid sequence , size and structure 20 , 21 . interestingly , casr receptors are not sensitive to glu or mglur agonists , but share amino acid identity of 28% with group i mglurs 22 . casrs are mainly involved in ca sensing and homeostasis in the parathyroid gland and detection of l - amino acid levels 20 . first , both mglurs and casrs form homodimers in vivo through disulfide bridging between crds and this event is critical for function 17 , 24 , 25 . in addition , unlike other gpcrs which typically have a short extracellular ligand binding domain , the atd is a large two - lobed structure allowing for glu binding between the two lobes ( figure 4 ) . for example , gpcrs which are activated by small molecules such as cations generally have a very small extracellular loop , while larger ligands require a larger lbd . the atd is related to bacterial periplasmic amino acid binding proteins , the structure of which has been extensively modeled and provides an excellent basis for models of the mglur atd 27 . reports have suggested that ca ( which can activate both mglurs and casrs ) and l - amino acids ( which can activate casr signaling ) may bind to the extracellular loops connecting the seven transmembrane domains , further separating group c gpcrs from the other groups 28 , 29 . the downstream signaling effects of glu binding and the pharmacology pharmacodynamics of agonist / antagonist binding to mglurs has been studied extensively due to this receptor group 's clear implications in both benign and malignant disease progression . this is not a rule , however , since all three receptor groups have been found on both pre - and postsynaptic cells . with regards to non - neural tissues , mglurs have been found in all of the major organ systems including epithelium , connective and muscle tissues . in many cases , the first member of the human mglur gene superfamily , mglur1 ( sometimes referred to as the glutamate receptor metabotropic 1 ; grm1 ) encodes five alternatively spliced isoforms ( figure 5 ) . a complete literature review on this gene is stymied by multiple confusing nomenclatures that have been used interchangeably throughout the decades . for example , the first isoform of mglur1 is synonymously referred to as the following : grm1a , mglur1a , mglur1 , gprc1a , and hmglu1a . surprisingly , a search for any of the synonyms excludes articles using the others , necessitating many searches to ensure completeness in collecting reliable and informative data in a review on this topic . we will use the simplest nomenclature which is the most utilized in recent studies , mglur1a , for this review and will always be referring to the homo sapiens gene unless explicitly noted . adding to this complexity is the fact that many of the studies on the mglur gene family have been performed using orthologues from the norway rat , rattus norvegicus . in fact , a large body of experimental data exists on the pharmacology , expression , mutational analysis and drug targeting of rodent mglur1s . this phenomenon is so pervasive that it is difficult to find a study in the last decade that has used the human gene or protein . the rodent orthologues have certainly facilitated our understanding of mglur signaling , but their translational value when interpreted in the context of non - malignant or malignant human diseases may remain questionable - at least in some cases . many of the initial studies on the human mglur1 gene cloning and isoform identification were published in the mid-1990s , before the human genome sequence became available and was regularly annotated with newly published information 36 - 41 . as a result , despite numerous publications on the human mglur1c and mglur1d isoforms , there are no references to them in the ncbi human genome database at either the mrna or protein levels . a nucleotide blast search with rat mglur1d found similarity with the human mglur1 and mglur5 precursor gene sequences , but since isoforms c and d were not annotated in the human genome databases , and human mglur1c and mglur1d do not exist there as either mrna or protein sequences . this issue is highlighted by the fact that annotation of isoforms a , b , c and d exists for the rattus norvegicus and mus musculus homologues of mglur1 in the ncbi databases while the human gene shows annotation of only isoforms a and b. the rat mglur1a protein sequence is 94% identical to the human mglur1a protein at the amino acid level ( figure 6 ) and is certainly an excellent model for studies on mglur1a function , however , it is a detriment to the field that genes with such a critical role in many organ systems , in ndds and in benign and malignant diseases have been far better characterized with modern bioinformatics in rodents than their human orthologues . figure 6 details the percent amino acid identity when the mouse and rat proteins are compared to the human protein . the highest homology between the mouse and rat protein and the human protein exists in the tmd , while the lowest percent identity is found in the crd ( figure 6 ) . although all of the cysteine residues in the crd are conserved , it is unclear whether or not the changes in the surrounding amino acids change the three dimensional structure of the domain or influence how it interacts with other mglur1a proteins during homodimerization . the localization ( diffuse cytoplasmic ) , size ( 145 kda ) and induction of receptor activity were assayed in the common rodent model , chinese hamster ovary ( cho ) cells 43 . it would be a great service to the field for a study to be undertaken in which full - length rodent and human mglur1a are expressed in a human cell line and downstream signaling and biological assays are shown to yield identical results in side - by - side experiments . we were unable to find such a study , however , recent work by esseltine and colleagues showed the result of human mglur1a expression in human embryonic kidney 293 ( hek293 ) cells 45 . the mglur1a protein that was expressed in in esseltine and colleagues ' study began at amino acid 19 , therefore removing the amino - terminal end of the protein to include their own localization domain 45 . although this study confirmed that the truncated mglur1a localized to the plasma membrane and was able to initiate downstream signaling cascades upon stimulation , no biological effects such as cell growth , migration , morphology , etc . excluding mglur1e , the human mglur1 isoforms are virtually identical except for alternative splicing changes near the 3 ' end of the mrna which result in much smaller cytoplasmic domains at their c - termini for the mglur1b , c and d proteins than the mglur1a isoform ( figure 5 ) . this difference in size is dramatic : an amino acid sequence alignment of human mglur1a and mglur1b are 100% identical until amino acid 886 . afterwards , a small exon containing a stop codon is incorporated in mglur1b that is not included in mglur1a , explaining the difference in size between the two isoforms ( figure 5 ) . this phenomenon is not limited to mglur1a since mglur5a ( a member of group i ) also possesses this domain , however , gpcr group c member , casr does not 21 . binding of full - length homer1isoforms to the consensus site found in the homer - binding motif of mglur1a/5a , ppxxf ( where x is any amino acid ) , has been shown to influence not only the trafficking of the receptor between the cytoplasm and the membrane , but also receptor clustering within the membrane through its scaffolding activity ( figure 4 ) . activation of mglur1a/5a results in ip3 production and rapid , localized activation of intracellular ca release through ip3r - the speed of which is facilitated by the close physical proximity of ip3r to active mglur1a/5a via homer1 scaffolding 52 . this short domain ( only 69 amino acids in mglur1 ) consists of three -pleated sheets and nine cysteine residues - all nine of which are very well - conserved in the other mglur proteins , irrespective of subgroup classifications and are also conserved in the human orthologues . activation of the mglur dimer has been shown to elicit a conformational change which brings the c - terminal regions of the crd in closer proximity to one another ( represented by a * in figure 4 ) and to involve a cysteine - cysteine interaction in the e2 loop of the tmd . the mutational analyses and crystallography that was used to obtain these models were all completed using the rat orthologues of the mglur family members and although all of the cysteine 's in the crd are conserved in the human protein , as shown in figure 6 , studies will need to be performed to confirm the residues of importance in the human orthologues . glu binding and aforementioned conformational changes can precipitate a number of varied signaling responses including : activation of coupled ca , k and na channels , activation of gq and consequent downstream signaling cascades , activation of extracellular - signal - regulated kinase ( erk ) 1/2 ( also known as mitogen activated protein kinase ; mapk ) and retrovirus ak thymoma [ akt ; also known as protein kinase b ( pkb ) ] signaling and even stimulation of sarcoma ( src ) kinase signaling ( figure 7 ) 4 , 15 , 53 - 56 . each of these second messenger systems and their functions in specific tissues will be expanded upon below . glu stimulation of both mglurs and iglurs in neural cells results in increased intracellular ca levels via differing signaling mechanisms . unlike iglur signaling , which directly involves the opening of an ion channel upon glu - binding , mglur signaling can lead to ca influx by indirect ion channel opening and/or by second messenger signaling systems . homer1 binding to the homer binding motif can recruit the scaffold protein , shank , which facilitates the direct interaction of mglurs and nmdars resulting in ca influx 58 . further , activation of plcs by g11 results in the cleavage of phosphatidylinositol 4,5-bisphosphate ( pip2 ) into two products : the phosphate head group ( ip3 ) and the fatty acid chains ( e.g. activated pi3k leads to the accumulation of phosphoinsitol 3,4,5-triphosphate ( pip3 ) and consequent recruitment of pleckstrin homology ( ph ) domain - containing proteins such as akt1 . fully active akt1 participates in a number of important downstream signaling events that certainly occur in neurons but are not limited to this cell type ( reviewed in 64 , 65 ) . it should be noted , however , that activation of mglur1 is also considered a pro - apoptosis signal in subsets of neural cells and in certain experimental paradigms 68 ( reviewed in 69 ) . therefore , the link between survival and mglur1 signaling may be complex . in signal transduction , calcium can act as either second messenger ( when influx or release is downstream of g - protein signaling ) or as direct effector of enzyme activity ( where influx occurs as a direct or indirect result of ion - channel opening ) . as outlined above further , ca binds and activates calmodulin ( cam ) , the main second messenger that transduces ca - related signals 79 . the binding of 4 ca molecules in cam 's 4 ef - hand motifs results in the activation of ca / cam - dependent kinases ( camk ) and subsequent signaling has effects on learning and memory and long - term potentiation of action potentials ( figure 7 ) 4 , 78 . indeed , an additional consequence of active mglurs appears to be the activation of src tyrosine kinase and downstream messengers such as erk 56 . studies have also demonstrated that activation of erk / mapk signaling in the cns is also critical for synaptic plasticity and memory formation - two processes that require long - term potentiation 56 , 81 , 82 . several overexpression models including cho , human embryonic kidney ( hek 293 ) and xenopus oocytes demonstrated that mglur1a can couple with gs to stimulate camp production 30 , 85 , 86 . a detailed review on the involvement of mglurs in malignant disease has been recently published by our group 90 . in addition to the described role for mglur signaling in normal nervous system function , glutamatergic signaling through the mglur family has been implicated in ndds and learning and memory disorders 91 . for example , in severe autism and alzeimer 's disease , secretion of amyloid precursor protein- ( sapp ) is a known contributor to disease severity 92 , 93 . many reports have postulated that targeting mglur signaling may provide symptomatic relief from alzheimer 's and autism 90 , 91 , 94 . the mechanism underlying this phenomenon has not been documented as of this review , but it clearly involves antagonizing or inhibition of the pi3k / akt / mtor pathway which is a known target of active mglur signaling 91 . much of the research done to characterize this complex network of receptors and second messenger systems has been performed using rodent models . this has led to a critical lack of information on the human orthologues of the mglur family - a fact which may have far - reaching impacts on drug discovery for ndds and other nervous system disorders as well as for anti - cancer therapies . for example , it is clear from an analysis of the literature on the crystal structure of the mglur proteins that much of this work has been done on the rodent orthologues . the exact amino acid sequence of the human mglur that are involved in the disulfide bridging between members of the receptor dimer are not known and are inferred from these studies . although it is true that in the majority of cases , the information obtained from rodent crystallography may be applicable to the human proteins , however , without mutational analysis , the question will remain .

intellectual disability is a developmental condition that is characterized by significant deficits in both intellectual functioning and adaptive behaviour including conceptual , social and practical skills . inhibiting participation in society for about 500 million people world - wide , intellectual disability is the most common developmental disorder affecting approximately 9 - 19% of the entire global population . there is increasing evidence that oral health , which is crucial for proper mastication , aesthetics , communication , appearance and quality of life , is deficient in the mentally disabled population [ 3 - 6 ] . aesthetically unacceptable and functionally inadequate dentitions affect self - esteem , confidence and psychological status , which is often already compromised in individuals affected by developmental disabilities . poor oral health in the intellectually disabled has been attributed to several causes , including the individual s inability to achieve proper oral hygiene ; the person s incapacity to express their pain and discomfort or by the parent or caregivers to evaluate their oral condition ; and to the lack of resources , organizational support and knowledgeable professionals [ 8 - 11 ] . there appears to be a general agreement that dental treatment is one of the greatest unattended health needs of disabled individuals but these needs are nonetheless grossly under - estimated by both caregivers and the dental profession . the reported associations between poor oral health in the intellectual disabled and the severity of the disability , parental education and socioeconomic status further highlight the social inequalities experienced by this vulnerable population . it has been estimated that up to 80% of intellectually disabled individuals reside in developing countries . yet , despite recent growing research interest in the oral health of disabled individuals globally , the oral health and treatment needs of these populations in low income and developing countries have not nearly been adequately assessed . although there are at least 27,500 intellectually disabled individuals in lebanon , an estimate that represents more than 28% of all disabled lebanese individuals , the oral health conditions and treatment needs of this population have not previously been assessed . the aim of the present study was to explore the dental status of institutionalized intellectually disabled individuals in lebanon , and to investigate the associations between the number of decayed , filled and missing teeth with selected background and health related behaviours and characteristics . participants constituted of 652 institutionalized lebanese individuals with intellectual disabilities residing across the 5 main lebanese governorates and divided into 4 age categories : 6 , 12 , 15 and 35 - 44 years old . ethical approval was obtained from the ethical research committee of the faculty of medical sciences at the lebanese university in addition to the ministry of social affairs in lebanon ( protocol no . 589 , 2 - 2 - 15 ) . written consent was obtained from participants parents , legal guardians or institution caretakers . disabled persons who were absent or unable to cope with the clinical examination or whose lack of cooperation prevented the adequate collection of all measures of interest were excluded from the study . all 49 registered institutions for the intellectual disability across the lebanese territory were approached and all but one , located in mount lebanon , approved participation in the study . out of 703 institutionalized individuals belonging to the 4 targeted age groups , 652 ( 92.7% ) were examined . data collection included two components : 1 ) a questionnaire filled by the participants caretakers that collected information on background characteristics ( age , sex , and governorate of residence ) , arrangement with the institution and characteristics of primary caretaker ( relation to disabled person and education ) , type of disability and severity , oral hygiene practices ( i.e. frequency of brushing ) and dietary habits ( i.e. consumption of sweets between meals ) and 2 ) a clinical examination that assessed each participant s oral health status as per the world health organization ( who ) oral assessment form ( 1997 ) . clinical examinations were carried out by a trained dental practitioner with more than 15 years of experience in providing oral care to disabled individuals ( the researcher ) , using appropriate protective infection control measures ( disposable gloves , masks and sterile equipment ) . each participant was seated under adequate room lighting and examined using sterile mouth mirrors and who probes . caries exposure was recorded using the decayed - missing - filled teeth ( dmft ) index which records all teeth with known decay experience , i.e. untreated decay ( d ) , missing teeth due to caries ( m ) , and filled ( f ) teeth . the notation decayed , filled teeth ( dft ) was used to distinguish primary teeth . this data constituted the four main outcomes of the study ( per individual ) : the total numbers of 1 ) decayed ; 2 ) missing and 3 ) filled teeth in addition to 4 ) the total number of decayed , missing and filled teeth ( dmft / dft index ) . the total number of missing teeth refers to missing permanent teeth only whereas the number of decayed and filled teeth and the dmft / dft index include pooled data for both primary and permanent teeth . descriptive statistics were generated to detail the distribution of the examined institutionalized participants across the various background , disability , health and nutritious practices . bivariate analyses assessing associations between the possible predictors and the four main outcomes were conducted using simple linear regressions . parametric data were expressed as mean and standard deviation ( m [ sd ] ) , degrees of freedom , f statistics and p values . variables associated with the outcomes studied at p < 0.2 at the bivariate analyses were included in multiple regression analyses . the variable teeth affected was included to the multivariate models to differentiate between primary and permanent teeth . regression coefficients , standard errors , 95% confidence intervals ( ci ) and two - sided p values were generated for the multivariate regression models . the ibm spss statistics 20.0 and stata / se 11.1 statistical packages were used to carry out all statistical analyses . background characteristics a total of 652 institutionalized individuals , 142 ( 21.8% ) children aged 6 years , 232 ( 35.6% ) aged 12 years , 152 ( 23.3% ) aged 15 years and 126 ( 19.3% ) adults between the ages of 35 to 44 years were examined ( table 1 ) . the majority of subjects were in institutions located in mount lebanon ( 44.8% ) , followed by the north ( 18.9% ) , bekaa ( 18.1% ) , the south ( 14.4% ) and finally beirut ( 3.8% ) . most examined subjects were affected by mild retardation ( 68.4% ) , 2% by severe and 29.4% by moderate retardation . the majority had a part - time arrangement with the institution ( 89% ) rather than a full - time arrangement , had a family member as their primary caretaker ( 89.7% ) rather than an institution caretaker and this caretaker had completed only secondary education or less ( 82.4% ) compared to 17.6% with college or university education . most subjects were not taking any medications for their disability at the time of their examination ( 81% ) . percent distribution of examined institutionalized disabled persons by age , gender and oral health indices ( n = 652 ) for subjects aged 6 years , the number of missing teeth is not recorded . dft = total number of decayed and filled primary teeth ; dmft = total number of decayed , missing , filled permanent teeth ; min = minimum ; max = maximum ; sd = standard deviation . mean refers to the mean number of teeth presenting with the specified condition per individual . overall , the dmft score was 5.86 ( 6 ) ; composed of 3.64 ( 4.05 ) decayed teeth , 1.71 ( 4.38 ) missing teeth and 0.87 ( 2.51 ) filled teeth ( table 1 ) . there was a clear trend of increasing overall index with increasing age : increasing from a dft = 3.5 ( 4.44 ) at 6 years to dmft = 4.8 ( 4.5 ) at 15 years and then to 12.71 ( 7.43 ) at 35 - 44 years . however , the number of untreated decays was actually lowest in the adult group ( 3.17 [ 4.11 ] ) and the highest contributors to the overall dmft in this age group were the missing ( 6.24 [ 7.02 ] ) then the filled ( 3.31 [ 4.56 ] ) teeth . on the other hand , the decayed teeth constituted the main component of the dmft / dft score for all other age groups ( table 1 ) . oral hygiene and nutrition practices the majority of participants brushed 2/day independently ( 37.4% ) followed by those that brushed 1/day without assistance ( 27.5% ) and only a minority ( 6% ) did not brush their teeth at all . therefore , at least 64.9% of the sample brushed independently without receiving assistance . similar proportions brushed either 2/day with help ( 10.6% ) , 1/day with help ( 7.8% ) and rarely brushed their teeth ( 10.7% ) . the majority consumed sweets between meals more than twice a day ( 32% ) and regularly consumed a combination of both home - cooked meals and fast food and pastries ( 64.1% ) . those consuming mainly home cooked meals were around one third ( 33.7% ) while those consuming predominantly only fast food and pastries were a small minority ( 2.2% ) . almost one fifth reported not to consume sugars between meals at all ( 17.2% ) and a similar proportion only 1/day ( 21.5% ) whereas only 1.5% reported to rarely consume sugars between meals . background predictors of oral health outcomes the distinction between primary and permanent teeth was not significant in any of the multivariate assessments of the combined or separate outcomes , suggesting that the predictors of these outcomes are similar regardless of the primary or permanent nature of the tooth ( p > 0.05 ) ( tables 2 and 3 ) . gender was not associated with any of the assessed outcomes , whereas age was a significant predictor of the number of missing teeth ( p < 0.001 ) , filled teeth ( p = 0.009 ) and overall dmft score ( p < 0.001 ) . in all cases , only adults had significantly higher scores than children - 12 and 15 year olds having similar d , m and dmft scores to 6 year old children ( p > 0.05 ) ( tables 2 and 3 ) . although the governorate of residence was not significantly associated with the total number of missing and filled teeth , it was a significant predictor of the total number of decayed teeth and the overall dmft scores when controlling for all other characteristics ( p 0.044 ) ( tables 2 and 3 ) . controlling for all other variables , subjects residing in the south , beirut and bekaa all had significantly higher numbers of decayed teeth than those in the north ( regression coefficient [ coef . ] with respect to overall dmft score , residents of beirut had significantly worse dmft scores than all those residing in any of the 4 other regions ( coef . multivariate analysis showing associations between selected variables and number of decayed , missing and filled teeth separately ( n = 652 ) statistically significant at p 0.05 ( multivariate analysis ) . statistically significant at p < 0.01 ( multivariate analysis ) . = regression coefficient ; ci = confidence interval ; f = the model s degrees of freedom and residual degrees of freedom ; rmse = root - mean - square - error . multivariate analysis showing associations between selected variables and number of decayed , missing and filled teeth ( n = 652 ) statistically significant at p 0.05 ( multivariate analysis ) . statistically significant at p < 0.01 ( multivariate analysis ) . coef . = regression coefficient ; ci = confidence interval ; f = the model s degrees of freedom and residual degrees of freedom ; rmse = root - mean - square - error . . predictors of oral health related to disability and arrangement the degree of intellectual disability was found to be a significant predictor only of the number of decayed teeth , and only when comparing moderate to mild disability in multivariate analysis ( table 2 ) . when controlling for all other factors , subjects with moderate disability had significantly higher numbers of decayed teeth than those with mild disability ( coef . = 0.87 ; p = 0.009 ) ( table 2 ) but the comparison between severe and mild disability was not statistically significant ( p = 0.561 ) . caretaker education was significantly associated with the number of decayed teeth but not with filled teeth , missing teeth or overall dmft score . subjects who had caretakers with college or university education had significantly lower numbers of decayed teeth than those whose caretakers had only secondary education or less ( coef . = -1.12 ; p = 0.008 ) ( table 2 ) . whether the primary caretaker was a parent or an institution caregiver and the type of arrangement with the institution did not significantly predict any of the four oral health outcomes ( p > 0.05 ) ( tables 2 and 3 ) . oral health and nutrition practices as predictors of oral health oral hygiene practices were significant predictors of all 4 oral health outcomes controlling for all remaining factors ( p < 0.05 ) ( tables 2 and 3 ) . all levels of brushing , with or without help , were associated with significantly better outcomes for decayed , missing , filled teeth and for overall dmft score . receiving assistance during brushing was associated with lower overall number of decayed teeth both in individuals brushing 1/day ( coef . = -1.94 with help compared to -1.68 without help ) and in those brushing 2/day ( coef . = -2.1 with help compared to -1.87 without help ) ( table 2 ) . for the number of filled teeth , receiving help during brushing was associated with lower total numbers of restorations both when subjects brushed 1/day ( coef . = 1.55 without help compared to 1.14 with help ) or 2/day ( coef . when controlling for tooth brushing and all other factors , the type of meals predominantly consumed ( fast food or home - cooked ) was not significantly associated with either decayed , missing , filled teeth or overall index . the consumption of sugars between meals , on the other hand , was a significant predictor of the number of carious teeth and the overall dmft score ( p < 0.001 ) ( tables 2 and 3 ) . both for carious teeth and dmft index , sweet consumption needed to be 2/day or more to be associated with significantly worse oral health compared to no sugar consumption between meals : decayed teeth , coef . = 3.13 , p < 0.001 for > 2/day ( table 2 ) ; and dmft score , coef . in a first assessment of its kind in lebanon , our study highlights the presence of poor oral health conditions in an intellectually disabled population in lebanon - corroborating the results of several global studies associating poor oral health with intellectual disability . the relatively high dmft scores described in our sample of children , adolescents and adults , averaging a value of almost 6 teeth , are similar to reports on intellectually disabled persons in several other populations [ 6,21 - 25 ] . the high caries activity noted in our sample may be attributed to their difficulty in maintaining oral hygiene , poor muscular co - ordination , and muscle weakness interfering with routine oral hygiene procedures . although assessments of dmft scores in younger age groups comparable to our 6 year old sample are very scarce , the oral health situation of this age group in our lebanese sample was worse than reported in a brazilian sample of 5 - 8 year old children . also report much lower dmft scores in their assessed population of indian children and young adults : 1.5 for 12 - 15 year olds and 2.75 for 26 - 30 year olds , and an overall mean of 2 . this finding may however be explained by the fact that the entire sample was collected from one special needs school which , according to the authors , likely provides very good oral health care and monitoring to their beneficiaries . even lower scores were reported in another study on indian children from special schools , with a mean dmft of only 0.5 teeth reported . however , the scarcity of such studies in comparison to the vast literature reporting poor dmft scores suggests an overall poor oral health among the intellectually disabled globally , albeit suggesting the ability to limit disease progression in well - controlled settings and with special care by appropriately trained personnel . as in our sample , several researchers have noted that the predominant contributor to the dmft score in various disabled populations was the decayed component [ 23 - 25,29 - 31 ] , although missing teeth were also reported to be significant contributors , especially in adult populations and with increasing disability severity . the low contribution of the filled component observed in our study is an observation that has also been reported by previous studies and may be reflective of the overall lower provision of dental treatment to the intellectually disabled than the general population ( and hence the greater progression of carious lesions ) and also to the tendency for extractions rather than restorations due to difficulties in the behavioural management of this population . exceptionally , in an assessment of more than 4,000 adult intellectually disabled persons in the united states , morgan et al . reported very low scores for decayed teeth and a high predominance of filled teeth . however , all assessed subjects were part of a large state - wide program specifically established to serve persons with special needs , thereby biasing these individuals towards greater access to and benefit from dental health care . our results bring additional data to previous research that proposes a number of individual , carer and social barriers contributing to oral health problems in the intellectually disabled . several researchers have linked greater disable severity with poorer caries scores , a finding that was present in our sample but only in association with the number of decayed teeth as a separate entity . although individuals with moderate disability had significantly more decayed teeth compared to those with mild disability , the comparison with severe disability failed to reach statistical significance , possibly because of the small sample size ( severe disability comprising only 2% of the assessed sample ) . the association between age and higher dmft scores observed in our sample has also been commonly reported in previous studies and the lack of association with gender has similarly been suggested . this trend was noted in our evaluation of overall dmft scores but also in evaluating the predictors of the decayed , missing and filled components separately , which , to our knowledge , have not previously been assessed as separate entities . notably , two modifiable behavioural factors were associated with poor dmft scores in our sample : oral hygiene practices and the consumption of sweets between meals , which were similarly predictors of the number of decayed and missing teeth separately . our observations support previous research on this topic were both oral hygiene practices and the degree of diet carcinogenicity predicted dmft scores . unfortunately , our results and the literature suggest that in the majority of cases , the oral health care of this risky population is often lacking . at least two thirds of our sample brushed their teeth independently without receiving any assistance from their caretakers , a phenomenon which is in agreement with reports from several other previous studies reporting . although this may be a manifestation of non - cooperation by the disabled persons , it also reflects poorly on the level of awareness and/or motivation among parents and caretakers regarding the importance of oral hygiene and their role as caretakers in its maintenance . the associations observed in our study between assisted brushing and lower numbers of decayed teeth and higher number of filled teeth emphasize the benefit of receiving assistance during brushing both in maintaining oral hygiene / plaque removal and perhaps in identifying carious lesions and therefore seeking treatment for the disabled person . although poor oral hygiene in the disabled has previously been associated with the type of caregiver and with institutional arrangement , none of the outcomes separately or combined were significantly associated with the type of arrangement with the institution ( part - time or full - time ) or whether the caretaker was a parent or an institution caregiver . caretaker education , on the other hand , was significantly associated with the total number of decayed teeth and is a finding that echoes the associations reported between dmft scores and the level of education of the parents in previous studies . previous studies have similarly highlighted the importance of socio - economic status ( ses ) as a predictor of oral health in intellectual disabled individuals , including variables such as parent education , geographical location , lack of organizational support and overall ses category . supporting previous research , our results similarly highlight significant socio - economic inequalities in lebanon with respect to oral health indices regardless of oral hygiene and nutrition factors . increased numbers of decayed teeth were noted in subjects residing in the south , bekaa and beirut compared to those in the north , the highest scores recorded in the capital city beirut . overall dmft score was similarly associated with the geographical location of the institution , subjects in beirut suffering from significantly higher dmft scores than those from all other lebanese governorates . the geographical location in our sample is likely to be a proxy variable for underlying socio - economic disadvantages that may include parental education , financial stability and inaccessibility to dental services , but the interesting finding of poor oral hygiene indices specifically in the capital is a finding that requires further investigation prior to generalization . it must be noted that only 5% of the entire sample were from institutions in beirut and all attended one institution . it is possible that specific factors relating to this institution are related to beneficiaries of lower socio - economic status and the results may not be generalized to the whole of beirut . the increased number of decayed teeth noted in the governorates of the south and bekaa , on the other hand , may be truly reflective of a wider scope of underlying social disadvantages and justify the implementation of targeted intervention programs that provide relief from existing disease and prevention of future disease . there is a definite need for preventive and treatment programs aiming to improve the oral health status of this vulnerable population . research suggests that individuals with intellectual disability would benefit from more frequent oral health assessments than would otherwise be needed in the general population . the significant role of behavioural factors disclosed by our study , including assisted oral hygiene and reduced sugar intake emphasizes the need to educate parents and caregivers on their active role in participating in preventive programs to ensure appropriate supervision of daily oral hygiene . raising caretaker awareness regarding the importance of supervised tooth brushing and limiting the intake of cariogenic substrates is a crucial step towards limiting the progression of carious diseases in the intellectually disabled population . it must be noted that despite the high response rate and large coverage of almost all registered institutions for the intellectual disability in lebanon , our results can not be generalized to the entire population of non - institutionalized intellectually disabled subjects in lebanon . the limitation of target age groups to ages 6 , 12 , 15 and 35 - 44 years , although for the purposes of practicality and comparability with similar studies , also limits generalizability to other generations . future studies both including wider ages groups and comparing the institutionalized to the non - institutionalized are imperative towards understanding the true state of oral health and dental treatment needs in the lebanese mentally disabled population at large . there was a clear trend of increasing overall dmft index with increasing age , characterized by low levels of untreated decayed teeth and predominance of missing teeth followed by filled teeth . increased brushing frequency predicted lower numbers of decayed , filled and missing teeth separately and overall dmft . sugar consumption between meals predicted worse scores for decayed teeth and dmft but type of meals consumed showed no association . the authors would like to thank the staff at charts - research design and biostatistics for their help in the statistical analysis and write up of the study . the authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article .

abstractobjectivesto assess the dental status of institutionalized intellectually disabled individuals in lebanon and the role of background and behavioural determinants.material and methodsoral health was recorded for 652 individuals ( 6 , 12 , 15 and 35 - 44 years old ) using the decayed , missing and filled teeth / decayed and filled teeth ( dmft / dft ) index . data collected was statistically analysed with statistical significance set at p = 0.05.resultsoverall , mean dmft index score was 5.86 ( sd 6 ) ; composed of 3.64 ( sd 4.05 ) decayed ( d ) teeth ; 1.71 ( sd 4.38 ) missing ( m ) teeth ; 0.87 ( sd 2.51 ) filled ( f ) teeth . dmft was highest in adults ( 12.71 [ sd 7.43 ] ) and had a large component of missing ( 6.24 [ sd 7.02 ] ) and filled ( 3.31 [ sd 4.56 ] ) teeth . dmft scores ranged between 3.5 ( sd 4.44 ) at 6 years and 4.8 ( sd 4.52 ) at 15 years and the decayed component was the main contributor . in multivariate analyses , governorate of residence was statistically significantly ( p 0.040 and p 0.044 ) associated with d and dmft , the degree of disability and caretaker educational level were statistically significantly ( p = 0.009 and p = 0.008 ) associated with d , oral hygiene practices were statistically significantly ( p 0.017 ; p < 0.001 ; p 0.017 ; p < 0.001 ) associated with d , m , f and dmft and sugar consumption was statistically significantly ( p 0.03 and p 0.019 ) associated with d and dmft.conclusionsin lebanon , preventive and treatment programs to improve the oral health status of institutionalized intellectually disabled subjects are needed .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

there is increasing evidence that oral health , which is crucial for proper mastication , aesthetics , communication , appearance and quality of life , is deficient in the mentally disabled population [ 3 - 6 ] . poor oral health in the intellectually disabled has been attributed to several causes , including the individual s inability to achieve proper oral hygiene ; the person s incapacity to express their pain and discomfort or by the parent or caregivers to evaluate their oral condition ; and to the lack of resources , organizational support and knowledgeable professionals [ 8 - 11 ] . there appears to be a general agreement that dental treatment is one of the greatest unattended health needs of disabled individuals but these needs are nonetheless grossly under - estimated by both caregivers and the dental profession . the reported associations between poor oral health in the intellectual disabled and the severity of the disability , parental education and socioeconomic status further highlight the social inequalities experienced by this vulnerable population . it has been estimated that up to 80% of intellectually disabled individuals reside in developing countries . yet , despite recent growing research interest in the oral health of disabled individuals globally , the oral health and treatment needs of these populations in low income and developing countries have not nearly been adequately assessed . although there are at least 27,500 intellectually disabled individuals in lebanon , an estimate that represents more than 28% of all disabled lebanese individuals , the oral health conditions and treatment needs of this population have not previously been assessed . the aim of the present study was to explore the dental status of institutionalized intellectually disabled individuals in lebanon , and to investigate the associations between the number of decayed , filled and missing teeth with selected background and health related behaviours and characteristics . participants constituted of 652 institutionalized lebanese individuals with intellectual disabilities residing across the 5 main lebanese governorates and divided into 4 age categories : 6 , 12 , 15 and 35 - 44 years old . ethical approval was obtained from the ethical research committee of the faculty of medical sciences at the lebanese university in addition to the ministry of social affairs in lebanon ( protocol no . all 49 registered institutions for the intellectual disability across the lebanese territory were approached and all but one , located in mount lebanon , approved participation in the study . data collection included two components : 1 ) a questionnaire filled by the participants caretakers that collected information on background characteristics ( age , sex , and governorate of residence ) , arrangement with the institution and characteristics of primary caretaker ( relation to disabled person and education ) , type of disability and severity , oral hygiene practices ( i.e. frequency of brushing ) and dietary habits ( i.e. consumption of sweets between meals ) and 2 ) a clinical examination that assessed each participant s oral health status as per the world health organization ( who ) oral assessment form ( 1997 ) . clinical examinations were carried out by a trained dental practitioner with more than 15 years of experience in providing oral care to disabled individuals ( the researcher ) , using appropriate protective infection control measures ( disposable gloves , masks and sterile equipment ) . caries exposure was recorded using the decayed - missing - filled teeth ( dmft ) index which records all teeth with known decay experience , i.e. untreated decay ( d ) , missing teeth due to caries ( m ) , and filled ( f ) teeth . the notation decayed , filled teeth ( dft ) was used to distinguish primary teeth . this data constituted the four main outcomes of the study ( per individual ) : the total numbers of 1 ) decayed ; 2 ) missing and 3 ) filled teeth in addition to 4 ) the total number of decayed , missing and filled teeth ( dmft / dft index ) . the total number of missing teeth refers to missing permanent teeth only whereas the number of decayed and filled teeth and the dmft / dft index include pooled data for both primary and permanent teeth . bivariate analyses assessing associations between the possible predictors and the four main outcomes were conducted using simple linear regressions . parametric data were expressed as mean and standard deviation ( m [ sd ] ) , degrees of freedom , f statistics and p values . variables associated with the outcomes studied at p < 0.2 at the bivariate analyses were included in multiple regression analyses . regression coefficients , standard errors , 95% confidence intervals ( ci ) and two - sided p values were generated for the multivariate regression models . background characteristics a total of 652 institutionalized individuals , 142 ( 21.8% ) children aged 6 years , 232 ( 35.6% ) aged 12 years , 152 ( 23.3% ) aged 15 years and 126 ( 19.3% ) adults between the ages of 35 to 44 years were examined ( table 1 ) . the majority of subjects were in institutions located in mount lebanon ( 44.8% ) , followed by the north ( 18.9% ) , bekaa ( 18.1% ) , the south ( 14.4% ) and finally beirut ( 3.8% ) . the majority had a part - time arrangement with the institution ( 89% ) rather than a full - time arrangement , had a family member as their primary caretaker ( 89.7% ) rather than an institution caretaker and this caretaker had completed only secondary education or less ( 82.4% ) compared to 17.6% with college or university education . percent distribution of examined institutionalized disabled persons by age , gender and oral health indices ( n = 652 ) for subjects aged 6 years , the number of missing teeth is not recorded . dft = total number of decayed and filled primary teeth ; dmft = total number of decayed , missing , filled permanent teeth ; min = minimum ; max = maximum ; sd = standard deviation . overall , the dmft score was 5.86 ( 6 ) ; composed of 3.64 ( 4.05 ) decayed teeth , 1.71 ( 4.38 ) missing teeth and 0.87 ( 2.51 ) filled teeth ( table 1 ) . there was a clear trend of increasing overall index with increasing age : increasing from a dft = 3.5 ( 4.44 ) at 6 years to dmft = 4.8 ( 4.5 ) at 15 years and then to 12.71 ( 7.43 ) at 35 - 44 years . however , the number of untreated decays was actually lowest in the adult group ( 3.17 [ 4.11 ] ) and the highest contributors to the overall dmft in this age group were the missing ( 6.24 [ 7.02 ] ) then the filled ( 3.31 [ 4.56 ] ) teeth . on the other hand , the decayed teeth constituted the main component of the dmft / dft score for all other age groups ( table 1 ) . oral hygiene and nutrition practices the majority of participants brushed 2/day independently ( 37.4% ) followed by those that brushed 1/day without assistance ( 27.5% ) and only a minority ( 6% ) did not brush their teeth at all . similar proportions brushed either 2/day with help ( 10.6% ) , 1/day with help ( 7.8% ) and rarely brushed their teeth ( 10.7% ) . the majority consumed sweets between meals more than twice a day ( 32% ) and regularly consumed a combination of both home - cooked meals and fast food and pastries ( 64.1% ) . almost one fifth reported not to consume sugars between meals at all ( 17.2% ) and a similar proportion only 1/day ( 21.5% ) whereas only 1.5% reported to rarely consume sugars between meals . background predictors of oral health outcomes the distinction between primary and permanent teeth was not significant in any of the multivariate assessments of the combined or separate outcomes , suggesting that the predictors of these outcomes are similar regardless of the primary or permanent nature of the tooth ( p > 0.05 ) ( tables 2 and 3 ) . gender was not associated with any of the assessed outcomes , whereas age was a significant predictor of the number of missing teeth ( p < 0.001 ) , filled teeth ( p = 0.009 ) and overall dmft score ( p < 0.001 ) . in all cases , only adults had significantly higher scores than children - 12 and 15 year olds having similar d , m and dmft scores to 6 year old children ( p > 0.05 ) ( tables 2 and 3 ) . although the governorate of residence was not significantly associated with the total number of missing and filled teeth , it was a significant predictor of the total number of decayed teeth and the overall dmft scores when controlling for all other characteristics ( p 0.044 ) ( tables 2 and 3 ) . with respect to overall dmft score , residents of beirut had significantly worse dmft scores than all those residing in any of the 4 other regions ( coef . multivariate analysis showing associations between selected variables and number of decayed , missing and filled teeth separately ( n = 652 ) statistically significant at p 0.05 ( multivariate analysis ) . statistically significant at p < 0.01 ( multivariate analysis ) . multivariate analysis showing associations between selected variables and number of decayed , missing and filled teeth ( n = 652 ) statistically significant at p 0.05 ( multivariate analysis ) . statistically significant at p < 0.01 ( multivariate analysis ) . predictors of oral health related to disability and arrangement the degree of intellectual disability was found to be a significant predictor only of the number of decayed teeth , and only when comparing moderate to mild disability in multivariate analysis ( table 2 ) . = 0.87 ; p = 0.009 ) ( table 2 ) but the comparison between severe and mild disability was not statistically significant ( p = 0.561 ) . caretaker education was significantly associated with the number of decayed teeth but not with filled teeth , missing teeth or overall dmft score . = -1.12 ; p = 0.008 ) ( table 2 ) . whether the primary caretaker was a parent or an institution caregiver and the type of arrangement with the institution did not significantly predict any of the four oral health outcomes ( p > 0.05 ) ( tables 2 and 3 ) . oral health and nutrition practices as predictors of oral health oral hygiene practices were significant predictors of all 4 oral health outcomes controlling for all remaining factors ( p < 0.05 ) ( tables 2 and 3 ) . all levels of brushing , with or without help , were associated with significantly better outcomes for decayed , missing , filled teeth and for overall dmft score . receiving assistance during brushing was associated with lower overall number of decayed teeth both in individuals brushing 1/day ( coef . = -1.94 with help compared to -1.68 without help ) and in those brushing 2/day ( coef . for the number of filled teeth , receiving help during brushing was associated with lower total numbers of restorations both when subjects brushed 1/day ( coef . when controlling for tooth brushing and all other factors , the type of meals predominantly consumed ( fast food or home - cooked ) was not significantly associated with either decayed , missing , filled teeth or overall index . the consumption of sugars between meals , on the other hand , was a significant predictor of the number of carious teeth and the overall dmft score ( p < 0.001 ) ( tables 2 and 3 ) . both for carious teeth and dmft index , sweet consumption needed to be 2/day or more to be associated with significantly worse oral health compared to no sugar consumption between meals : decayed teeth , coef . = 3.13 , p < 0.001 for > 2/day ( table 2 ) ; and dmft score , coef . in a first assessment of its kind in lebanon , our study highlights the presence of poor oral health conditions in an intellectually disabled population in lebanon - corroborating the results of several global studies associating poor oral health with intellectual disability . the relatively high dmft scores described in our sample of children , adolescents and adults , averaging a value of almost 6 teeth , are similar to reports on intellectually disabled persons in several other populations [ 6,21 - 25 ] . the high caries activity noted in our sample may be attributed to their difficulty in maintaining oral hygiene , poor muscular co - ordination , and muscle weakness interfering with routine oral hygiene procedures . although assessments of dmft scores in younger age groups comparable to our 6 year old sample are very scarce , the oral health situation of this age group in our lebanese sample was worse than reported in a brazilian sample of 5 - 8 year old children . also report much lower dmft scores in their assessed population of indian children and young adults : 1.5 for 12 - 15 year olds and 2.75 for 26 - 30 year olds , and an overall mean of 2 . this finding may however be explained by the fact that the entire sample was collected from one special needs school which , according to the authors , likely provides very good oral health care and monitoring to their beneficiaries . even lower scores were reported in another study on indian children from special schools , with a mean dmft of only 0.5 teeth reported . however , the scarcity of such studies in comparison to the vast literature reporting poor dmft scores suggests an overall poor oral health among the intellectually disabled globally , albeit suggesting the ability to limit disease progression in well - controlled settings and with special care by appropriately trained personnel . as in our sample , several researchers have noted that the predominant contributor to the dmft score in various disabled populations was the decayed component [ 23 - 25,29 - 31 ] , although missing teeth were also reported to be significant contributors , especially in adult populations and with increasing disability severity . the low contribution of the filled component observed in our study is an observation that has also been reported by previous studies and may be reflective of the overall lower provision of dental treatment to the intellectually disabled than the general population ( and hence the greater progression of carious lesions ) and also to the tendency for extractions rather than restorations due to difficulties in the behavioural management of this population . exceptionally , in an assessment of more than 4,000 adult intellectually disabled persons in the united states , morgan et al . reported very low scores for decayed teeth and a high predominance of filled teeth . however , all assessed subjects were part of a large state - wide program specifically established to serve persons with special needs , thereby biasing these individuals towards greater access to and benefit from dental health care . our results bring additional data to previous research that proposes a number of individual , carer and social barriers contributing to oral health problems in the intellectually disabled . although individuals with moderate disability had significantly more decayed teeth compared to those with mild disability , the comparison with severe disability failed to reach statistical significance , possibly because of the small sample size ( severe disability comprising only 2% of the assessed sample ) . the association between age and higher dmft scores observed in our sample has also been commonly reported in previous studies and the lack of association with gender has similarly been suggested . this trend was noted in our evaluation of overall dmft scores but also in evaluating the predictors of the decayed , missing and filled components separately , which , to our knowledge , have not previously been assessed as separate entities . notably , two modifiable behavioural factors were associated with poor dmft scores in our sample : oral hygiene practices and the consumption of sweets between meals , which were similarly predictors of the number of decayed and missing teeth separately . our observations support previous research on this topic were both oral hygiene practices and the degree of diet carcinogenicity predicted dmft scores . unfortunately , our results and the literature suggest that in the majority of cases , the oral health care of this risky population is often lacking . although this may be a manifestation of non - cooperation by the disabled persons , it also reflects poorly on the level of awareness and/or motivation among parents and caretakers regarding the importance of oral hygiene and their role as caretakers in its maintenance . the associations observed in our study between assisted brushing and lower numbers of decayed teeth and higher number of filled teeth emphasize the benefit of receiving assistance during brushing both in maintaining oral hygiene / plaque removal and perhaps in identifying carious lesions and therefore seeking treatment for the disabled person . although poor oral hygiene in the disabled has previously been associated with the type of caregiver and with institutional arrangement , none of the outcomes separately or combined were significantly associated with the type of arrangement with the institution ( part - time or full - time ) or whether the caretaker was a parent or an institution caregiver . caretaker education , on the other hand , was significantly associated with the total number of decayed teeth and is a finding that echoes the associations reported between dmft scores and the level of education of the parents in previous studies . previous studies have similarly highlighted the importance of socio - economic status ( ses ) as a predictor of oral health in intellectual disabled individuals , including variables such as parent education , geographical location , lack of organizational support and overall ses category . supporting previous research , our results similarly highlight significant socio - economic inequalities in lebanon with respect to oral health indices regardless of oral hygiene and nutrition factors . increased numbers of decayed teeth were noted in subjects residing in the south , bekaa and beirut compared to those in the north , the highest scores recorded in the capital city beirut . overall dmft score was similarly associated with the geographical location of the institution , subjects in beirut suffering from significantly higher dmft scores than those from all other lebanese governorates . the geographical location in our sample is likely to be a proxy variable for underlying socio - economic disadvantages that may include parental education , financial stability and inaccessibility to dental services , but the interesting finding of poor oral hygiene indices specifically in the capital is a finding that requires further investigation prior to generalization . it is possible that specific factors relating to this institution are related to beneficiaries of lower socio - economic status and the results may not be generalized to the whole of beirut . there is a definite need for preventive and treatment programs aiming to improve the oral health status of this vulnerable population . research suggests that individuals with intellectual disability would benefit from more frequent oral health assessments than would otherwise be needed in the general population . the significant role of behavioural factors disclosed by our study , including assisted oral hygiene and reduced sugar intake emphasizes the need to educate parents and caregivers on their active role in participating in preventive programs to ensure appropriate supervision of daily oral hygiene . raising caretaker awareness regarding the importance of supervised tooth brushing and limiting the intake of cariogenic substrates is a crucial step towards limiting the progression of carious diseases in the intellectually disabled population . it must be noted that despite the high response rate and large coverage of almost all registered institutions for the intellectual disability in lebanon , our results can not be generalized to the entire population of non - institutionalized intellectually disabled subjects in lebanon . the limitation of target age groups to ages 6 , 12 , 15 and 35 - 44 years , although for the purposes of practicality and comparability with similar studies , also limits generalizability to other generations . future studies both including wider ages groups and comparing the institutionalized to the non - institutionalized are imperative towards understanding the true state of oral health and dental treatment needs in the lebanese mentally disabled population at large . there was a clear trend of increasing overall dmft index with increasing age , characterized by low levels of untreated decayed teeth and predominance of missing teeth followed by filled teeth . increased brushing frequency predicted lower numbers of decayed , filled and missing teeth separately and overall dmft . sugar consumption between meals predicted worse scores for decayed teeth and dmft but type of meals consumed showed no association .

dn , as one complication of diabetes , is one of the leading causes of end - stage renal disease ( esrd ) worldwide . although great progress has been made in the study of animal models , none of the models can reproduce all the structural and functional changes of human dn . murine models have substantial advantages over other species in the studies on pathogenesis of dn , including lower cost , murine repositories that bear multiple mutations , plentiful inbred strains , and an available map of murine genomic sequence on the internet . the animal models of diabetic complications consortium ( amdcc ) proposes the following three criteria for a desirable murine model of dn : ( 1 ) more than 50% decline in glomerular filtration rate ( gfr ) over the lifetime of the animal ; ( 2 ) greater than 10-fold increase in albuminuria compared with controls for that strain at the same age and gender ; ( 3 ) histopathology findings which include mesangial sclerosis ( a 50% increase in mesangial volume ) , any degree of arteriolar hyalinosis , glomerular basement membrane ( gbm ) thickening ( a > 25% increase compared with baseline by electron microscopy morphometry ) , and tubulointerstitial fibrosis . in fact , there are no murine models that meet all of the three criteria . here we review the identified murine models of dn including artificially induced , spontaneous , and genetically engineered ( knockout and transgenic ) animal models and compare their advantages and deficiencies . some of the key issues , such as strain , genetic background , type of diabetes , method of induction , gene deficiency , animal age and gender , kidney histopathology , and phenotypic alterations , are all included in this review . we hope this review could enhance our understanding of genetic susceptibility and molecular mechanisms responsible for dn , as well as provide new clues as to how to choose appropriate animal models of dn . alloxan and streptozotocin ( stz ) are widely used for producing artificially induced t1 dm which causes kidney damage with similarities to human dn . both of them are glucose analogues that enter the insulin - producing beta - cells via a glucose transporter . the diabetogenic actions of alloxan and stz are mediated by reactive oxygen species ( ros ) . however , they are generated through different approaches in the case of alloxan and stz . in the presence of glutathione , alloxan and its reduction product , dialuric acid , generate superoxide radicals in a cyclic redox reaction which undergo dismutation to hydrogen peroxide afterwards . then fenton reaction ensues with the formation of highly reactive hydroxyl radicals . after being taken into pancreatic beta - cells dna damage causes the activation of poly adp - ribosylation which leads to depletion of cellular nad and atp . , superoxide radicals are formed . ros and a simultaneous cytosolic calcium overload lead to acute necrosis of pancreatic beta - cells [ 1 , 2 ] . so both of the two diabetogenic agents induce diabetes secondary to the necrosis of pancreatic beta - cells . these stz - treated models develop a modest degree of proteinuria and serum creatinine increase , as well as minimal mesangial matrix expansion , depending on the genetic background [ 3 , 4 ] . animal models of stz - induced dn are usually performed in mice , sprague - dawley and wistar - kyoto rats . given the mouse strains which are resistant to a single dose of stz ( 200 mg / kg ) or a two - dose regimen of stz ( 2 1002 125 mg / kg ) , greater cytotoxicity to pancreatic beta - cells and collateral tissue occurs , resulting in a higher incidence and severity of diabetes [ 47 ] . because of the severe hyperglycaemia , the diabetic models need to be monitored for blood glucose and administrated insulin . some investigators optimize the animal models of stz - induced dn by uninephrectomy in advance which lead to compensatory hypertrophy of the remaining kidney and acceleration of the disease progression . however , owing to the nephrotoxicity of stz , it is difficult to differentiate between the direct toxic effect of stz and lesions caused by hyperglycaemia [ 8 , 9 ] . some studies on mouse models of dn show that high - dose stz - treated mice exhibit more albuminuria than those which receive low - dose stz , despite of similar blood glucose . besides , evidence for acute kidney injury caused by high - dose stz in mice and rats has been reported [ 9 , 11 ] . although stz - treated models result in hypoinsulinemia and hyperglycaemia , they do not share autoimmune features like patients with t1 dm . in order to reduce nonspecific nephrotoxicity of stz , the regimen of multiple low - dose injections of stz to induce diabetes has been performed , that is daily intraperitoneally injections of 4060 mg / kg stz for five consecutive days [ 4 , 1214 ] which usually induces repetitive low - grade beta cell damage accompanied by secondary autoimmune insulitis [ 15 , 16 ] . it is quite different among inbred strains of mice in susceptibility to both pancreatic beta cell toxicity and the direct nephrotoxicity of low - dose stz . it is reported by gurley et al . that there is a hierarchical response of blood glucose level to multiple low doses of stz ( dba/2 > c57bl/6 > mrl / mp > his research results also show that males are more susceptible to diabetes induced by this stz regimen than females . generally , given strain - appropriate doses of stz , mice that receive low - dose stz develop parallel levels of hyperglycemia to those that receive high - dose stz ; the levels of albuminuria , by contrast , are generally lower as a result of reduced direct nephrotoxicity of stz [ 19 , 20 ] . the us - based amdcc also recommends a standard low - dose model for stz - induced diabetic complications which include dn . according to this recommendation , mouse models of dn should be induced by daily intraperitoneally injections of 50 mg / kg stz for five consecutive days . however , studies show that only 50% of c57bl/6 mice develop overt diabetes through this approach . therefore , whether it will be adopted by relevant experts is not yet determined . the protocol of high - fat diet is widely used to induce insulin resistance and obesity [ 2123 ] . it is also of great use for the research of accelerated atherosclerosis [ 2426 ] , although inbred strains of mice exhibit significant differences in response to the effect of high - fat diet . c57bl6 mice respond strongly to high - fat diet ; a / j mice , by contrast , are relatively resistant . sugano et al . have reported a new rat model of dn induced by high - fat diet , multiple low - dose injections of stz , and uninephrectomy . this model exhibits most features of human dn from t2 dm including hyperglycemia , hypoinsulinemia , hyperlipidaemia , hypertension , and microalbuminuria followed by overt albuminuria , mesangial expansion , and terminal glomerular sclerosis . spontaneous animal models of dn are established by selective breeding from animals which spontaneously develop dn due to genetic abnormality . renal abnormalities in these models resemble human diseases ; therefore , these models provide an experimental platform for studying pathogenesis and genetic susceptibility responsible for dn . although these models are difficult to feed and breed , not widely available , and with long modeling cycle and higher cost , the application of them is becoming increasingly extensive . the spontaneous murine model of t1 dm that has been studied most extensively is the nod mouse . due to pathogenic and genetic similarities to the human disease , the model serves as a useful tool to study the etiology , pathology , and progression of disease . nod mouse was derived from the jcl : icr cataract mouse 30 years ago in japan . these mice develop spontaneous insulitis at the age of 4 - 5 weeks , and overt diabetes emerges at the age of 2430 weeks when most of pancreatic -cells are destroyed . this model exhibits a number of clinical features of human t1 dm including hyperglycaemia , glycosuria , polyuria , and polydipsia ; however , it is more resistant to ketoacidosis . without insulin administration , nod mice usually die of dehydration , rather than ketoacidosis . like in humans , the major histocompatibility complex ( mhc ) alleles some mhc alleles must be accompanied by other non - mhc genes for the development of t1 dm [ 29 , 30 ] , which is also the same in human disease . even so , nod mice are not the perfect animal model for human t1 dm . as an inbred strain , they have a fixed genetic risk for t1 dm and develop t1 dm in a predictable fashion , whereas human t1 dm develops as a result of deleterious interactions between relevant genes and from external environmental factors . despite of the extensive study of the genetic and immunologic pathogenesis of t1 dm in nod mouse , few investigators choose nod mouse to do research on dn because of the complicated genetics , the late and variable age of onset of t1 dm , and the requirement for insulin administration . even so , one of the few studies shows that the amount of albuminuria in hyperglycemic nod mice is seven times higher than that in nod mice before the development of hyperglycemia . the results of renal lesions in acute phase of t1 dm in nod mice show mild changes in glomeruli and structural alteration of the proximal straight tubules . the study also points out that increased neuronal nitric oxide synthase may represent one of the pathogenic factors of dn . besides , there are also studies using nod mice demonstrating that transforming growth factor- ( tgf- ) and advanced glycosylation endproducts ( age ) take an important role in mesangial proliferation and sclerosis [ 7173 ] , which is also the same in human dn . the mutation leads to the misfolding of insulin protein , which is toxic to pancreatic -cell . the ins-2 akita mutation which is autosomal dominant was originally found in c57bl/6 mice in akita , japan . mice heterozygous for the mutation develop significant hyperglycemia as a result of severe insulin deficiency at 3 to 4 weeks of age , but homozygotus mice usually die in perinatal period . mice with ins-2 akita mutation exhibit modest levels of albuminuria and mild - to - moderate glomerular mesangial expansion . nonetheless , akita mice have higher levels of hyperglycemia , albuminuria , blood pressure , and more consistent structural changes of kidney compared with stz - induced dn . gurley et al . have found that renal phenotype of akita mice is largely dependent on their genetic background strains . it means that genetic factors might influence susceptibility to dn in akita mice which is the same in human disease . thus , mice bearing ins-2 akita mutation have significant advantages as a model of t1 dm . the db / db mouse which has a g - to - t mutation in the gene coding the leptin receptor develops obesity , insulin resistance , and t2 dm spontaneously . the db / db mutation which is autosomal recessive was initially recognized from an obese and hyperphagic mouse in the c57blks / j strain and was subsequently backcrossed to a pure c57bl/6j background . mice in the c57blks / j stain exhibit hyperinsulinemia at 10 days of age and slight hyperglycemia at 1 month of age . manifestations of dn are albuminuria , glomerular hypertrophy , mesangial matrix expansion , and gbm thickening . albuminuria can be detected as early as 3 to 4 weeks after the onset of hyperglycemia . the level of albuminuria in the db / db male mouse is 68600 g/24 h [ 20 , 3437 ] which is only 421 g/24 h [ 34 , 37 ] in the age - matched heterozygous littermate . the db / db mice display an increase in glomerular size and mesangial matrix by 5 - 6 months of age . by 1820 months , the glomerular and mesangial matrix enlargements become more remarkable , and thickening of the gbm is observed . in general , db / db mice do not develop mesangiolysis , nodular mesangial sclerosis , and progressive renal insufficiency . hyperglycemia , and renal changes of db / db mice in c57blks / j stain are usually worse than in the c57bl/6 background . hence , investigations of dn in db / db mice are more widely conducted with the c57blks / j strain . nonetheless , db / db mice in the c57bl/6 stain which have been intercrossed with gene knockout and transgenic mice [ 74 , 75 ] provide new strains to identify pathogenesis of dn . the model is characterized by hyperphagia , mild obesity , late - onset hyperglycemia , hypertension , dyslipidemia and advanced dn . multiple recessive genes are associated with the induction of diabetes , including odb-1 on x - chromosome of oletf rats . it is also reported that a major quantitative trait locus colocalizing with cholecystokinin type a receptor gene influences poor pancreatic proliferation in oletf rats . the progression of t2 dm in oletf rats can be prevented by exercise and calorie - restricted diet as human disease . at 1220 weeks of age late - onset hyperglycemia is noted by 18 weeks of age . at 22 weeks of age , the oletf rats develop overt albuminuria , and at 54 weeks , advanced renal changes such as macroalbuminuria , nodular lesions , diffuse glomerulosclerosis , and tubulointerstitial fibrosis are noted like human dn . therefore , oletf rat is considered as one of the best murine models to study dn . goto kakizaki ( gk ) rat , a spontaneous polygenic model of t2 dm , is established by repeated inbreeding of glucose - intolerant wistar rats over many generations . this model is characterized by moderate hyperglycemia , peripheral insulin resistance , and nonhyperlipidemia , and nonobese phenotype . it is recognized that t2 dm in gk rat is primarily caused by beta - cell deficit . in order to identify the origin of the abnormality , miralles and porthe compared the development of the embryo in gk and wistar rats . they found a decrease in pancreatic cell proliferation from embryonic day 16 to 20 ( e16e20 ) and a wave of pancreatic cell apoptosis from e16 to e18 . by e16 , the number of pancreatic beta - cells in the gk rats is half of the wistar rats , and this difference was sustained until birth . gk rat exhibits morphological changes which can be seen in early stage of human dn such as glomerular hypertrophy and gbm thickening . it does not develop overt proteinuria or progressive nephropathy by 8 months of age , even being treated with some initiators to promote renal injury [ 42 , 43 ] . however , sato et al . have reported advanced renal changes in gk rats such as segmental glomerulosclerosis and tubulointerstitial fibrosis at 24 months of age . at that time , albuminuria increases notably . thus , they draw the conclusion that renal changes in gk rats at a late stage were similar to those of progressive human dn . therefore , gk rat serves a useful tool for studying t2 dm and dn . compared with db / db mouse , ob / ob mouse develops t2 dm caused by the spontaneous recessive mutation in leptin , the ligand for the leptin receptor . the ob / ob mutation exists in c57bl/6j , dba2/j , and fvb strains . thus , it is not widely used as an animal model of dn . however , a new mouse model that mimics progressive dn has been developed in btbr strain with the ob / ob mutation . its characteristics are insulin resistance , hyperinsulinemia , pancreatic islet hypertrophy , severe hyperglycemia , obesity , hypercholesterolemia , and elevated triglycerides . the btbr ob / ob mice are largely resistant to the hypoglycemic effect of insulin administration and rapidly develop pathological changes of both early and advanced human dn . characteristics of early dn such as glomerular hypertrophy , accumulation of mesangial matrix , and loss of podocytes are detectable by 8 weeks of age . glomerular lesions of progressive , advanced dn are present by 20 weeks . by 22 weeks , morphological characteristics of renal injury has 20% increase in gbm thickness , 50% increase in mesangial matrix , mesangiolysis , diffuse mesangial sclerosis , focal arteriolar hyalinosis , and focal mild interstitial fibrosis . on the one hand , the advantage of btbr ob / ob mice over other animal models is the relatively short period for the development of advanced dn . on the other hand , compared with db / db mice ( with deficiency of the leptin receptor ) , btbr ob / ob mice provide a new tool for testing therapeutic effect of leptin administration in dn , whereas the mice have apparent limitations , such as their high cost and infertility . besides , due to the limited study on btbr strain , investigators have to pay great attention to the time - consuming backcrossing strategies in order to induce specific genetic mutations into these mice . nonetheless , btbr ob / ob mouse is fairly valuable for testing therapeutic interventions . the nzo mouse is established by selective breeding from polygenic mice of obesity and t2 dm in new zealand . it is characterized by obesity , t2 dm , and low - titer igm antibodies to the insulin receptor . the qtls on chromosomes 1 , 2 , 4 , 5 , 6 , 7 , 11 , 12 , 13 , 15 , 17 , and 18 [ 8183 ] are responsible for the disease . nzo mice are prone to autoimmune disease and develop circulating antibodies to both native dna and single - stranded dna . by 6 months of age , the antibody levels in nzo mice are comparable to those found in the mouse models of systemic lupus erythematosus . nzo mice exhibit morphological features of both diabetic and lupus nephropathies , such as glomerular proliferation , mesangial deposits , mild gbm thickening , glomerulosclerosis , eosinophilic nodules in some glomeruli , occasional hyalinization of the glomerular arterioles , and healing arteriolar inflammation . thus , given the evidence of immune disorder , nzo mouse offers a unique opportunity to study the relationship among t2 dm , autoimmunity , and obesity . kk - ay mice develop t2 dm caused by the dominant mutation in agouti yellow ( ay ) gene . the ay gene is expressed in the hair follicle , where the gene product acts as an antagonist of melanocyte stimulating hormone receptor resulting in the inhibition of melanogenesis and yellow fur . kk - ay mice spontaneously exhibit severe , early - onset hyperinsulinemia , hyperglycemia , obesity , hypertriglyceridemia , fatty liver , and albuminuria . kk - ay mice develop morphological changes of early dn , such as glomerular hypertrophy , mild and moderated mesangial matrix expansion , and segmental proliferative glomerular nephritis . therefore , kk - ay mouse might be a useful murine model of the early stage of dn . it is notable , though , that most of male kk - ay mice die of obstructive uropathy associated with hydronephrosis between 7 and 14 months of age without unknown causes . partly for this reason , kk - ay mouse is not widely used as animal model of dn . the zdf rat which has a missense mutation in the gene coding the leptin receptor ( fa / fa ) spontaneously develops insulin resistance , t2 dm , hyperlipidemia , both moderate hypertension and obesity , and progressive renal injury . although female zdf rats have similar levels of insulin resistance and degrees of obesity to male zdf rats , they develop hyperglycaemia only when administered with diabetogenic diet . thus , the zdf male rats are more widely used as the animal models of t2 dm and dn . histopathologic changes of kidney [ 5560 ] have been described as focal segmental glomerulosclerosis , mild mesangial expansion , macrophage infiltration , and interstitial fibrosis . these rats develop albuminuria at 14 weeks and focal segmental glomerulosclerosis at 1820 weeks of age [ 56 , 57 ] . the zdf rat exhibits a physiological and metabolic profile similar to human t2 dm and functional and morphological renal lesions that resemble human dn . thus , zdf rat is considered as an excellent animal model of t2 dm and dn . all the above studies indicate the importance of genetic factors in differential susceptibility to dn , whereas limited progress has been made in identifying specific genetic factors that contribute to dn due to genetic heterogeneity and multigenic pathogenesis . recently , investigators have developed genetically engineered murine models in combination with genetic manipulations , including transgenic and knockout mice bearing defined alterations in a single gene or in a series of candidate genes . studies in vivo using these models show that the genes coding tgf- , plasma prorenin , inducible camp early repressor , receptor for advanced glycation endproducts ( rage ) , endothelial nitric oxide synthase ( enos ) , and aldose reductase involve in the origin and progression of dn , corroborating experimental findings from human association studies . genetically engineered murine models provide valuable insight into the role of pathogenetic genes and molecular mechanisms responsible for dn , thus opening new avenues to develop novel therapeutic strategies . table 2 lists some of the genetically engineered murine models that develop advanced dn - like human disease . here we emphasize more on the ove26 and the enos / db / db mice , of type 1 and type 2 diabetes , that develop kidney injury most resembling that seen in human . the ove26 mouse on the fvb inbred strain is a transgenic mouse model of severe early - onset type 1 diabetes . these mice exhibit severe hyperglycemia 2 - 3 weeks after birth due to -cell - specific damage in response to overexpression of calmodulin transgene regulated by the insulin promoter . has reported progressively increasing albuminuria which is 305 g/24 h by 2 months and 15,000 g/24 h by 9 months of age . they also detect hypoalbuminemia , increased gfr from 2 - 3 months , and the following decreased gfr from 59 months , as well as hypertension which coincided with increasing albuminuria . the ove26 mice develop morphological changes of advanced dn including enlarged glomeruli , enlarging mesangium with diffuse and nodular expansion of mesangial matrix , gbm thickening , diffuse and nodular glomerulosclerosis , nodules similar to typical k - w nodules , expansion of the tubules , atrophy of tubular cells , interstitial infiltration of mononuclear cells , and tubulointerstitial fibrosis . a low level of pancreatic beta cell survival allows ove26 mice to survive well over a year with no insulin treatment and maintain near normal body weight . therefore , ove26 mice provide a valuable model of advanced human dn . however , severe albuminuria in ove26 mice highly depends on fvb background . albuminuria , mesangial matrix expansion , and fibrosis are all significantly diminished when ove26 mice were crossbred with c57bl6 or dba2 mice . this strain dependence makes it difficult to introduce other genetic mutations from other backgrounds into this model . the enos / db / db mouse is a model of type 2 diabetes generated by backcrossing of enos knockout mouse on the c57/b6 background with db / db mouse on the c57blks / j ( bks ) background . the enos / lepr double - knockout mice exhibit obesity , hyperglycemia , hyperinsulinemia , hypertension , dramatic albuminuria , and decreased gfr [ 67 , 68 ] . these mice develop histopathological changes of dn - like human disease such as mesangial expansion , gbm thickening , mesangiolysis , focal segmental and nodular glomerulosclerosis , nodules that resemble k - w nodules , striking fibronectin accumulation in glomeruli , arteriolar hyalinosis , minimal tubulointerstitial fibrosis , and microaneurysms . these features establish this mouse model as one of the very few to develop features of advanced dn . with increasing incidence of dn , development of an ideal animal model becomes one of the top priorities in combating this health crisis . impact of genetic background on several murine models of dn is evident on the susceptibility to diabetes - associated renal injury , the severity and histopathology of renal lesions . stz - treated mice and rats are widely used as animal models of early dn due to their cost effectiveness and the absence of advanced pathological lesions . genetic engineering enables us to insert or delete a specific gene or a series of candidate genes , providing valuable insight into the role of pathogenetic genes and molecular mechanisms responsible for dn , as well as opening new avenues to develop novel therapeutic strategies . however , it is difficult to establish an animal model that recapitulates all the features of human dn . few models develop morphologically advanced dn ; among these , oletf rats , ove26 mice , btbr ob / ob mice , enos / db / db mice , rage / inos mice , and megsin / rage / inos mice seem to be the most robust . perhaps the last four models suggest to us that more robust murine models of dn can be established by superimposed genetic mutations or crossbreeding with an entirely different strain . novel animal models that reproduce human dn have yet to be established in the future .

diabetic nephropathy ( dn ) is one of the microvascular complications of both type 1 and type 2 diabetes , which is also associated with a poor life expectancy of diabetic patients . however , the pathogenesis of dn is still unclear . thus , it is of great use to establish appropriate animal models of dn for doing research on pathogenesis and developing novel therapeutic strategies . although a large number of murine models of dn including artificially induced , spontaneous , and genetically engineered ( knockout and transgenic ) animal models have been developed , none of them develops renal changes sufficiently reflecting those seen in humans . here we review the identified murine models of dn from the aspects of genetic background , type of diabetes , method of induction , gene deficiency , animal age and gender , kidney histopathology , and phenotypic alterations in the hope of enhancing our comprehension of genetic susceptibility and molecular mechanisms responsible for this disease and providing new clues as to how to choose appropriate animal models of dn .

1. Introduction 2. Artificially Induced Murine Models of DN 3. Spontaneous Murine Models of DN 4. Genetically Engineered (Knockout and Transgenic) Murine Models of DN 5. Conclusions

dn , as one complication of diabetes , is one of the leading causes of end - stage renal disease ( esrd ) worldwide . although great progress has been made in the study of animal models , none of the models can reproduce all the structural and functional changes of human dn . murine models have substantial advantages over other species in the studies on pathogenesis of dn , including lower cost , murine repositories that bear multiple mutations , plentiful inbred strains , and an available map of murine genomic sequence on the internet . the animal models of diabetic complications consortium ( amdcc ) proposes the following three criteria for a desirable murine model of dn : ( 1 ) more than 50% decline in glomerular filtration rate ( gfr ) over the lifetime of the animal ; ( 2 ) greater than 10-fold increase in albuminuria compared with controls for that strain at the same age and gender ; ( 3 ) histopathology findings which include mesangial sclerosis ( a 50% increase in mesangial volume ) , any degree of arteriolar hyalinosis , glomerular basement membrane ( gbm ) thickening ( a > 25% increase compared with baseline by electron microscopy morphometry ) , and tubulointerstitial fibrosis . in fact , there are no murine models that meet all of the three criteria . here we review the identified murine models of dn including artificially induced , spontaneous , and genetically engineered ( knockout and transgenic ) animal models and compare their advantages and deficiencies . some of the key issues , such as strain , genetic background , type of diabetes , method of induction , gene deficiency , animal age and gender , kidney histopathology , and phenotypic alterations , are all included in this review . we hope this review could enhance our understanding of genetic susceptibility and molecular mechanisms responsible for dn , as well as provide new clues as to how to choose appropriate animal models of dn . alloxan and streptozotocin ( stz ) are widely used for producing artificially induced t1 dm which causes kidney damage with similarities to human dn . both of them are glucose analogues that enter the insulin - producing beta - cells via a glucose transporter . however , they are generated through different approaches in the case of alloxan and stz . in the presence of glutathione , alloxan and its reduction product , dialuric acid , generate superoxide radicals in a cyclic redox reaction which undergo dismutation to hydrogen peroxide afterwards . so both of the two diabetogenic agents induce diabetes secondary to the necrosis of pancreatic beta - cells . these stz - treated models develop a modest degree of proteinuria and serum creatinine increase , as well as minimal mesangial matrix expansion , depending on the genetic background [ 3 , 4 ] . animal models of stz - induced dn are usually performed in mice , sprague - dawley and wistar - kyoto rats . given the mouse strains which are resistant to a single dose of stz ( 200 mg / kg ) or a two - dose regimen of stz ( 2 1002 125 mg / kg ) , greater cytotoxicity to pancreatic beta - cells and collateral tissue occurs , resulting in a higher incidence and severity of diabetes [ 47 ] . because of the severe hyperglycaemia , the diabetic models need to be monitored for blood glucose and administrated insulin . some investigators optimize the animal models of stz - induced dn by uninephrectomy in advance which lead to compensatory hypertrophy of the remaining kidney and acceleration of the disease progression . however , owing to the nephrotoxicity of stz , it is difficult to differentiate between the direct toxic effect of stz and lesions caused by hyperglycaemia [ 8 , 9 ] . some studies on mouse models of dn show that high - dose stz - treated mice exhibit more albuminuria than those which receive low - dose stz , despite of similar blood glucose . in order to reduce nonspecific nephrotoxicity of stz , the regimen of multiple low - dose injections of stz to induce diabetes has been performed , that is daily intraperitoneally injections of 4060 mg / kg stz for five consecutive days [ 4 , 1214 ] which usually induces repetitive low - grade beta cell damage accompanied by secondary autoimmune insulitis [ 15 , 16 ] . it is quite different among inbred strains of mice in susceptibility to both pancreatic beta cell toxicity and the direct nephrotoxicity of low - dose stz . it is reported by gurley et al . according to this recommendation , mouse models of dn should be induced by daily intraperitoneally injections of 50 mg / kg stz for five consecutive days . it is also of great use for the research of accelerated atherosclerosis [ 2426 ] , although inbred strains of mice exhibit significant differences in response to the effect of high - fat diet . have reported a new rat model of dn induced by high - fat diet , multiple low - dose injections of stz , and uninephrectomy . this model exhibits most features of human dn from t2 dm including hyperglycemia , hypoinsulinemia , hyperlipidaemia , hypertension , and microalbuminuria followed by overt albuminuria , mesangial expansion , and terminal glomerular sclerosis . spontaneous animal models of dn are established by selective breeding from animals which spontaneously develop dn due to genetic abnormality . renal abnormalities in these models resemble human diseases ; therefore , these models provide an experimental platform for studying pathogenesis and genetic susceptibility responsible for dn . although these models are difficult to feed and breed , not widely available , and with long modeling cycle and higher cost , the application of them is becoming increasingly extensive . due to pathogenic and genetic similarities to the human disease , the model serves as a useful tool to study the etiology , pathology , and progression of disease . nod mouse was derived from the jcl : icr cataract mouse 30 years ago in japan . this model exhibits a number of clinical features of human t1 dm including hyperglycaemia , glycosuria , polyuria , and polydipsia ; however , it is more resistant to ketoacidosis . like in humans , the major histocompatibility complex ( mhc ) alleles some mhc alleles must be accompanied by other non - mhc genes for the development of t1 dm [ 29 , 30 ] , which is also the same in human disease . despite of the extensive study of the genetic and immunologic pathogenesis of t1 dm in nod mouse , few investigators choose nod mouse to do research on dn because of the complicated genetics , the late and variable age of onset of t1 dm , and the requirement for insulin administration . even so , one of the few studies shows that the amount of albuminuria in hyperglycemic nod mice is seven times higher than that in nod mice before the development of hyperglycemia . the results of renal lesions in acute phase of t1 dm in nod mice show mild changes in glomeruli and structural alteration of the proximal straight tubules . the study also points out that increased neuronal nitric oxide synthase may represent one of the pathogenic factors of dn . besides , there are also studies using nod mice demonstrating that transforming growth factor- ( tgf- ) and advanced glycosylation endproducts ( age ) take an important role in mesangial proliferation and sclerosis [ 7173 ] , which is also the same in human dn . the mutation leads to the misfolding of insulin protein , which is toxic to pancreatic -cell . nonetheless , akita mice have higher levels of hyperglycemia , albuminuria , blood pressure , and more consistent structural changes of kidney compared with stz - induced dn . have found that renal phenotype of akita mice is largely dependent on their genetic background strains . thus , mice bearing ins-2 akita mutation have significant advantages as a model of t1 dm . the db / db mouse which has a g - to - t mutation in the gene coding the leptin receptor develops obesity , insulin resistance , and t2 dm spontaneously . the db / db mutation which is autosomal recessive was initially recognized from an obese and hyperphagic mouse in the c57blks / j strain and was subsequently backcrossed to a pure c57bl/6j background . mice in the c57blks / j stain exhibit hyperinsulinemia at 10 days of age and slight hyperglycemia at 1 month of age . manifestations of dn are albuminuria , glomerular hypertrophy , mesangial matrix expansion , and gbm thickening . the level of albuminuria in the db / db male mouse is 68600 g/24 h [ 20 , 3437 ] which is only 421 g/24 h [ 34 , 37 ] in the age - matched heterozygous littermate . by 1820 months , the glomerular and mesangial matrix enlargements become more remarkable , and thickening of the gbm is observed . in general , db / db mice do not develop mesangiolysis , nodular mesangial sclerosis , and progressive renal insufficiency . hyperglycemia , and renal changes of db / db mice in c57blks / j stain are usually worse than in the c57bl/6 background . hence , investigations of dn in db / db mice are more widely conducted with the c57blks / j strain . nonetheless , db / db mice in the c57bl/6 stain which have been intercrossed with gene knockout and transgenic mice [ 74 , 75 ] provide new strains to identify pathogenesis of dn . multiple recessive genes are associated with the induction of diabetes , including odb-1 on x - chromosome of oletf rats . it is also reported that a major quantitative trait locus colocalizing with cholecystokinin type a receptor gene influences poor pancreatic proliferation in oletf rats . at 22 weeks of age , the oletf rats develop overt albuminuria , and at 54 weeks , advanced renal changes such as macroalbuminuria , nodular lesions , diffuse glomerulosclerosis , and tubulointerstitial fibrosis are noted like human dn . therefore , oletf rat is considered as one of the best murine models to study dn . this model is characterized by moderate hyperglycemia , peripheral insulin resistance , and nonhyperlipidemia , and nonobese phenotype . it is recognized that t2 dm in gk rat is primarily caused by beta - cell deficit . by e16 , the number of pancreatic beta - cells in the gk rats is half of the wistar rats , and this difference was sustained until birth . gk rat exhibits morphological changes which can be seen in early stage of human dn such as glomerular hypertrophy and gbm thickening . have reported advanced renal changes in gk rats such as segmental glomerulosclerosis and tubulointerstitial fibrosis at 24 months of age . thus , they draw the conclusion that renal changes in gk rats at a late stage were similar to those of progressive human dn . thus , it is not widely used as an animal model of dn . however , a new mouse model that mimics progressive dn has been developed in btbr strain with the ob / ob mutation . the btbr ob / ob mice are largely resistant to the hypoglycemic effect of insulin administration and rapidly develop pathological changes of both early and advanced human dn . characteristics of early dn such as glomerular hypertrophy , accumulation of mesangial matrix , and loss of podocytes are detectable by 8 weeks of age . by 22 weeks , morphological characteristics of renal injury has 20% increase in gbm thickness , 50% increase in mesangial matrix , mesangiolysis , diffuse mesangial sclerosis , focal arteriolar hyalinosis , and focal mild interstitial fibrosis . on the one hand , the advantage of btbr ob / ob mice over other animal models is the relatively short period for the development of advanced dn . on the other hand , compared with db / db mice ( with deficiency of the leptin receptor ) , btbr ob / ob mice provide a new tool for testing therapeutic effect of leptin administration in dn , whereas the mice have apparent limitations , such as their high cost and infertility . it is characterized by obesity , t2 dm , and low - titer igm antibodies to the insulin receptor . the qtls on chromosomes 1 , 2 , 4 , 5 , 6 , 7 , 11 , 12 , 13 , 15 , 17 , and 18 [ 8183 ] are responsible for the disease . nzo mice are prone to autoimmune disease and develop circulating antibodies to both native dna and single - stranded dna . by 6 months of age , the antibody levels in nzo mice are comparable to those found in the mouse models of systemic lupus erythematosus . nzo mice exhibit morphological features of both diabetic and lupus nephropathies , such as glomerular proliferation , mesangial deposits , mild gbm thickening , glomerulosclerosis , eosinophilic nodules in some glomeruli , occasional hyalinization of the glomerular arterioles , and healing arteriolar inflammation . thus , given the evidence of immune disorder , nzo mouse offers a unique opportunity to study the relationship among t2 dm , autoimmunity , and obesity . the ay gene is expressed in the hair follicle , where the gene product acts as an antagonist of melanocyte stimulating hormone receptor resulting in the inhibition of melanogenesis and yellow fur . kk - ay mice spontaneously exhibit severe , early - onset hyperinsulinemia , hyperglycemia , obesity , hypertriglyceridemia , fatty liver , and albuminuria . kk - ay mice develop morphological changes of early dn , such as glomerular hypertrophy , mild and moderated mesangial matrix expansion , and segmental proliferative glomerular nephritis . therefore , kk - ay mouse might be a useful murine model of the early stage of dn . it is notable , though , that most of male kk - ay mice die of obstructive uropathy associated with hydronephrosis between 7 and 14 months of age without unknown causes . partly for this reason , kk - ay mouse is not widely used as animal model of dn . the zdf rat which has a missense mutation in the gene coding the leptin receptor ( fa / fa ) spontaneously develops insulin resistance , t2 dm , hyperlipidemia , both moderate hypertension and obesity , and progressive renal injury . thus , the zdf male rats are more widely used as the animal models of t2 dm and dn . histopathologic changes of kidney [ 5560 ] have been described as focal segmental glomerulosclerosis , mild mesangial expansion , macrophage infiltration , and interstitial fibrosis . thus , zdf rat is considered as an excellent animal model of t2 dm and dn . all the above studies indicate the importance of genetic factors in differential susceptibility to dn , whereas limited progress has been made in identifying specific genetic factors that contribute to dn due to genetic heterogeneity and multigenic pathogenesis . recently , investigators have developed genetically engineered murine models in combination with genetic manipulations , including transgenic and knockout mice bearing defined alterations in a single gene or in a series of candidate genes . studies in vivo using these models show that the genes coding tgf- , plasma prorenin , inducible camp early repressor , receptor for advanced glycation endproducts ( rage ) , endothelial nitric oxide synthase ( enos ) , and aldose reductase involve in the origin and progression of dn , corroborating experimental findings from human association studies . genetically engineered murine models provide valuable insight into the role of pathogenetic genes and molecular mechanisms responsible for dn , thus opening new avenues to develop novel therapeutic strategies . table 2 lists some of the genetically engineered murine models that develop advanced dn - like human disease . here we emphasize more on the ove26 and the enos / db / db mice , of type 1 and type 2 diabetes , that develop kidney injury most resembling that seen in human . the ove26 mouse on the fvb inbred strain is a transgenic mouse model of severe early - onset type 1 diabetes . they also detect hypoalbuminemia , increased gfr from 2 - 3 months , and the following decreased gfr from 59 months , as well as hypertension which coincided with increasing albuminuria . the ove26 mice develop morphological changes of advanced dn including enlarged glomeruli , enlarging mesangium with diffuse and nodular expansion of mesangial matrix , gbm thickening , diffuse and nodular glomerulosclerosis , nodules similar to typical k - w nodules , expansion of the tubules , atrophy of tubular cells , interstitial infiltration of mononuclear cells , and tubulointerstitial fibrosis . albuminuria , mesangial matrix expansion , and fibrosis are all significantly diminished when ove26 mice were crossbred with c57bl6 or dba2 mice . the enos / db / db mouse is a model of type 2 diabetes generated by backcrossing of enos knockout mouse on the c57/b6 background with db / db mouse on the c57blks / j ( bks ) background . the enos / lepr double - knockout mice exhibit obesity , hyperglycemia , hyperinsulinemia , hypertension , dramatic albuminuria , and decreased gfr [ 67 , 68 ] . these mice develop histopathological changes of dn - like human disease such as mesangial expansion , gbm thickening , mesangiolysis , focal segmental and nodular glomerulosclerosis , nodules that resemble k - w nodules , striking fibronectin accumulation in glomeruli , arteriolar hyalinosis , minimal tubulointerstitial fibrosis , and microaneurysms . these features establish this mouse model as one of the very few to develop features of advanced dn . with increasing incidence of dn , development of an ideal animal model becomes one of the top priorities in combating this health crisis . impact of genetic background on several murine models of dn is evident on the susceptibility to diabetes - associated renal injury , the severity and histopathology of renal lesions . stz - treated mice and rats are widely used as animal models of early dn due to their cost effectiveness and the absence of advanced pathological lesions . genetic engineering enables us to insert or delete a specific gene or a series of candidate genes , providing valuable insight into the role of pathogenetic genes and molecular mechanisms responsible for dn , as well as opening new avenues to develop novel therapeutic strategies . however , it is difficult to establish an animal model that recapitulates all the features of human dn . few models develop morphologically advanced dn ; among these , oletf rats , ove26 mice , btbr ob / ob mice , enos / db / db mice , rage / inos mice , and megsin / rage / inos mice seem to be the most robust . perhaps the last four models suggest to us that more robust murine models of dn can be established by superimposed genetic mutations or crossbreeding with an entirely different strain . novel animal models that reproduce human dn have yet to be established in the future .

most motile animals show some form of active foraging behavior to locate resources they need in their environment , and will actively avoid stimuli that are harmful to them . these basic behavioral responses have been used to provide a simple operational definition of whether stimuli are rewarding or punishing to an animal . in the field of animal behavior research , rewarding and punishing stimuli are often defined simply by the nature of the responses they elicit : rewards elicit approach behavior , whereas punishing stimuli elicit avoidance behavior ( skinner , 1938 ) . rewards also have a reinforcing property , in that almost all motile animals studied will learn to repeat actions that bring about ( or bring closer ) a rewarding outcome . actions that result in attaining the reward ( wise and rompre , 1989 ; wise , 2004 ) . given this operational definition of reward , it can be said that reward - seeking and reward learning are fundamental aspects of animal behavior . reward - seeking was recognized as fundamental to even the earliest explanatory models of behavior : sherrington ( 1906 ) , tinbergen ( 1951 ) , and lorenz ( 1965 ) all developed behavioral models that incorporated an assumed innate drive to seek rewards . exploring the neurobiology of reward responses and reward processing has long been a major focus of neuroscience research . understandably the vast majority of studies have considered humans and other mammals , while more recently , mechanisms of reward processing have been studied in a wider range of animal model systems . mammalian research has clearly established that the circuit connecting midbrain dopaminergic neurons to the ventral striatum and prefrontal cortex is central to mammalian brain reward systems ( wise and rompre , 1989 ; koob and le moal , 1997 ; schultz , 2000 , 2007 ; watson and platt , 2008 ) . dopamine is a key modulator of this circuit , and of behavioral responses to rewards ( berridge and robinson , 1998 ; roitman et al . , 2004 ; wise , 2004 ; berridge et al . , 2009 ) . more recent comparative studies with diverse species scattered across a divergent range of animal phyla have also repeatedly demonstrated roles for the biogenic amines , and especially dopamine , in reward - seeking and reward learning . this raises the question of whether the function of dopamine in reward - seeking and learning has been broadly conserved across animal phyla , or whether this is a case of convergent evolution . in this review we discuss the evidence that dopamine affects reward responses across the nematoda , vertebrata , platyhelminthes and mollusca , representing highly divergent phyla . we then discuss how phylum arthropoda appear to be an exception in that most studies have shown reward responses and reward learning to be affected by octopamine rather than dopamine . dopamine has been shown to affect responses to reward in extremely diverse animal groups , but the documented effects of dopamine vary . most studies with invertebrates have used food stimuli as rewards since these tend to elicit the most robust behavioral responses and learning . nematodes ( e.g. , caenorhabditis elegans ) possess one of the simplest animal nervous systems , but even in this animal , dopamine affects the expression of a form of food - seeking behavior . c. elegans possesses dopaminergic mechanosensory neurons that release dopamine in the presence of bacterial food ( sawin et al . , 2000 ; rivard et al . , dopamine release from these neurons reduced crawling speed ( sawin et al . , 2000 ; rivard et al . , 2010 ) , and dopamine modulation of the locomotor circuit also increased turning behavior ( hills et al . , 2004 ) . such behavioral responses to food are greater in starved worms than fed worms ( sawin et al . , 2000 ) . these changes in locomotor behavior in response to food are a very simple form of reward - seeking behavior . area - restricted search pattern of locomotion , which ensures that the animal explores and dwells longer in an area containing food ( hills et al . , 2004 ) , and the outcome of their change in crawling behavior is that they are more likely to locate food . it is striking that even this elementary form of reward - seeking behavior is modulated by dopamine . caenorhabditis elegans learned to locate food faster in a simple t - maze after repeated training trials with the apparatus ( qin and wheeler , 2007 ) . dopamine deficient cat-2 mutant worms , which lack the enzyme tyrosine - hydroxylase necessary for the biosynthesis of dopamine ( figure 1 ) , learned the maze task less well , and the memory did not persist as long as in wild - type animals ( qin and wheeler , 2007 ) . this suggests that dopamine affects memory systems involved in food - seeking behavior in nematodes . chemical relationships and biosynthetic pathways linking dopamine , tyramine , octopamine , and norepinephrine ( enzymes in italics ) . dopamine , tyramine , octopamine , and norepinephrine are all derived from tyrosine . in order to synthesize dopamine , tyrosine is first converted to dopa by tyrosine - hydroxylase , which is then decarboxylated by dopa - decarboxylase to yield dopamine . tyramine is either produced directly from tyrosine by tyrosine - decarboxylase , or ( more rarely ) dehydroxylated from dopamine by dopamine - dehydroxylase . this figure summarizes the most common synthesis pathways , but there are variations among the phyla . in some groups , octopamine is a trace amine and synthesized from tyramine by dopamine--hydroxylase , while in other phyla , norepinephrine is physiologically irrelevant and not present at any biologically meaningful level . they have no body cavities and a simple nervous system , but they do have rudimentary cephalization , and they can learn both classical and operant conditioning tasks ( best and rubinstein , 1962 ; shafer and corman , 1963 ) . dopamine seems to be involved in the mechanism of reinforcement in the flatworm dugesia japonica . kusayama and watanabe ( 2000 ) developed a conditioned - place - preference assay for d. japonica , and were able to induce in the flatworms a preference for an environment in which they had been treated with methamphetamine ( which increases extracellular biogenic amine levels ) . this preference could be eliminated by treatment with three different antagonists characterized in mammals as active against dopamine receptors ( kusayama and watanabe , 2000 ) , suggesting that dopamine is involved in reinforcement in planarians . far more is known about the role of dopamine in reinforcement in the mollusk aplysia ( lechner et al . the ingestion of seaweed involves rhythmic and coordinated movements of the foregut and peri - oral structures . lechner et al . ( 2000a ) developed a training protocol for the classical conditioning of biting in aplysia so that animals could be trained to associate a light touch to the lips with a paintbrush with the presentation of seaweed reward . after conditioning , the number of biting responses to the lip tickle was increased ( lechner et al . , 2000a,2000b ) . lesioning the esophageal nerve blocked this form of conditioning , showing that the esophageal nerve mediates the reinforcing property of the unconditioned food reward during classical conditioning ( lechner et al . ( 2002 ) were able to train the biting response in a slightly different operant paradigm that paired biting with direct electrical stimulation of the esophageal nerve , providing more evidence that the esophageal nerve conveys the reinforcement signal . the esophageal nerve contains many processes that are dopaminergic ( kabotyanski et al . , 2000 ) , and ( 2002 ) were able to replace electrical stimulation of the esophageal nerve with iontophoretic application of dopamine onto selected post - synaptic neurons to achieve effective training . this showed conclusively that dopamine is the neurochemical mediator of the reinforcement signal in operant conditioning . in the classical conditioning paradigm , association of a tactile stimulus with food could also be blocked by treatment with the dopamine receptor antagonist , methylergonovine ( reyes et al . , 2005 ) . therefore evidence from both classical and operant conditioning studies in aplysia suggest a role for dopamine in reinforcement and reward learning . the nematoda , platyhelminthes and mollusca are representatives of three different superphyla within the protostomes ( figure 2 ) . the chordata are deuterostomes , and a separate lineage from the protostome groups ( figure 2 ) . despite all this taxonomic diversity dopamine affects behavioral responses to reward and reinforcement in a similar manner . the affects of dopamine on mammalian reward responses have been well reviewed recently ( schultz , 2007 ) , and hence here we consider only the main findings as relevant to this comparative review . phylogeny of groups discussed in this paper , based on nearly complete ribosomal rna gene analyses ( mallatt et al . , 2010 ) . discussed phyla represent examples of protostome , deuterostome , and diploblastic groups , yet dopamine is a modulator in the motor systems of all these diverse phyla , and affects responses to rewarding or punishing stimuli in mollusks , platyhelminths , arthropods , nematodes , and chordates . dopamine has long been identified with motor function in mammals , but the first evidence linking dopamine to reward - seeking came from the observation that moderate dopamine receptor antagonist treatments attenuated the motivation to respond to a food reward before compromising the ability to respond ( wise and schwartz , 1981 ; wise , 2004 ) . this effect appeared to be caused in part by dopamine receptor antagonists eliminating the reinforcing properties of rewards ( wise and schwartz , 1981 ) . animals will work at lever - press and other tasks for electrical stimulation of dopaminergic midbrain regions such as the ventral tegmental area and lateral hypothalamus ( routtenberg and lindy , 1965 ; carlezon and chartoff , 2007 ; watson and platt , 2008 ) . such stimulation can be chosen in preference over food or water reward ( routtenberg and lindy , 1965 ) . dopamine regulates learning of stimuli associated with reward : it is essential for both the establishment and expression of conditioned reinforcement via associative learning ( wise , 2004 ) . dopamine - selective lesions of the nucleus accumbens can block responding to reward - associated stimuli ( taylor and robbins , 1986 ) , whereas amphetamine injection into the nucleus accumbens , to elevate extracellular dopamine in this area , enhances responding ( taylor and robbins , 1984 ) . recordings from midbrain dopamine neurons in mammals have shown strong responses to both primary rewards ( such as food and water ) , and also conditioned stimuli associated with rewards ( schultz , 1998 ; schultz , 2001 ) . most midbrain dopaminergic neurons projecting to the nucleus accumbens and frontal cortex are tonically active ( meaning a relatively constant baseline level of firing ) , but show phasic activation ( bursts of enhanced firing ) following primary food rewards , or stimuli learned to be predictive of reward ( schultz , 2000 , 2007 ) . these reward responses are not unconditional ; rather the intensity of the phasic activation is modulated by reward predictability ( hollerman and schultz , 1998 ; schultz , 1998 ) . most midbrain dopaminergic neurons have a tonic firing rate that is strongly enhanced by unexpected rewards far more than expected rewards , while the neuronal firing rate drops below baseline in response to expected rewards that do not appear ( schultz , 2001 , 2007 ) . this pattern of activity appears to represent the reward prediction error : that being the difference between predicted and obtained rewards . wagner model of learning ( rescorla and wagner , 1972 ; schultz , 2000 ; pessiglione et al . , subsecond changes in the amount of dopamine released into the nucleus accumbens appear to directly modulate reward - seeking behavior ( roitman et al . , 2004 ) . short pulses of dopamine released into the nucleus accumbens were recorded in rats trained to lever - press for sucrose in response to stimuli signaling the start of a lever - pressing session . lever - presses were coincident with the peaks of the dopamine surge ( roitman et al . the taste of sugar evoked a similar short pulse of dopamine release into the nucleus accumbens , whereas quinine ( an aversive taste ) suppressed dopamine release ( roitman et al . , 2008 ) . together , these findings show that the phasic responses of dopamine neurons signal an assessment of the current value of reward stimuli , and that these dopamine signals directly modulate behavioral responses to rewards . so far , we have discussed examples from four phyla of highly diverse animals in which dopamine dominates reward learning and the reinforcing properties of rewards , but the arthropoda do not seem to fit this pattern . the arthropoda are ecdysozoan protostomes most closely related to nematoda ( figure 2 ) , but within this group evidence from both insects and crustaceans has shown that octopamine affects reward learning and behavioral responses to rewards ( hammer , 1997 ; hammer and menzel , 1998 ; schwaerzel et al . , 2003 ; unoki et al . , 2005 ; vergoz et al . , 2007 ; kaczer and maldonado , 2009 ; selcho et al . , octopamine ( chemically similar to both dopamine and noradrenaline , figure 1 ) is a trace amine whose physiological importance is presently not well established ( burchett and hicks , 2006 ) . by contrast , in the arthropods , octopamine is a major regulator of behavior and physiology ( roeder et al . , 2003 ; roeder , 2005 ) . the similarities between the octopamine receptor subtypes in protostomes and adrenergic receptor subtypes in vertebrates suggest these two systems may have diverged from a common evolutionary origin ( evans and maqueira , 2003 ; maqueira et al . , 2005 ; pfluger and stevenson , 2005 ) . as detailed below , pharmacological studies with crustacea and insecta kaczer and maldonado ( 2009 ) showed that in the crab chasmagnathus granulates , octopamine treatments influenced expression of a learned exploratory response triggered by experiencing food in a novel environment ( kaczer and maldonado , 2009 ) . octopamine injection enhanced the exploratory response to food , whereas injection of two octopamine receptor antagonists reduced this response ( kaczer and maldonado , 2009 ) . diverse studies with honey bees ( apis mellifera , hymenoptera ) have shown that octopamine treatment affects behavioral responses to sucrose reward ( mercer and menzel , 1982 ; hammer and menzel , 1998 ; scheiner et al . , 2002 ; schulz et al . , 2002 ; barron et al . , 2007 ) . a robust and widely used assay for appetitive conditioning in honey bees is proboscis extension response conditioning where bees learn to extend their proboscis in response to a novel odor paired with the presentation of sucrose reward ( kuwabara , 1957 ; bitterman et al . , 1983 ) . dopamine microinjection into the brain reduced performance in appetitive conditioning of proboscis extension ( mercer and menzel , 1982 ) , whereas microinjection of octopamine into either the mushroom bodies or antennal lobe could substitute for sucrose presentation in training ( hammer and menzel , 1998 ) . some of the vum ( ventral unpaired median)neurons respond to sucrose ( hammer , 1993 ; schroeter et al . , 2007 ) , and one of these ( vummx1 ) has been shown to mediate sucrose reinforcement ( hammer , 1993 ) . it is of interest to note that thoracic octopamine injection increased reflexive proboscis extension responsiveness to sucrose in an unconditioned paradigm in honey bees , whereas dopamine receptor agonist treatment reduced responsiveness ( scheiner et al . , 2002 ) . this suggests an opponent relationship between octopamine and dopamine systems in response to sucrose reward . more recent pharmacological studies with the cricket ( gryllus bimaculatus , orthoptera ) ( unoki et al . , 2005 , 2006 ; mizunami et al . , have shown that treatments with octopamine receptor antagonists and agonists affected performance in reward learning assays , but treatment with dopamine receptor antagonists and agonists affected performance in aversive learning assays . as a result of these studies a commonly held view is that for the arthropods , octopamine and dopamine modulate different motivational systems with octopamine modulating appetitive learning and dopamine modulating aversive learning ( beggs et al . however , new research with drosophila ( described below ) suggests that this interpretation may be an oversimplification . the arthropod studies described so far have relied heavily on pharmacological tools to manipulate biogenic amine systems . a difficulty with this approach is that the affinities of most of the available biogenic amine receptor agonists and antagonists to all the biogenic amine receptors in the different experimental insect species are incompletely known . consequently , it is difficult to experimentally manipulate a single receptor system in isolation or to be completely confident that nominated agonists or antagonists do not affect more than one biogenic amine system . currently , the only solution to this problem is to use several different antagonists or agonists against the same receptor system(s ) , and hopefully show the same behavioral effects ( unoki et al . , 2005 ; vergoz et al . , also , in many cases pharmacological treatments have been applied to the whole organism or to the whole brain , which has limited a circuit - level analysis of reinforcement systems in arthropods . the genetic tools available for drosophila melanogaster ( diptera ) have enabled very different approaches to investigate the roles of octopamine and dopamine in reward responses . in drosophila , several studies have used different genetic tools to manipulate all ( or most ) dopaminergic or octopaminergic neurons in the fly brain . the conclusions of these studies are consistent in that they have shown that dopamine is required for aversive learning , but not reward learning , and octopamine is required for reward learning but not aversive learning ( schwaerzel et al . , 2003 ; schroll et al . , 2006 ; claridge - chang et al . , however , more recent studies have used more selective genetic manipulations to target specific dopamine receptors , or specific small groups of dopamine neurons . these have shown that some dopamine signals may also modulate reward responses in drosophila ( kim et al . we first review studies that have manipulated all dopaminergic or octopaminergic neurons in the fly brain , and then studies that have selectively targeted specific populations of dopamine neurons , or dopamine receptor systems . we then discuss how findings from genetic studies with drosophila can be reconciled with pharmacological studies with other arthropods . ( 2003 ) explored the role of octopamine and dopamine in appetitive and aversive conditioning using strains of drosophila melanogaster in which the enzymes responsible for the synthesis of different biogenic amines were under the control of heat - shock sensitive promoters . flies in which the tyramine--hydroxylase ( figure1 ) gene had been knocked out could not synthesize octopamine ( monastirioti et al . , these flies performed normally in an aversive learning task associating electric shock with a novel odor , but did not learn to associate a sugar reward with an odor ( schwaerzel et al . , 2003 ) . this defect could be rescued by a transgene containing the wild - type tyramine--hydroxylase gene downstream of a heat - shock promoter , such that after heat - shock to activate the promoter and restore octopamine synthesis , flies performed normally in both the appetitive and aversive learning tasks ( schwaerzel et al . , 2003 ) . to examine the role of dopamine signaling in the two learning assays schwaerzel et al . ( 2003 ) used a sophisticated gene construct that enabled neurotransmitter release from dopaminergic neurons to be blocked by maintaining flies at an elevated temperature . at the restrictive temperature , flies performed poorly in the aversive learning paradigm , but normally in an appetitive learning paradigm ( schwaerzel et al . , 2003 ) . similar findings have been reported for drosophila larvae ( honjo and furukubo - tokunaga , 2009 ) . the conclusion is that for adult and larval drosophila , octopamine affects learning of rewarding stimuli and dopamine affects learning of aversive stimuli . relatively new genetic tools allow neuronal activity to be modulated by light pulses , which has allowed researchers to study the behavioral changes that result when octopaminergic or dopaminergic cell populations are activated in association with different environmental stimuli . to investigate the roles of octopamine and dopamine in learning by drosophila larvae , schroll et al . ( 2006 ) used channelrhodopsin gene constructs that allowed different neuronal populations to be activated by pulses of blue light . larvae learned to avoid an odor that had been paired with light activation of dopaminergic neurons , but they became attracted to odors paired with light activation of octopaminergic and tyraminergic neurons ( schroll et al . , 2006 ) . the inference is that activity of dopaminergic neurons mediates punishment , whereas activity of octopaminergic or tyraminergic neuron populations mediates the reinforcing properties of reward ( schroll et al . , 2006 ) . ( 2009 ) were able to optically activate populations of dopaminergic neurons in transgenic adult flies with a burst of laser light , by driving the expression of atp - gated p2x channels in dopaminergic neurons , and using laser light to trigger atp release from a previously microinjected caged precursor ( claridge - chang et al . , 2009 ) . when laser - activation of dopaminergic neurons was associated with a specific odor cue , flies learned aversion to the odor . the consistent message from the drosophila studies discussed so far is that for both larval and adult flies , octopamine is necessary for the learning of food reward , and dopamine is necessary for aversive learning . this is in agreement with the main findings from pharmacological studies performed with other arthropods ( unoki et al . , however , more targeted genetic manipulations of specific dopamine signals in drosophila suggest that this understanding of dopamine 's role in insects is an oversimplification . in the insect brain , the mushroom bodies are a protocerebral higher brain center known for their roles in olfactory processing and learning and memory ( farris , 2008 ) . in drosophila , the mushroom bodies are necessary for associative learning ( de belle and heisenberg , 1994 ; heisenberg , 1998 ; schwaerzel et al . , 2003 ; margulies et al . , 2005 ; krashes et al . , 2007 ) . the dopamine receptor dda1 ( a d1-like dopamine receptor that activates adenylyl cyclase ) is highly expressed in adult drosophila mushroom bodies , and also other regions of the brain . kim et al . ( 2003 ) identified two mutants dumb and dumb that eliminated expression of dda1 in the adult mushroom bodies and central complex . both dumb mutants completely failed to learn the association of an odor stimulus with electric shock , and also showed partial impairment of learning of an odor associated with sucrose reward ( kim et al . , 2007 ) . these defects could be rescued by restoring dda1 expression in the mushroom bodies ( kim et al . , 2007 ) . a study with larval drosophila also reported that dumb and dumb mutants were defective in both aversive and appetitive learning assays ( selcho et al . , 2009 ) , supporting the conclusion that signaling via the dda1 receptor in the mushroom bodies modulates learning of both rewarding and punishing stimuli . krashes and waddell ( 2008 ) have shown that the level of satiation of drosophila influences the performance of flies in assays of appetitive memory . in fed flies , appetitive memory performance is low because mushroom body neurons are inhibited by tonic dopamine release from a population of dopaminergic neurons innervating the medial lobe and pedunculus of the mushroom body ( the mb - mp neurons krashes et al . , 2009 ) . stimulation of neurons expressing neuropeptide f ( ddpf ) promoted appetitive memory performance in flies , mimicking the performance levels seen in hungry flies ( krashes et al . , 2009 ) . dnpf is an ortholog of mammalian neuropeptide y that regulates food - seeking in mammals ( tatemoto et al . , 1982 ; kaira , 1997 ) . one action of dnpf is to suppress the inhibitory mb - mp neurons , which then enables the expression of food - associated conditioned responses ( krashes et al . , 2009 ) . therefore , a specific dopamine signal inhibits mushroom body neurons , and reduces the expression of appetitive memory . to conclude the discussion of the arthropods ; both pharmacological treatments and genetic manipulations of brain octopamine and dopamine systems have suggested different behavioral roles for octopamine and dopamine . octopamine affects reward responses and dopamine affects punishment responses ( schwaerzel et al . , 2003 ; unoki et al . , 2005 ; but new genetic studies that have manipulated specific dopamine signals have shown that in drosophila , different dopamine signals affect expression of learned responses to both rewarding and punishing stimuli ( kim et al . , 2007 ; krashes et al . , 2009 ; selcho et al . , it would seem that pharmacological or genetic manipulations of the whole brain might not have been selective or precise enough to reveal all the behavioral effects of different dopamine signals . the arthropods are different from the other phyla discussed so far in that octopamine has been shown by most studies to play a dominant role in mediating reward responses and reward learning , but it now seems likely that in drosophila different dopamine signals affect expression of learned responses to both rewarding and punishing stimuli . this complexity parallels what is known of the many different behavioral roles of dopamine in mammals . in the mammalian brain , dopamine is most well known for its important role in the reward systems , but distinct mesolimbic dopamine signals mediate behavioral responses to aversive events and stress also ( ikemoto and panksepp , 1999 ; pruessner et al . , 2004 ; alcaro et al . , 2007 ; schultz , 2007 ; fadok et al . , 2009 ; diaconescu et al . , from a phylogenetic perspective , the link between dopamine and behavioral responses to reward is extremely broad ( figure 2 ) . in this section we consider the implications of the similar behavioral roles of dopamine in various different phyla for the evolution of brain reward systems . a consideration of the general behavioral functions of the biogenic amines across animal phyla suggests that dopamine could have been predisposed to evolve functions in reward processing from an ancestral role as a signaling molecule modulating motor circuits in response to salient environmental stimuli . in one of the simplest metazoans , the nematode c. elegans , dopamine functions to modulate motor output and locomotor behavior , and is released in response to environmental stimuli that signal the local abundance of food ( hills et al . , as far as we know , dopamine modulation of motor circuits has been reported for every animal phylum in which it has been investigated : nematoda ( sawin et al . , 2000 ; rivard et al . , 2010 ) , platyhelminthes ( buttarelli et al . , 2000 , 2008 ; 2001 ) , annelida ( esch and kristan , 2001 , 2002 ; friesen and kristan , 2007 ) , mollusca ( pavlova , 2001 ) , arthropoda ( burrows , 1996 ) , chordata ( grillner et al . , 1995 ; jordan et al . , 2008 ) and also diploblastic cnidaria ( chung and spencer , 1996 ; kass - simon and pierobon , 2007 ) . the cnidaria represent perhaps the simplest animal nervous systems , and molecular and morphological evidence places the cnidaria as basal among metazoans ( mallatt et al . , 2010 ) . in the cnidarian hydra japonica , dopamine affects the extent of mouth opening in response to food stimuli ( hanai and kitajima , 1984 ) . it seems likely that modulation of motor circuits in response to environmental stimuli could be one of the ancestral functions of dopamine as a signaling molecule in simple nervous systems . from this proposed ancestral role , different biogenic amine systems could have evolved progressively more specialized functions in behavioral responses to rewarding or aversive stimuli as increasing levels of behavioral complexity evolved along with the evolution of more complex nervous systems ( hills , 2006 ) . this hypothesis would explain why dopamine and other biogenic amines have roles in aversive responses ( schwaerzel et al . , 2003 ; schroll et al . , 2006 ; alcaro et al . , 2007 ; schultz , 2007 ; claridge - chang et al . , 2009 ) and in setting the general level of arousal ( andretic et al . , 2005 ; kume et al . , 2005 ; monti and monti , 2007 ; krashes et al . , 2009 ) , as well as in reward responses across many phyla . there are now several examples of genes , gene pathways or signaling molecules that appear to have cyclic amp - dependent protein kinase - related proteins are involved in learning and memory across diverse vertebrate and invertebrate groups ( dubnau et al . , 2003 ; kandel , 2006 ) . cyclic gmp - protein kinases affect various form of foraging behavior across nematodes and arthropods ( fitzpatrick and sokolowski , 2004 ; toth and robinson , 2007 ) and serotonin has a role in aggression across vertebrates and invertebrates ( kravitz , 2000 ) . the roles of dopamine in reward responses across phyla is another example of what seems to be a general behavioral mechanism , but is this the product of conservation or convergent evolution ? when considering traits shared across phyla distinguishing between conservation and convergence is not simple . the difficulty is illustrated by considering the case of eye evolution ( fernald , 2006 ) . based on morphological evidence it was thought that vertebrate and invertebrate eyes evolved independently , and their similarities were the result of convergent evolution . but detailed molecular genetic analyses of the process of eye development have shown that eye development in vertebrate and invertebrates is organized by homologous gene families ( pichaud and desplan , 2002 ) . this has renewed debate over whether eyes have evolved repeatedly , or once from an ancestral light - sensitive structure . in the field of evolutionary development toolkit for development that is broadly conserved across diverse taxa ( carroll , 2005 ) . concept recognizes that common genomic elements can be involved in the development of different structures across phyla , even if the way the tools are used and the structures formed are very different between groups ( carroll , 2005 ) . similarly toth and robinson ( 2007 ) have argued that the toolkit concept can be extended to aid in understanding the evolution of different forms of behavior . a core toolkit of genes and signaling molecules could have been adapted and used in different ways as various complex forms of behavior evolved ( toth and robinson , 2007 ) . ancestrally biogenic amines may have functioned as signaling molecules in nervous systems released in response to environmental stimuli , but these simple behavioral elements have been adapted and modified in various ways as new and more complex behavioral responses to reward and punishment evolved . developmental and molecular evidence indicates that higher brain centers have evolved independently in different phyla ( farris , 2008 ) . here , higher brain centers are defined as multimodal areas that gather and integrate information from lower unimodal regions for integration of sensory information / associations , behavioral flexibility and cognitive behavior ( farris , 2008 ) . reward systems in arthropods and vertebrates both extensively involve higher brain centers ( hammer , 1993 ; hammer and menzel , 1998 ; schwaerzel et al . , 2003 ; roitman et al . , 2004 ; wise , 2004 ) . since the vertebrate cortex and insect mushroom bodies are structures that have evolved independently ( farris , 2008 ) brain reward systems almost certainly evolved independently in these groups . but in both cases the evolutionary process may have made use of a common molecular toolkit , which included the biogenic amines as signaling molecules . even the simplest motile animals change their behavior in response to the perception of stimuli they need to survive or reproduce , and most animals display active reward - seeking behavior . this is a major organizer and driver of animal behavior ( tinbergen , 1951 ) , and research with mammals has emphasized dopamine as a key neurochemical that modulates reinforcement , reward - seeking and reward learning ( wise and rompre , 1989 ; schultz et al . , 1993 ; berridge and robinson , 1998 ; schultz , 2007 ; berridge et al . , 2009 ) . effects of biogenic amines , especially dopamine , on behavioral responses to reward have been reported across diverse animal phyla , but the reported functions of the biogenic amines do differ between groups . in nematodes , dopamine is a modulator of motor neurons . by changing locomotor behavior in response to food stimuli dopamine , 2000 ; hills , 2006 ; rivard et al . , 2010 ) , and learned changes in locomotor behavior in response to food ( qin and wheeler , 2007 ) . in mollusks , dopamine not only modulates the motor neurons involved in feeding behavior , but also plays a role in reinforcement and reward learning ( lechner et al . , octopamine has been shown to be necessary for reward learning ( hammer and menzel , 1998 ; schwaerzel et al . , 2003 ; unoki et al . , 2005 ) , but dopamine signals also affect reward responses ( kim et al . , 2007 ; krashes et al . , 2009 ; selcho et al . , while the link between the biogenic amines and reward responses is clearly strong across diverse phyla , it is unlikely that this indicates a true homology of brain reward systems . it is possible that an ancestral role for the biogenic amines as modulators of motor circuits in response to environmental stimuli meant that these neurochemical systems were predisposed to be adapted in the course of evolution for more specialized functions in reward - seeking behavior and reward learning as higher levels of brain complexity evolved . 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 .

motile animals actively seek out and gather resources they find rewarding , and this is an extremely powerful organizer and motivator of animal behavior . mammalian studies have revealed interconnected neurobiological systems for reward learning , reward assessment , reinforcement and reward - seeking ; all involving the biogenic amine dopamine . the neurobiology of reward - seeking behavioral systems is less well understood in invertebrates , but in many diverse invertebrate groups , reward learning and responses to food rewards also involve dopamine . the obvious exceptions are the arthropods in which the chemically related biogenic amine octopamine has a greater effect on reward learning and reinforcement than dopamine . here we review the functions of these biogenic amines in behavioral responses to rewards in different animal groups , and discuss these findings in an evolutionary context .

Introduction Dopamine and Reward Responses in Nematoda, Platyhelminthes, Mollusca, and Vertebrata Roles of Dopamine and Octopamine in Reward Responses in Arthropoda Genetic Analyses of the Function of the Biogenic Amines in Reward and Aversive Learning in The Evolution of Brain Reward Systems: Inferences from Comparative Neurochemistry Conclusion Conflict of Interest Statement

most motile animals show some form of active foraging behavior to locate resources they need in their environment , and will actively avoid stimuli that are harmful to them . these basic behavioral responses have been used to provide a simple operational definition of whether stimuli are rewarding or punishing to an animal . in the field of animal behavior research , rewarding and punishing stimuli are often defined simply by the nature of the responses they elicit : rewards elicit approach behavior , whereas punishing stimuli elicit avoidance behavior ( skinner , 1938 ) . rewards also have a reinforcing property , in that almost all motile animals studied will learn to repeat actions that bring about ( or bring closer ) a rewarding outcome . given this operational definition of reward , it can be said that reward - seeking and reward learning are fundamental aspects of animal behavior . reward - seeking was recognized as fundamental to even the earliest explanatory models of behavior : sherrington ( 1906 ) , tinbergen ( 1951 ) , and lorenz ( 1965 ) all developed behavioral models that incorporated an assumed innate drive to seek rewards . exploring the neurobiology of reward responses and reward processing has long been a major focus of neuroscience research . understandably the vast majority of studies have considered humans and other mammals , while more recently , mechanisms of reward processing have been studied in a wider range of animal model systems . dopamine is a key modulator of this circuit , and of behavioral responses to rewards ( berridge and robinson , 1998 ; roitman et al . more recent comparative studies with diverse species scattered across a divergent range of animal phyla have also repeatedly demonstrated roles for the biogenic amines , and especially dopamine , in reward - seeking and reward learning . this raises the question of whether the function of dopamine in reward - seeking and learning has been broadly conserved across animal phyla , or whether this is a case of convergent evolution . we then discuss how phylum arthropoda appear to be an exception in that most studies have shown reward responses and reward learning to be affected by octopamine rather than dopamine . dopamine has been shown to affect responses to reward in extremely diverse animal groups , but the documented effects of dopamine vary . most studies with invertebrates have used food stimuli as rewards since these tend to elicit the most robust behavioral responses and learning . , caenorhabditis elegans ) possess one of the simplest animal nervous systems , but even in this animal , dopamine affects the expression of a form of food - seeking behavior . , 2010 ) , and dopamine modulation of the locomotor circuit also increased turning behavior ( hills et al . such behavioral responses to food are greater in starved worms than fed worms ( sawin et al . these changes in locomotor behavior in response to food are a very simple form of reward - seeking behavior . area - restricted search pattern of locomotion , which ensures that the animal explores and dwells longer in an area containing food ( hills et al . , 2004 ) , and the outcome of their change in crawling behavior is that they are more likely to locate food . it is striking that even this elementary form of reward - seeking behavior is modulated by dopamine . dopamine deficient cat-2 mutant worms , which lack the enzyme tyrosine - hydroxylase necessary for the biosynthesis of dopamine ( figure 1 ) , learned the maze task less well , and the memory did not persist as long as in wild - type animals ( qin and wheeler , 2007 ) . this suggests that dopamine affects memory systems involved in food - seeking behavior in nematodes . chemical relationships and biosynthetic pathways linking dopamine , tyramine , octopamine , and norepinephrine ( enzymes in italics ) . dopamine , tyramine , octopamine , and norepinephrine are all derived from tyrosine . this figure summarizes the most common synthesis pathways , but there are variations among the phyla . in some groups , octopamine is a trace amine and synthesized from tyramine by dopamine--hydroxylase , while in other phyla , norepinephrine is physiologically irrelevant and not present at any biologically meaningful level . they have no body cavities and a simple nervous system , but they do have rudimentary cephalization , and they can learn both classical and operant conditioning tasks ( best and rubinstein , 1962 ; shafer and corman , 1963 ) . kusayama and watanabe ( 2000 ) developed a conditioned - place - preference assay for d. japonica , and were able to induce in the flatworms a preference for an environment in which they had been treated with methamphetamine ( which increases extracellular biogenic amine levels ) . after conditioning , the number of biting responses to the lip tickle was increased ( lechner et al . , 2000 ) , and ( 2002 ) were able to replace electrical stimulation of the esophageal nerve with iontophoretic application of dopamine onto selected post - synaptic neurons to achieve effective training . this showed conclusively that dopamine is the neurochemical mediator of the reinforcement signal in operant conditioning . therefore evidence from both classical and operant conditioning studies in aplysia suggest a role for dopamine in reinforcement and reward learning . the chordata are deuterostomes , and a separate lineage from the protostome groups ( figure 2 ) . despite all this taxonomic diversity dopamine affects behavioral responses to reward and reinforcement in a similar manner . the affects of dopamine on mammalian reward responses have been well reviewed recently ( schultz , 2007 ) , and hence here we consider only the main findings as relevant to this comparative review . discussed phyla represent examples of protostome , deuterostome , and diploblastic groups , yet dopamine is a modulator in the motor systems of all these diverse phyla , and affects responses to rewarding or punishing stimuli in mollusks , platyhelminths , arthropods , nematodes , and chordates . dopamine has long been identified with motor function in mammals , but the first evidence linking dopamine to reward - seeking came from the observation that moderate dopamine receptor antagonist treatments attenuated the motivation to respond to a food reward before compromising the ability to respond ( wise and schwartz , 1981 ; wise , 2004 ) . dopamine - selective lesions of the nucleus accumbens can block responding to reward - associated stimuli ( taylor and robbins , 1986 ) , whereas amphetamine injection into the nucleus accumbens , to elevate extracellular dopamine in this area , enhances responding ( taylor and robbins , 1984 ) . recordings from midbrain dopamine neurons in mammals have shown strong responses to both primary rewards ( such as food and water ) , and also conditioned stimuli associated with rewards ( schultz , 1998 ; schultz , 2001 ) . most midbrain dopaminergic neurons projecting to the nucleus accumbens and frontal cortex are tonically active ( meaning a relatively constant baseline level of firing ) , but show phasic activation ( bursts of enhanced firing ) following primary food rewards , or stimuli learned to be predictive of reward ( schultz , 2000 , 2007 ) . , subsecond changes in the amount of dopamine released into the nucleus accumbens appear to directly modulate reward - seeking behavior ( roitman et al . together , these findings show that the phasic responses of dopamine neurons signal an assessment of the current value of reward stimuli , and that these dopamine signals directly modulate behavioral responses to rewards . so far , we have discussed examples from four phyla of highly diverse animals in which dopamine dominates reward learning and the reinforcing properties of rewards , but the arthropoda do not seem to fit this pattern . the arthropoda are ecdysozoan protostomes most closely related to nematoda ( figure 2 ) , but within this group evidence from both insects and crustaceans has shown that octopamine affects reward learning and behavioral responses to rewards ( hammer , 1997 ; hammer and menzel , 1998 ; schwaerzel et al . by contrast , in the arthropods , octopamine is a major regulator of behavior and physiology ( roeder et al . octopamine injection enhanced the exploratory response to food , whereas injection of two octopamine receptor antagonists reduced this response ( kaczer and maldonado , 2009 ) . diverse studies with honey bees ( apis mellifera , hymenoptera ) have shown that octopamine treatment affects behavioral responses to sucrose reward ( mercer and menzel , 1982 ; hammer and menzel , 1998 ; scheiner et al . , 2007 ) , and one of these ( vummx1 ) has been shown to mediate sucrose reinforcement ( hammer , 1993 ) . it is of interest to note that thoracic octopamine injection increased reflexive proboscis extension responsiveness to sucrose in an unconditioned paradigm in honey bees , whereas dopamine receptor agonist treatment reduced responsiveness ( scheiner et al . , have shown that treatments with octopamine receptor antagonists and agonists affected performance in reward learning assays , but treatment with dopamine receptor antagonists and agonists affected performance in aversive learning assays . as a result of these studies a commonly held view is that for the arthropods , octopamine and dopamine modulate different motivational systems with octopamine modulating appetitive learning and dopamine modulating aversive learning ( beggs et al . the arthropod studies described so far have relied heavily on pharmacological tools to manipulate biogenic amine systems . a difficulty with this approach is that the affinities of most of the available biogenic amine receptor agonists and antagonists to all the biogenic amine receptors in the different experimental insect species are incompletely known . consequently , it is difficult to experimentally manipulate a single receptor system in isolation or to be completely confident that nominated agonists or antagonists do not affect more than one biogenic amine system . currently , the only solution to this problem is to use several different antagonists or agonists against the same receptor system(s ) , and hopefully show the same behavioral effects ( unoki et al . , also , in many cases pharmacological treatments have been applied to the whole organism or to the whole brain , which has limited a circuit - level analysis of reinforcement systems in arthropods . in drosophila , several studies have used different genetic tools to manipulate all ( or most ) dopaminergic or octopaminergic neurons in the fly brain . the conclusions of these studies are consistent in that they have shown that dopamine is required for aversive learning , but not reward learning , and octopamine is required for reward learning but not aversive learning ( schwaerzel et al . , however , more recent studies have used more selective genetic manipulations to target specific dopamine receptors , or specific small groups of dopamine neurons . we first review studies that have manipulated all dopaminergic or octopaminergic neurons in the fly brain , and then studies that have selectively targeted specific populations of dopamine neurons , or dopamine receptor systems . ( 2003 ) explored the role of octopamine and dopamine in appetitive and aversive conditioning using strains of drosophila melanogaster in which the enzymes responsible for the synthesis of different biogenic amines were under the control of heat - shock sensitive promoters . flies in which the tyramine--hydroxylase ( figure1 ) gene had been knocked out could not synthesize octopamine ( monastirioti et al . , these flies performed normally in an aversive learning task associating electric shock with a novel odor , but did not learn to associate a sugar reward with an odor ( schwaerzel et al . at the restrictive temperature , flies performed poorly in the aversive learning paradigm , but normally in an appetitive learning paradigm ( schwaerzel et al . larvae learned to avoid an odor that had been paired with light activation of dopaminergic neurons , but they became attracted to odors paired with light activation of octopaminergic and tyraminergic neurons ( schroll et al . the inference is that activity of dopaminergic neurons mediates punishment , whereas activity of octopaminergic or tyraminergic neuron populations mediates the reinforcing properties of reward ( schroll et al . ( 2009 ) were able to optically activate populations of dopaminergic neurons in transgenic adult flies with a burst of laser light , by driving the expression of atp - gated p2x channels in dopaminergic neurons , and using laser light to trigger atp release from a previously microinjected caged precursor ( claridge - chang et al . the consistent message from the drosophila studies discussed so far is that for both larval and adult flies , octopamine is necessary for the learning of food reward , and dopamine is necessary for aversive learning . this is in agreement with the main findings from pharmacological studies performed with other arthropods ( unoki et al . , however , more targeted genetic manipulations of specific dopamine signals in drosophila suggest that this understanding of dopamine 's role in insects is an oversimplification . in the insect brain , the mushroom bodies are a protocerebral higher brain center known for their roles in olfactory processing and learning and memory ( farris , 2008 ) . the dopamine receptor dda1 ( a d1-like dopamine receptor that activates adenylyl cyclase ) is highly expressed in adult drosophila mushroom bodies , and also other regions of the brain . both dumb mutants completely failed to learn the association of an odor stimulus with electric shock , and also showed partial impairment of learning of an odor associated with sucrose reward ( kim et al . dnpf is an ortholog of mammalian neuropeptide y that regulates food - seeking in mammals ( tatemoto et al . therefore , a specific dopamine signal inhibits mushroom body neurons , and reduces the expression of appetitive memory . to conclude the discussion of the arthropods ; both pharmacological treatments and genetic manipulations of brain octopamine and dopamine systems have suggested different behavioral roles for octopamine and dopamine . , 2005 ; but new genetic studies that have manipulated specific dopamine signals have shown that in drosophila , different dopamine signals affect expression of learned responses to both rewarding and punishing stimuli ( kim et al . the arthropods are different from the other phyla discussed so far in that octopamine has been shown by most studies to play a dominant role in mediating reward responses and reward learning , but it now seems likely that in drosophila different dopamine signals affect expression of learned responses to both rewarding and punishing stimuli . in the mammalian brain , dopamine is most well known for its important role in the reward systems , but distinct mesolimbic dopamine signals mediate behavioral responses to aversive events and stress also ( ikemoto and panksepp , 1999 ; pruessner et al . , from a phylogenetic perspective , the link between dopamine and behavioral responses to reward is extremely broad ( figure 2 ) . a consideration of the general behavioral functions of the biogenic amines across animal phyla suggests that dopamine could have been predisposed to evolve functions in reward processing from an ancestral role as a signaling molecule modulating motor circuits in response to salient environmental stimuli . in one of the simplest metazoans , the nematode c. elegans , dopamine functions to modulate motor output and locomotor behavior , and is released in response to environmental stimuli that signal the local abundance of food ( hills et al . , as far as we know , dopamine modulation of motor circuits has been reported for every animal phylum in which it has been investigated : nematoda ( sawin et al . the cnidaria represent perhaps the simplest animal nervous systems , and molecular and morphological evidence places the cnidaria as basal among metazoans ( mallatt et al . in the cnidarian hydra japonica , dopamine affects the extent of mouth opening in response to food stimuli ( hanai and kitajima , 1984 ) . it seems likely that modulation of motor circuits in response to environmental stimuli could be one of the ancestral functions of dopamine as a signaling molecule in simple nervous systems . from this proposed ancestral role , different biogenic amine systems could have evolved progressively more specialized functions in behavioral responses to rewarding or aversive stimuli as increasing levels of behavioral complexity evolved along with the evolution of more complex nervous systems ( hills , 2006 ) . this hypothesis would explain why dopamine and other biogenic amines have roles in aversive responses ( schwaerzel et al . there are now several examples of genes , gene pathways or signaling molecules that appear to have cyclic amp - dependent protein kinase - related proteins are involved in learning and memory across diverse vertebrate and invertebrate groups ( dubnau et al . cyclic gmp - protein kinases affect various form of foraging behavior across nematodes and arthropods ( fitzpatrick and sokolowski , 2004 ; toth and robinson , 2007 ) and serotonin has a role in aggression across vertebrates and invertebrates ( kravitz , 2000 ) . the roles of dopamine in reward responses across phyla is another example of what seems to be a general behavioral mechanism , but is this the product of conservation or convergent evolution ? based on morphological evidence it was thought that vertebrate and invertebrate eyes evolved independently , and their similarities were the result of convergent evolution . a core toolkit of genes and signaling molecules could have been adapted and used in different ways as various complex forms of behavior evolved ( toth and robinson , 2007 ) . ancestrally biogenic amines may have functioned as signaling molecules in nervous systems released in response to environmental stimuli , but these simple behavioral elements have been adapted and modified in various ways as new and more complex behavioral responses to reward and punishment evolved . developmental and molecular evidence indicates that higher brain centers have evolved independently in different phyla ( farris , 2008 ) . since the vertebrate cortex and insect mushroom bodies are structures that have evolved independently ( farris , 2008 ) brain reward systems almost certainly evolved independently in these groups . but in both cases the evolutionary process may have made use of a common molecular toolkit , which included the biogenic amines as signaling molecules . even the simplest motile animals change their behavior in response to the perception of stimuli they need to survive or reproduce , and most animals display active reward - seeking behavior . this is a major organizer and driver of animal behavior ( tinbergen , 1951 ) , and research with mammals has emphasized dopamine as a key neurochemical that modulates reinforcement , reward - seeking and reward learning ( wise and rompre , 1989 ; schultz et al . , 2009 ) . effects of biogenic amines , especially dopamine , on behavioral responses to reward have been reported across diverse animal phyla , but the reported functions of the biogenic amines do differ between groups . by changing locomotor behavior in response to food stimuli dopamine , 2000 ; hills , 2006 ; rivard et al . , 2010 ) , and learned changes in locomotor behavior in response to food ( qin and wheeler , 2007 ) . in mollusks , dopamine not only modulates the motor neurons involved in feeding behavior , but also plays a role in reinforcement and reward learning ( lechner et al . , octopamine has been shown to be necessary for reward learning ( hammer and menzel , 1998 ; schwaerzel et al . , 2005 ) , but dopamine signals also affect reward responses ( kim et al . , while the link between the biogenic amines and reward responses is clearly strong across diverse phyla , it is unlikely that this indicates a true homology of brain reward systems . it is possible that an ancestral role for the biogenic amines as modulators of motor circuits in response to environmental stimuli meant that these neurochemical systems were predisposed to be adapted in the course of evolution for more specialized functions in reward - seeking behavior and reward learning as higher levels of brain complexity evolved .

the dedifferentiation and proliferation / apoptosis of smooth muscle cells ( smcs ) in the arterial intima represent one of the changes found in early atherosclerotic lesions , when the disease is still reversible [ 13 ] . cultured smcs from an atherosclerotic plaque showed a greater susceptibility to apoptosis that did not vary with subculture . this finding suggests that stable and cell - intrinsic changes in the expression of proapoptotic or antiapoptotic genes exert a greater control over apoptosis in smcs than do cell - cell interactions or the microenvironment in the plaque , indicating that this susceptibility develops in initial stages of the disease . if the sum of signals gives apoptosis as a result , the whole protein machinery for destruction is unleashed . the bcl-2 protein family also regulates the apoptosis pathways to influence cell survival ; this family forms heterodimers between apoptosis - inhibiting proteins such as bcl-2 , bcl - xl , and a1 , and inducing proteins such as bax , bad , bid , regulating cell survival . the ratio of anti- to proapoptotic proteins determines susceptibility to apoptosis , especially the bcl-2/bax ratio . smcs express low levels of bcl-2 in vitro and in vivo [ 4 , 7 ] . although smcs in an atherosclerotic process show no major changes in bcl-2 expression , the balance between antiapoptotic and proapoptotic proteins can change in favor of the latter and trigger apoptosis , mainly mediated by bax [ 8 , 9 ] . the most numerous cholesterol oxides found in human atheromatous plaque are 7-hydroxycholesterol and 7-ketocholesterol , while 7-ketocholesterol , and 25-hydroxycholesterol are the most abundant in the aortic wall of experimental animals after a cholesterol - rich diet and are the most effective to induce cell death , especially 25-hydroxycholesterol . thus , chickens have been reported to readily develop hypercholesterolemia with a cholesterol - rich diet , producing atherosclerotic lesions in some cases [ 1416 ] . preliminary studies demonstrated that a 20-day diet enriched with 5% cholesterol is sufficient to cause hypercholesterolemia and gives rise to lipid deposits in the main arteries that are similar to the fatty streaks observed in the first stages of atherosclerosis . because the presence of apoptosis in atherosclerotic lesions can have a major impact on the progression of the disease , characterization of oxysteroid - induced cell death is important to understand the development of atherosclerosis , since it is not clear how cholesterol and its oxides induce apoptosis in smcs . therefore , the objective of this study was to investigate the apoptotic pathways induced by oxysterols , using a cell model in which smcs were exposed to atherogenic factors ( cholesterol - rich diet ) or antiatherogenic factors ( fish oil - rich diet ) . these cells were then exposed in vitro to 25-hydroxycholesterol , studying levels of apoptosis and apoptotic proteins bcl-2 , bcl - xl , and bax and the expression of genes that encode the proteins involved in apoptosis , bcl-2 , bcl - xl . the protocol of this study was approved by the animal laboratory service of the university of granada ( spain ) and chickens received humane treatment according to the regulations for animal research of the european union . newborn white leghorn male chicks ( gallus domesticus ) , supplied by the animal laboratory service of the university of granada , were kept in a chamber with a light cycle from 09.00 to 21.00 hours and controlled temperature of 2931c and allowed free access to food and water . the diet was started at hatching and kept on until the chicks were killed ( 20 days after ) . none of the chicks died a natural death during the treatment nor developed any illness . three groups of chicks twenty days old were used , the control diet group ( c - group ) was kept on a standard diet ( sanders a-00 ) while treated group ( ch - group ) was fed on the same diet supplemented with 5% w / w powdered cholesterol mixed homogeneously ( panreac reagent barcelona , pure grade ) . the third group ( ch - fo - group ) was fed for 10 days with a diet of 5% cholesterol and then cholesterol diet was withdrawn and the standard diet was supplemented with 10% of fish ( menhaden ) oil for 10 days more . standard diet free of cholesterol contained ( w / w ) 42% carbohydrate ( mainly starch ) , 3.5% fat , and 20.5% protein . experimental diets were obtained by supplementation to de standard diet with 5% of cholesterol ( ch diet ) and 10% fish ( menhaden ) oil ( fo diet ) . no significant differences were observed in fatty acid composition of each diet during the experiments . animals did not suffer at all at any stage of the experiment because our laboratory usually uses the anesthetic ketamine ( 60 mg / kg of body weight ) and sodium pentobarbital ( 50 mg / kg of body weight ) according to the regulations of the animal research of the european union . the research staff had the preparation and the certification to do experimental work with animals . smcs were isolated from the aortic arch of the chicks as described elsewhere with slight modifications and cultured in dulbecco 's modification of eagle 's medium ( dmem ) supplemented with d - glucose ( 4.5 g / l ) , l - glutamate ( 0.584 g / ml ) , antibiotic cocktail composed of penicillin ( 100 g / ml ) and amphotericin ( 0.25 g / ml ) ( sigma - aldrich , inc . ) , as well as 10% ( v / v ) fetal bovine serum ( fbs ) . medium was buffered with bicarbonate and cultures were kept at 37c in humidified atmosphere of 95% air and 5% co2 . secondary cultures were initiated after either low or high passages using 0.05%/0.02% trypsin - edta solution . cells were determined to be vascular smcs by their hill - and - valley configuration at confluence and positive fluorescence staining for smooth muscle actin and myosin . smcs were plated in 96-well plates at a density of 25000 cells / well . after adhering during overnight culture , cells were treated for 24 and 48 hours with 25-hydroxycholesterol ( 540 g / ml ) dissolved in absolute ethanol . the final concentration of ethanol in the culture medium never exceeded 0.8% and no effect on culture was observed at or below this concentration . an amount of this solution equal to 10% of the culture medium volume was added to cell cultures . the formazan crystals were solubilized by adding 200 l l of solubilization solution ( 0.05 n hcl in isopropanol ) . metabolic activity was quantified by subtracting light absorbance at 630 nm from absorbance at 570 nm . total rna was isolated with tri - reagent / trizol ( invitrogen , ltd , uk ) . single - stranded cdna was synthesized from 4 g total rna using an oligo(dt)1218 as primer and powerscript reverse transcriptase ( clontech laboratories , inc . , real - time pcr was performed with the fast start dna master sybr green i kit ( roche ) and light cycler system ( roche ) . for the light cycler reaction , a master mix of the following reaction components was prepared to the indicated final concentration : 12.6 l l h2o , 2.4 l l mgcl2 ( 4 mm ) , 1 l l forward primer ( 0.5 m ) , 1 l l reverse primer ( 0.5 m ) , and 2.0 l l of the fast start dna master sybr green i mix ( roche ) . the primer sequences used in this study are given in table 2 and optimized at an annealing temperature of 55c . the cdna of the genes studied in the different samples ( treated in vivo and in vitro ) were diluted 1 : 100 and amplified to obtain the cp value for each sample . light cycler products of the different gene expressions were analyzed by agarose gel electrophoresis and a light cycler melting curve was constructed to test for a single product at the end of each pcr reaction . a mathematical model developed by pfaffl was used for the relative quantification of bcl-2 , bcl - xl , c - myc , and p53 mrna expression in real - time pcr with respect to the reference -actin gene transcript . the efficiency of the assay for each studied gene was -actin : 1.86 ; bcl-2 : 1.89 ; bcl - x : 1.9 ; c - myc : 1.91 ; p53 : 1.81 . cell monolayer ( 1 10 cells ) was disrupted by incubation with ripa buffer ( 1 pbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , 0.1% sds , 0.1 mg / ml pmsf , 2 g / ml aprotinin , 0.18 mg / ml sodium orthovanadate ) . protein concentration was determined by using the micro - bca procedure ( bio - rad laboratories , inc . , calif proteins were denatured in 1 sample buffer with 5% 2-mercaptoethanol at 95 for 10 minutes . total cell lysate was separated under reducing conditions by 12% sds - polyacrylamide gel electrophoresis ( mini protean ii , bio - rad ) . gel - resolved proteins were then transferred onto a polyvinylidene difluoride membrane ( pvdf ) using a mini trans - blot cell apparatus ( bio - rad ) . membranes were blocked for 1 hour at room temperature in blocking reagent ( 5% milk power , 0,05% tween-20 in tbs or pbs , ph 7.4 ) and probed at 4c overnight with the primary antibody : anti - bcl - xl ( cell signaling technology , inc . , mass , usa ) 1 : 1000 , anti - bax 1 : 100 ( santa cruz biotechnology , inc . , santa cruz , calif , usa ) , and anti - bcl-2 1 : 500 ( bd biosciences , calis , usa ) . the immunocomplex was detected with the ecl - plus kit ( amersham , buckinghamshire , uk ) , and the band density was analyzed using quantiscan software ( biosoft , cambridge , uk ) . data were analyzed by student 's t - test when the variability was the same in each group , because the t - test assumes that standard deviations of two datasets are equal . a difference between groups was considered significant when the p - value was .05 . to evaluate the cytotoxicity of 25-hidroxycholesterol on smc - c , smc - ch , and smc - ch - fo cultures , cells in third passage were incubated for 24 hours in 25-hidroxycholesterol at concentrations of 0 , 5 , 10 , 20 , 40 , and 80 g / ml , were used . the 25-hidroxycholesterol was previously dissolved in the ethanol and was diluted in cultures in order to obtain the concentrations of previous experiments . the concentration of ethanol in cultures never exceeded 0.8% , wich previously verified that it was not toxic for the cells . as figure 1 shows , the citotoxicity of 25-hidroxycholesterol during 24 hours is the same in the three types of cultures . even then , a slightly greater viability was observed in the smc - ch - fo cultures with respect to the control . with a concentration of 20 g / ml expression of proteins of the bcl-2 family of oncogenes ( bax , bcl - xl , and bcl-2 ) was studied ( figure 2 ) . because of the homodimerization and heterodimerization capacity of bax , bcl - xl , and bcl-2 , the relationship between expression levels of bcl-2/bax and bcl - xl / bax determines whether the cells will undergo apoptosis or not after an apoptotic stimulus . table 3 shows the antiapoptotic / proapoptotic ratios at baseline ( after in vivo treatments ) and after 25-hydroxycholesterol in vitro treatment . at baseline , expression of bcl-2 and bax ( figure 2 ) was similar among the three culture types , but expression of bcl - xl was higher in smc - ch and smc - ch - fo than in smc - c . hence , the bcl-2/bax ratios were very similar among cultures , whereas the bcl - xl / bax ( table 3 ) was significantly higher in smc - ch - fo . the smc - ch - fo ( table 3 ) showed a highly significant increase in the bcl - xl / bax ratio but a similar bcl-2/bax ratio to that of the control cells , indicating some resistance to apoptosis . therefore , the replacement of a cholesterol - rich diet with a dish oil - rich diet not only reverses the effects of cholesterol but also protects the smcs from apoptotic stimuli . the addition of 25-hydroxycholesterol produced a highly significant increase in bax levels in all three culture types ( figure 2 ) but no change in bcl - xl , except for a nonsignificant increase in the smc - c . moreover , a large reduction in the bcl - xl / bax ratio was observed for the smc - c and an intermediate reduction for the smc - ch - fo ( table 3 ) . we also studied the expression of genes that encode proteins implicated in apoptosis ( bcl-2 , bcl - xl ) . at baseline ( after in vivo experiments ) , the amount of mrna of bcl-2 gene remained unchanged in the smc - c and smc - ch ( figure 3 ) . however , the smc - ch - fo showed a slight increase in bcl-2 ( p < .05 ) . no major differences were observed in baseline expression of the bcl - xl gene ( figure 4 ) in the smc - c , smc - ch , and smc - ch - fo cells , as also found for bcl-2 and protein levels . incubation of the cultures with 25-hydroxycholesterol increased the mrna of bcl-2 ( p < .001 ) to a greater degree in smc - ch than in smc - c ( p < .05 ) , see figure 3 . moreover , the expression of bcl - xl markedly increased in the three culture types after the addition of 25-hydroxycholesterol ( p < .001 ) , see figure 4 . hyperlipemia and similar conditions , in particular , high cholesterol , change the expression of genes in endothelial cells and smcs , inducing atherosclerotic lesions in which proliferation and apoptosis are both present [ 1 , 8 , 21 ] . the dedifferentiation and proliferation / apoptosis of smcs in the arterial intima represent one of the changes found in early atherosclerotic lesions , when the disease is still reversible [ 2 , 3 ] . in light of this , it is predictable that smc culture models from animals fed with fish oil diet could produce some reversal of the changes induced by cholesterol in apoptotic proteins bcl-2 , bcl - xl , and bax and the expression of bcl-2 and bcl - xl genes [ 22 , 23 ] . the smc culture model generated in our laboratory by isolating the cells from control and cholesterol - fed chicks produces changes in smcs that make them more susceptible to 25-hydroxycholesterol - mediated apoptosis . replacement of a cholesterol - rich diet with a fish oil - rich diet produces some reversal of the cholesterol - induced changes , increasing the resistance of smcs to apoptosis . the very low level of apoptosis showed in smcs is similar to the 4% death rate usually observed in cultured cells . these data are in agreement with previous reports that cultured smc from the arterial media , even those from an atherosclerotic plaque , showing no apoptosis in culture , unlike observations in smc from the intima . in early atherosclerosis , the cells in the arterial media that will proliferate and migrate , modulating their phenotype , are in some way protected against apoptosis . in our experimental cell model , bcl-2 expression was very low in smcs from chicks after a 20-day calorie - rich diet , whereas bax expression showed virtually no change . these results are consistent with previous reports that bcl-2 is undetectable in most smcs from atherosclerotic plaque or healthy arteries [ 25 , 26 ] , explaining its poorly defined role , and that the expression of bax does not differ between smcs with synthetic and contractile phenotypes . however , other studies suggested that bcl-2 is expressed in smcs with contractile phenotype but not in those with synthetic phenotype . it has , therefore , been postulated that bcl-2 may be repressed throughout the modulation from contractile to synthetic phenotype . this modulation in smcs of the arterial intima is observed in early atherosclerotic lesions , when the disease is still reversible . cells in fatty streaks have lipid deposits in small vacuoles in the cytoplasm [ 2729 ] . they subsequently become so - called foam cells [ 30 , 31 ] , and both cell types show a high expression of bax . therefore , although the increase in bax expression in our cells did not reach significance , it suggests that the lesion may still be in an early and reversible state , as are initial adaptive streaks in which bax is not detectable . in contrast , the bcl - xl / bax ratio increased in the smc - ch . the expression of bcl - xl has been reported in smcs from atherosclerotic plaques , and is believed to play the main role in preventing apoptosis in atherosclerosis . hence , it is thought to act largely in initial stages of atherosclerosis , in which proliferation must predominate . in fact , an increase in bcl - xl was observed in smc from the intima in early atherosclerotic lesions . other members of the bcl-2 family , for example , bak , are abundantly expressed in smcs from atherosclerotic plaques and tend to heterodimerize with bcl - xl . oxysterols , especially 25-hydroxycholesterol , can reduce the expression of bcl-2 y bcl - xl [ 3436 ] , activate bad , and increase the expression of genes that encode bh3-only proapoptotics , for example , bim , which in turn activates bax / bak . these actions appear to be mediated by the inhibition of akt survival kinases . beside the protein levels , we also studied the expression of genes of bcl-2 family that encode proteins implicated in apoptosis ( bcl-2 , bcl - xl ) , the amount of mrna of bcl-2 gene ( prototype of antiapoptotic genes ) remained unchanged in the smc - c and smc - ch , consistent with previous findings of no modifications in the expression of bcl-2 during atherosclerotic alterations [ 9 , 25 , 26 ] . however , the smc - ch - fo showed a slight increase in bcl-2 , which may imply some resistance to apoptotic stimuli . in fact , many genes involved in the response to oxidative stress , for example , nf-b survival factor genes , are activated by a fish oil - rich diet and are also involved in the regulation of bcl-2 [ 3840 ] . no major differences observed in baseline expression of the bcl - xl gene in the smc - c , smc - ch and smc - ch - fo cells , as also found for bcl-2 and protein levels . the bcl - xl gene is thought to play a critical role in the prevention of cell death in atherosclerotic plaques , and a reduction in bcl - xl expression levels may be a prerequisite for apoptotic induction . it has also been reported that the inhibition of nf-b ( activator of cell survival genes reduces the expression of bcl - xl , which makes the cells more susceptible to apoptosis ) . the increase in the mrna of bcl-2 to a greater degree in smc - ch than in smc - c may appear contradictory , since bcl-2 protein levels are known to be reduced by oxysterol treatment of the cells . however , this reduction is largely produced by dimerization of this protein with bax , which increases in the presence of oxysterols , as demonstrated by the results of our protein studies . the marked increase of the expression of bcl - xl in the three culture types after the addition of 25-hydroxycholesterol again appears contradictory because bcl - xl protein levels are reduced , which may be explained by the dimerization of bcl - xl with bax and especially with bak . the aortic arches of male chick were chosen for this study for two reasons : first , for the ability of chicks to absorb high amount of dietary cholesterol [ 14 , 42 ] ; and second , because intimal thickening in the aortas from these hypercholesterolemic chicks can be detected after only 20 days [ 43 , 44 ] . this short diet period is very convenient and contrasts with those reported for other experimental animal models , mainly non - human primates and rabbits , which must be a cholesterol - containing diet for between 1 and 18 months to obtain an atherosclerotic lesion . the short time of the chicken model is comparable with that necessary to induce mitotic activity in aortas of white carneau pigeons or swine . serum cholesterol levels observed in 10-day - old control chicks are normal because newly hatchet chicken reabsorb the yolk during the first week of live , thus developing hypercholesterolemia , which becomes almost three times higher in cholesterol - fed chicks . the significantly elevated cholesterol and triglyceride plasma levels are in accordance with previously reported plasma lipids levels . avian models were used in trials of atherosclerosis , and , in fact , cholesterol feeding has been described elsewhere as producing atherosclerotic lesions in fowl species . we have also used chicks previously to study lipids metabolism after cholesterol administration and to develop a cell - culture avian model to look into , in vitro , the effect of a cholesterol diet , in vivo , on the transformation of smc . thus , in our laboratory , we isolated smcs from cholesterol - fed chicks ( smc - ch cultures ) which are very proliferative in culture compared with smc isolated from control - fed chicks ( smc - c cultures ) , and with identical maintenance in culture of the two lines of smc . in this way , dna synthesis in the s phase was 4-fold higher and after 20 days of culture , smc - ch increased their cholesterol content to double that of smc - c , giving smc - ch cultures under conditions mimicking such a cholesterol diet and a very early atherosclerosis in vivo / in vitro model at the smc level . we have studied the cholesterol synthesis and hmg - coa reductase gene expression in these cultures , showing great differences between smc - c and smc - ch [ 50 , 51 ] . also we demonstrated the existence of cyclic fluctuations of hmg - coa reductase activity in nonsynchronized smc cultures not correlated to the cultured feeding and not with the increase of mrna , suggesting the posttranscriptional modulation of the hmg - coa reductase and the relationships between hmg - coa reductase activity and cell division . finally , we have examined the morphological , molecular , and proliferation change in arterial smc mimicking such a cholesterol diet . consequentially , this transformed smc is a good model to study the alterations of the differentiated state of smc , caused not only by cholesterol - rich diet but also by fish oil - rich diet as well as the apoptotic pathway induced by oxysteroid in smcs . in conclusion , we investigated the apoptotic pathway induced by oxysteroid in smcs isolated from male chicks exposed to a control diet ( smc - c ) and an atherogenic cholesterol - rich diet ( smc - ch ) or an antiatherogenic fish oil - rich diet ( smc - ch - fo ) . we found that 25-hydroxycholesterol - induced apoptosis in smcs , mediated by a high increase in bax protein and bax gene expression . these changes were more evident in smc isolated from chicks exposed to an atherogenic than to an antiatherogenic one . thus , we deducted that the replacement of a cholesterol - rich diet with a fish - oil rich diet might produce some reversal of cholesterol - induced changes in oxysterol - activated apoptotic pathway , making smc more resistant to apoptosis . moreover , high cholesterol diet and fish - oil rich diet in the animals , before obtaining the smcs cultures , induced changes in the apoptotic gene expression studied . thus , the mrna concentration was downregulated in smc - ch - fo versus smc - ch . since we reported previously that , the nutritional culture conditions , the intracellular cholesterol concentration , and the ultrastructural morphology were the same during the first days of cultures ; the change in gene expression must have been induced by the cholesterol or fish oil diet in the aortic smc in vivo . in this way , this in vivo / in vitro model is relevant to study the nutritional control and gene regulation of the differentiation and apoptosis in smc . new studies would be necessary to demonstrate the implication of the cell signalling pathways , as well as to show if new protein synthesis is required for the bax increase or if some peaks of gene expression correlate with other regulatory events .

smooth muscle cells ( smcs ) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis . recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease . the present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in smcs cultures , using an in vivo / in vitro cell model in which smcs were isolated and culture from chicken exposed to an atherogenic cholesterol - rich diet ( smc - ch ) and/or an antiatherogenic fish oil - rich diet ( smc - ch - fo ) . cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins bcl-2 , bcl - xl and bax and the expression of bcl-2 and bcl - xl , genes . the quantitative real - time reverse transcriptase - polymerase chain reaction and the immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in smcs , mediated by a high increase in bax protein and bax gene expression . these changes were more marked in smc - ch than in smc - ch - fo , indicating that dietary cholesterol produces changes in smcs that make them more susceptible to 25-hydroxycholesterol - mediated apoptosis . our results suggest that the replacement of a cholesterol - rich diet with a fish oil - rich diet produces some reversal of cholesterol - induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in smcs cultures , making smcs more resistant to apoptosis .

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

the dedifferentiation and proliferation / apoptosis of smooth muscle cells ( smcs ) in the arterial intima represent one of the changes found in early atherosclerotic lesions , when the disease is still reversible [ 13 ] . this finding suggests that stable and cell - intrinsic changes in the expression of proapoptotic or antiapoptotic genes exert a greater control over apoptosis in smcs than do cell - cell interactions or the microenvironment in the plaque , indicating that this susceptibility develops in initial stages of the disease . the bcl-2 protein family also regulates the apoptosis pathways to influence cell survival ; this family forms heterodimers between apoptosis - inhibiting proteins such as bcl-2 , bcl - xl , and a1 , and inducing proteins such as bax , bad , bid , regulating cell survival . smcs express low levels of bcl-2 in vitro and in vivo [ 4 , 7 ] . although smcs in an atherosclerotic process show no major changes in bcl-2 expression , the balance between antiapoptotic and proapoptotic proteins can change in favor of the latter and trigger apoptosis , mainly mediated by bax [ 8 , 9 ] . the most numerous cholesterol oxides found in human atheromatous plaque are 7-hydroxycholesterol and 7-ketocholesterol , while 7-ketocholesterol , and 25-hydroxycholesterol are the most abundant in the aortic wall of experimental animals after a cholesterol - rich diet and are the most effective to induce cell death , especially 25-hydroxycholesterol . thus , chickens have been reported to readily develop hypercholesterolemia with a cholesterol - rich diet , producing atherosclerotic lesions in some cases [ 1416 ] . because the presence of apoptosis in atherosclerotic lesions can have a major impact on the progression of the disease , characterization of oxysteroid - induced cell death is important to understand the development of atherosclerosis , since it is not clear how cholesterol and its oxides induce apoptosis in smcs . therefore , the objective of this study was to investigate the apoptotic pathways induced by oxysterols , using a cell model in which smcs were exposed to atherogenic factors ( cholesterol - rich diet ) or antiatherogenic factors ( fish oil - rich diet ) . these cells were then exposed in vitro to 25-hydroxycholesterol , studying levels of apoptosis and apoptotic proteins bcl-2 , bcl - xl , and bax and the expression of genes that encode the proteins involved in apoptosis , bcl-2 , bcl - xl . newborn white leghorn male chicks ( gallus domesticus ) , supplied by the animal laboratory service of the university of granada , were kept in a chamber with a light cycle from 09.00 to 21.00 hours and controlled temperature of 2931c and allowed free access to food and water . three groups of chicks twenty days old were used , the control diet group ( c - group ) was kept on a standard diet ( sanders a-00 ) while treated group ( ch - group ) was fed on the same diet supplemented with 5% w / w powdered cholesterol mixed homogeneously ( panreac reagent barcelona , pure grade ) . the third group ( ch - fo - group ) was fed for 10 days with a diet of 5% cholesterol and then cholesterol diet was withdrawn and the standard diet was supplemented with 10% of fish ( menhaden ) oil for 10 days more . standard diet free of cholesterol contained ( w / w ) 42% carbohydrate ( mainly starch ) , 3.5% fat , and 20.5% protein . experimental diets were obtained by supplementation to de standard diet with 5% of cholesterol ( ch diet ) and 10% fish ( menhaden ) oil ( fo diet ) . smcs were isolated from the aortic arch of the chicks as described elsewhere with slight modifications and cultured in dulbecco 's modification of eagle 's medium ( dmem ) supplemented with d - glucose ( 4.5 g / l ) , l - glutamate ( 0.584 g / ml ) , antibiotic cocktail composed of penicillin ( 100 g / ml ) and amphotericin ( 0.25 g / ml ) ( sigma - aldrich , inc . ) cells were determined to be vascular smcs by their hill - and - valley configuration at confluence and positive fluorescence staining for smooth muscle actin and myosin . the final concentration of ethanol in the culture medium never exceeded 0.8% and no effect on culture was observed at or below this concentration . single - stranded cdna was synthesized from 4 g total rna using an oligo(dt)1218 as primer and powerscript reverse transcriptase ( clontech laboratories , inc . , real - time pcr was performed with the fast start dna master sybr green i kit ( roche ) and light cycler system ( roche ) . the cdna of the genes studied in the different samples ( treated in vivo and in vitro ) were diluted 1 : 100 and amplified to obtain the cp value for each sample . a mathematical model developed by pfaffl was used for the relative quantification of bcl-2 , bcl - xl , c - myc , and p53 mrna expression in real - time pcr with respect to the reference -actin gene transcript . the efficiency of the assay for each studied gene was -actin : 1.86 ; bcl-2 : 1.89 ; bcl - x : 1.9 ; c - myc : 1.91 ; p53 : 1.81 . membranes were blocked for 1 hour at room temperature in blocking reagent ( 5% milk power , 0,05% tween-20 in tbs or pbs , ph 7.4 ) and probed at 4c overnight with the primary antibody : anti - bcl - xl ( cell signaling technology , inc . the immunocomplex was detected with the ecl - plus kit ( amersham , buckinghamshire , uk ) , and the band density was analyzed using quantiscan software ( biosoft , cambridge , uk ) . to evaluate the cytotoxicity of 25-hidroxycholesterol on smc - c , smc - ch , and smc - ch - fo cultures , cells in third passage were incubated for 24 hours in 25-hidroxycholesterol at concentrations of 0 , 5 , 10 , 20 , 40 , and 80 g / ml , were used . as figure 1 shows , the citotoxicity of 25-hidroxycholesterol during 24 hours is the same in the three types of cultures . even then , a slightly greater viability was observed in the smc - ch - fo cultures with respect to the control . with a concentration of 20 g / ml expression of proteins of the bcl-2 family of oncogenes ( bax , bcl - xl , and bcl-2 ) was studied ( figure 2 ) . because of the homodimerization and heterodimerization capacity of bax , bcl - xl , and bcl-2 , the relationship between expression levels of bcl-2/bax and bcl - xl / bax determines whether the cells will undergo apoptosis or not after an apoptotic stimulus . table 3 shows the antiapoptotic / proapoptotic ratios at baseline ( after in vivo treatments ) and after 25-hydroxycholesterol in vitro treatment . at baseline , expression of bcl-2 and bax ( figure 2 ) was similar among the three culture types , but expression of bcl - xl was higher in smc - ch and smc - ch - fo than in smc - c . hence , the bcl-2/bax ratios were very similar among cultures , whereas the bcl - xl / bax ( table 3 ) was significantly higher in smc - ch - fo . the smc - ch - fo ( table 3 ) showed a highly significant increase in the bcl - xl / bax ratio but a similar bcl-2/bax ratio to that of the control cells , indicating some resistance to apoptosis . therefore , the replacement of a cholesterol - rich diet with a dish oil - rich diet not only reverses the effects of cholesterol but also protects the smcs from apoptotic stimuli . the addition of 25-hydroxycholesterol produced a highly significant increase in bax levels in all three culture types ( figure 2 ) but no change in bcl - xl , except for a nonsignificant increase in the smc - c . moreover , a large reduction in the bcl - xl / bax ratio was observed for the smc - c and an intermediate reduction for the smc - ch - fo ( table 3 ) . we also studied the expression of genes that encode proteins implicated in apoptosis ( bcl-2 , bcl - xl ) . at baseline ( after in vivo experiments ) , the amount of mrna of bcl-2 gene remained unchanged in the smc - c and smc - ch ( figure 3 ) . however , the smc - ch - fo showed a slight increase in bcl-2 ( p < .05 ) . no major differences were observed in baseline expression of the bcl - xl gene ( figure 4 ) in the smc - c , smc - ch , and smc - ch - fo cells , as also found for bcl-2 and protein levels . incubation of the cultures with 25-hydroxycholesterol increased the mrna of bcl-2 ( p < .001 ) to a greater degree in smc - ch than in smc - c ( p < .05 ) , see figure 3 . moreover , the expression of bcl - xl markedly increased in the three culture types after the addition of 25-hydroxycholesterol ( p < .001 ) , see figure 4 . hyperlipemia and similar conditions , in particular , high cholesterol , change the expression of genes in endothelial cells and smcs , inducing atherosclerotic lesions in which proliferation and apoptosis are both present [ 1 , 8 , 21 ] . in light of this , it is predictable that smc culture models from animals fed with fish oil diet could produce some reversal of the changes induced by cholesterol in apoptotic proteins bcl-2 , bcl - xl , and bax and the expression of bcl-2 and bcl - xl genes [ 22 , 23 ] . the smc culture model generated in our laboratory by isolating the cells from control and cholesterol - fed chicks produces changes in smcs that make them more susceptible to 25-hydroxycholesterol - mediated apoptosis . replacement of a cholesterol - rich diet with a fish oil - rich diet produces some reversal of the cholesterol - induced changes , increasing the resistance of smcs to apoptosis . the very low level of apoptosis showed in smcs is similar to the 4% death rate usually observed in cultured cells . these data are in agreement with previous reports that cultured smc from the arterial media , even those from an atherosclerotic plaque , showing no apoptosis in culture , unlike observations in smc from the intima . in early atherosclerosis , the cells in the arterial media that will proliferate and migrate , modulating their phenotype , are in some way protected against apoptosis . in our experimental cell model , bcl-2 expression was very low in smcs from chicks after a 20-day calorie - rich diet , whereas bax expression showed virtually no change . these results are consistent with previous reports that bcl-2 is undetectable in most smcs from atherosclerotic plaque or healthy arteries [ 25 , 26 ] , explaining its poorly defined role , and that the expression of bax does not differ between smcs with synthetic and contractile phenotypes . this modulation in smcs of the arterial intima is observed in early atherosclerotic lesions , when the disease is still reversible . they subsequently become so - called foam cells [ 30 , 31 ] , and both cell types show a high expression of bax . therefore , although the increase in bax expression in our cells did not reach significance , it suggests that the lesion may still be in an early and reversible state , as are initial adaptive streaks in which bax is not detectable . in contrast , the bcl - xl / bax ratio increased in the smc - ch . the expression of bcl - xl has been reported in smcs from atherosclerotic plaques , and is believed to play the main role in preventing apoptosis in atherosclerosis . in fact , an increase in bcl - xl was observed in smc from the intima in early atherosclerotic lesions . other members of the bcl-2 family , for example , bak , are abundantly expressed in smcs from atherosclerotic plaques and tend to heterodimerize with bcl - xl . oxysterols , especially 25-hydroxycholesterol , can reduce the expression of bcl-2 y bcl - xl [ 3436 ] , activate bad , and increase the expression of genes that encode bh3-only proapoptotics , for example , bim , which in turn activates bax / bak . beside the protein levels , we also studied the expression of genes of bcl-2 family that encode proteins implicated in apoptosis ( bcl-2 , bcl - xl ) , the amount of mrna of bcl-2 gene ( prototype of antiapoptotic genes ) remained unchanged in the smc - c and smc - ch , consistent with previous findings of no modifications in the expression of bcl-2 during atherosclerotic alterations [ 9 , 25 , 26 ] . however , the smc - ch - fo showed a slight increase in bcl-2 , which may imply some resistance to apoptotic stimuli . in fact , many genes involved in the response to oxidative stress , for example , nf-b survival factor genes , are activated by a fish oil - rich diet and are also involved in the regulation of bcl-2 [ 3840 ] . no major differences observed in baseline expression of the bcl - xl gene in the smc - c , smc - ch and smc - ch - fo cells , as also found for bcl-2 and protein levels . the bcl - xl gene is thought to play a critical role in the prevention of cell death in atherosclerotic plaques , and a reduction in bcl - xl expression levels may be a prerequisite for apoptotic induction . it has also been reported that the inhibition of nf-b ( activator of cell survival genes reduces the expression of bcl - xl , which makes the cells more susceptible to apoptosis ) . the increase in the mrna of bcl-2 to a greater degree in smc - ch than in smc - c may appear contradictory , since bcl-2 protein levels are known to be reduced by oxysterol treatment of the cells . the marked increase of the expression of bcl - xl in the three culture types after the addition of 25-hydroxycholesterol again appears contradictory because bcl - xl protein levels are reduced , which may be explained by the dimerization of bcl - xl with bax and especially with bak . the aortic arches of male chick were chosen for this study for two reasons : first , for the ability of chicks to absorb high amount of dietary cholesterol [ 14 , 42 ] ; and second , because intimal thickening in the aortas from these hypercholesterolemic chicks can be detected after only 20 days [ 43 , 44 ] . this short diet period is very convenient and contrasts with those reported for other experimental animal models , mainly non - human primates and rabbits , which must be a cholesterol - containing diet for between 1 and 18 months to obtain an atherosclerotic lesion . serum cholesterol levels observed in 10-day - old control chicks are normal because newly hatchet chicken reabsorb the yolk during the first week of live , thus developing hypercholesterolemia , which becomes almost three times higher in cholesterol - fed chicks . we have also used chicks previously to study lipids metabolism after cholesterol administration and to develop a cell - culture avian model to look into , in vitro , the effect of a cholesterol diet , in vivo , on the transformation of smc . thus , in our laboratory , we isolated smcs from cholesterol - fed chicks ( smc - ch cultures ) which are very proliferative in culture compared with smc isolated from control - fed chicks ( smc - c cultures ) , and with identical maintenance in culture of the two lines of smc . in this way , dna synthesis in the s phase was 4-fold higher and after 20 days of culture , smc - ch increased their cholesterol content to double that of smc - c , giving smc - ch cultures under conditions mimicking such a cholesterol diet and a very early atherosclerosis in vivo / in vitro model at the smc level . we have studied the cholesterol synthesis and hmg - coa reductase gene expression in these cultures , showing great differences between smc - c and smc - ch [ 50 , 51 ] . also we demonstrated the existence of cyclic fluctuations of hmg - coa reductase activity in nonsynchronized smc cultures not correlated to the cultured feeding and not with the increase of mrna , suggesting the posttranscriptional modulation of the hmg - coa reductase and the relationships between hmg - coa reductase activity and cell division . consequentially , this transformed smc is a good model to study the alterations of the differentiated state of smc , caused not only by cholesterol - rich diet but also by fish oil - rich diet as well as the apoptotic pathway induced by oxysteroid in smcs . in conclusion , we investigated the apoptotic pathway induced by oxysteroid in smcs isolated from male chicks exposed to a control diet ( smc - c ) and an atherogenic cholesterol - rich diet ( smc - ch ) or an antiatherogenic fish oil - rich diet ( smc - ch - fo ) . we found that 25-hydroxycholesterol - induced apoptosis in smcs , mediated by a high increase in bax protein and bax gene expression . these changes were more evident in smc isolated from chicks exposed to an atherogenic than to an antiatherogenic one . thus , we deducted that the replacement of a cholesterol - rich diet with a fish - oil rich diet might produce some reversal of cholesterol - induced changes in oxysterol - activated apoptotic pathway , making smc more resistant to apoptosis . moreover , high cholesterol diet and fish - oil rich diet in the animals , before obtaining the smcs cultures , induced changes in the apoptotic gene expression studied . thus , the mrna concentration was downregulated in smc - ch - fo versus smc - ch . since we reported previously that , the nutritional culture conditions , the intracellular cholesterol concentration , and the ultrastructural morphology were the same during the first days of cultures ; the change in gene expression must have been induced by the cholesterol or fish oil diet in the aortic smc in vivo . in this way , this in vivo / in vitro model is relevant to study the nutritional control and gene regulation of the differentiation and apoptosis in smc . new studies would be necessary to demonstrate the implication of the cell signalling pathways , as well as to show if new protein synthesis is required for the bax increase or if some peaks of gene expression correlate with other regulatory events .

these complexes are required for a variety of processes , including chromatin organization , transcriptional regulation , decatenation of chromatids during mitosis , and dna repair . the mammalian switch / sucrose nonfermentable ( swi / snf ) complex is one of four mammalian chromatin remodelling complexes . recurrent inactivating mutations in certain subunits of this complex have been identified in different cancers . despite its known roles in tumor suppression , the mammalian swi / snf complex this stems from the recognition that residual swi / snf complexes are critical for the growth of genetically defined cancers , including swi / snf mutant and max mutant tumors as well as acute leukemias . in acute leukemias , it was found that the swi / snf complex supports an oncogenic transcriptional program . in the absence of the swi / snf atpase a recent study highlighted a role of another swi / snf subunit , brd9 , in leukemia growth . the brd9 bromodomain ( bd ) was shown to be required for the proliferation of acute myeloid leukemia ( aml ) cells . over the past decade , chemical probe compounds have been shown to be invaluable in the elucidation of protein function . we set out to develop a probe compound targeting the bd of brd9 in order to evaluate the function of this domain within the swi / snf complex . bds are protein - binding domains with an affinity to lysine - acetylated target proteins . the acetyl - lysine binding pockets of these domains have been shown to be amenable to inhibition by drug - like small molecules , and the activity of several inhibitors directed against bromodomain and extra - terminal motif ( bet ) containing proteins ( brd2 , brd3 , brd4 , and brd - t ) is being clinically assessed in cancer , including hematopoietic malignancies , and atherosclerosis ( http://www.resverlogix.com/blog/tag/atherosclerosis/ ) . a key selectivity parameter in designing our tool compounds was to avoid activity against bet family proteins because of the pleiotropic effects that bet inhibitors exert on various cellular processes . recently , three brd9 inhibitors have been published in the literature : lp99,i - brd9 , and ketone compound 28 ( supporting information table 3 ) . lp99 is the first published potent and selective inhibitor of brd9 and brd7 [ kd ( brd9 , itc ) = 99 nm vs kd ( brd7 , itc ) = 909 nm ] ; its structure is based on a methylquinolinone scaffold . cellular target engagement was demonstrated using a quantitative nanobret assay [ ic50 ( brd9-h3.3 ) = 5.1 m and ic50 ( brd9-h4 ) = 6.2 m ] . i - brd9 , which is derived from a thienopyridone scaffold , is a potent cell - active selective binder to brd9 [ kd ( brd9 , discoverx ) = 1.9 nm vs kd ( brd7 , discoverx ) = 380 nm ; ic50 ( brd9-h3.3 , nanobret ) = 158 nm ] , but it presents some residual affinity toward other bet family members [ kd ( brd4-bd1 , discoverx ) = 1400 nm ] . additionally , i - brd9 was shown to bind to endogenous brd9 in a chemoproteomic assay . finally , ketone compound 28 was developed starting from a keto - indolizine baz2a / b chemical probe ; the compound is potent and selective toward brd9 and brd7 [ kd ( brd9 , itc ) = 68 nm vs kd ( brd7 , itc ) = 368 nm ] and shows cellular activity at 1 m in a fluorescence recovery after photobleaching ( frap ) assay . in this article , we describe the discovery and development of a potent and selective brd9 bd inhibitor series based on a new scaffold arising from two parallel screening approaches consisting of fragment - based screening and virtual screening of proprietary libraries . in particular , we report the structure - based design of brd9 inhibitor 1 ( bi-7273 ) , which was previously demonstrated to mimic genetic perturbation of brd9.1 also targets brd7 bd , which is a bd protein that has been found in a subclass of swi / snf remodelling complexes ( pbaf ) and shares high sequence homology with brd9 . we further describe a second brd9 inhibitor , 2 ( bi-9564 ) , which displays enhanced selectivity against the brd7 bd as well as improved pharmacokinetic properties when compared to those of 1 . these two chemical probes , 1 and 2 , should prove to be useful in further probing brd9 bd biology in both in vitro and in vivo settings . three parallel biophysical assays , a differential scanning fluorimetry ( dsf ) assay , a surface plasmon resonance ( spr ) assay , and a microscale thermophoresis ( mst ) assay , were used to screen our proprietary fragment library of around 1700 compounds against the brd9 bd ( supporting information figures 1 and 2 and table 1 ) . it is of note that this is one of the first fragment screening applications described for the mst technology . the primary screening hits identified by these three screening methods were validated in an orthogonal binding assay using heteronuclear single quantum coherence nuclear magnetic resonance ( n hsqc nmr ) ( supporting information figures 13 ) . seventy - seven hits showed significant cross peak shifts in the 2d h/ n hsqc nmr spectra , and 55 of these compounds were successfully soaked into the crystals of the brd9 bd . the observed hit rate in mst screening was significantly higher than that with the other two techniques ( 124 primary hits for mst vs 36 and 45 for dsf and spr , respectively ) , with only a 10% overlap in the hits identified by spr and dsf . alternative assay formats or sensitivities of the mst instrument ( monolith nt.015 prototype ) may underlie the observed differences in the hit rate . however , despite the higher primary hit rate , mst screening yielded 38 hits validated by hsqc nmr , from which 29 were identified solely by this technology . from these 29 hits , 14 co - crystal structures could be obtained , indicating the significant value of pursuing single - technology hits in addition to hits identified by several orthogonal primary screening techniques . after quantification of the binding affinity of the 77 compounds by spr , 12 compounds displayed a dissociation constant ( kd ) below 100 m . additionally , a proprietary high - concentration screening ( hicos ) library of 74 500 compounds was screened by glide docking , followed by brd9 bd pharmacophore mapping and finally filtering based on molecular weight ( mw < 280 ) and lipophilicity ( clogp < 2 ) ( supporting information figure 4 ) . this virtual screening led to the selection of 208 available compounds , which were measured in dsf and spr ( % ctrl ) assays . the binding affinity ( kd ) of the hits was quantified using spr ( supporting information figure 1 ) , leading to the discovery of 23 additional candidates , of which 11 compounds had their binding mode elucidated by x - ray co - crystal structure determination . all 23 compounds were resynthesized and had their binding affinity confirmed by spr . eleven compounds had a kd below 100 m by spr ( supporting information figure 1 ) . structure - based medicinal chemistry was then initiated using the x - ray co - crystal structures of the fragments obtained in the brd9 bd ( supporting information figures 58 and table 2 ) . the methylpyridopyrimidinone or dimethylpyridinone scaffold compound series was selected as a promising starting point based on their potential exit vectors and binding affinities [ kd ( 3 , spr ) = 37.5 m , kd ( 4 , spr ) = 9.1 m ] ( figure 1a , with the scaffold highlighted ) . the binding mode of the two compounds showed similar binding in the brd9 anchor region ( figures 1b , c and supporting information figures 58 ) . the carbonyl of the pyridinone functionality makes two hydrogen bonds with the protein : a direct h - bond with the n h2 side chain of asn100 and an h - bond to tyr57 via a conserved water molecule . a methyl group ( either from the n - methyl or from the methyl alpha to the carbonyl ) occupies a small lipophilic pocket surrounded by four conserved water molecules . the binding mode of these small acetylated lysine mimic binders is typical of those reported for other bds . indeed , similar binding activity was observed toward bet family members [ e.g. , ic50 ( 3 , brd4-bd1 ) = 80.2 m and ic50 ( 4 , brd4-bd1 ) = 3.7 m ] for both fragments , highlighting the challenge of achieving high selectivity against brd4 . additionally , the aromatic pyridinone core in compounds 3 and 4 makes a -stacking interaction with the tyr106 in the brd9 bd . the distance between the aromatic core and tyr106 was measured at 3.4 and 3.7 for 3 and 4 , respectively ( optimal aromatic c aromatic c parallel offset stacking is 3.43.6 ) . finally , in the specific case of compound 4 , we observe a c h -interaction between ile53 and the phenyl moiety ( distance ch3phenyl = 3.6 ) , which could be beneficial for improving potency toward brd9 ( figure 1c ) . binding mode of the methylpyridopyrimidinone 3 or dimethylpyridinone 4 scaffold in brd9 bd ( compound 3 : pdb code 5f2p ; compound 4 : pdb code 5f25 ) . ( a ) structures and binding affinities of compounds 3 and 4 identified by fbs screening and virtual screening of hicos library , respectively . ( b , c ) binding mode of ( b ) 3 and ( c ) 4 in the brd9 bd : h - bond to asn100 , water - bridged interaction with tyr57 , -stacking with tyr106 , and c h -interaction with ile53 . za channel , anchor region , and n - side are indicated in blue . in parallel to the biophysical spr assay , we developed a biochemical assay measuring the inhibitory effect of our compounds on the binding between acetylated histone h3 and brd9 bd . this assay showed good correlation with the data obtained by spr [ kd ( 3 , spr ) = 37.5 m vs ic50 ( 3 ) = 48.9 m ; kd ( 4 , spr ) = 9.1 m vs ic50 ( 4 ) = 9.4 m ] . as the aim of the project was to develop a potent and selective inhibitor , we developed in vitro peptide displacement assays to routinely monitor the inhibitory effect of our compounds on the binding between brd9 , brd7 , brd4-bd1 , brd4-bd2 , or brd2-bd1 bd and acetylated histone h3 or h4 . acetylated histones bind to their bd partners via their n - acetylated lysine tails , in particular interacting with a conserved asparagine ( for brd9 bd it is asn100 ) in the anchor region and along a region previously referred to as the n - side ( figure 1b ) . considering that this binding mode is conserved among all bds , we decided to concentrate on improving potency by growing the initial fragment toward the less conserved za channel area ( figure 1b ) , as we anticipated that this approach would give us a greater chance of successfully achieving selectivity against bet family members . in order to optimize the za channel linker , we used the dimethylpyridinone scaffold as an anchor binder . both methyl and carbonyl functions on this scaffold are essential for effective binding to the brd9 bd ; indeed , removal of one of the methyl groups [ position 1 ( n - me ) or 3 ] or removal of the carbonyl moiety ( e.g. , replacement of anchor binder by 2,6-dimethylpyridine ) led to a loss of binding . in the za channel of the brd9 bd , we identified phe44 ( figures 1b , c ) as an amino acid of interest and hypothesized that addressing this interaction could improve potency . compound 4 showed a suboptimal edge - to - face interaction ( also referred to as t - stacking ) with phe44 [ distance phe44phenyl ( 4 ) = 4.67 vs optimal aromatic c aromatic c edge - to - face distance = 3.63.9 ] . it is reported that introduction of electron - donating groups on the facially substituted phenyl improves t - stacking . following this principle , we replaced the amide functional group by a methylene dimethylamine ( compound 5 , table 1 ) , which led to an 8-fold increase in potency . introduction of additional electron - donating groups onto the phenyl ring further enhanced potency by improving t - stacking with phe44 ( compounds 610 , table 1 ) . using 4-(dimethylamino)methyl-3,5-dimethoxyphenyl as a za channel linker ( compound 7 ) increased the potency 170-fold compared to that of initial starting hit 4 [ ic50 ( 7 , brd9 ) = 54 nm vs ic50 ( 4 , brd9 ) = 9338 nm ] . it was possible to further boost potency by addressing the backbone carbonyl of his42 with either a hydroxyl moiety or an amine moiety ( compounds 11 and 12 , table 1 ) . the azetidine substituent provided the best vector to the his42 carbonyl without interfering with the optimal t - stacking of the dimethoxyphenyl za channel linker with phe44 . 4-(3-aminoazedinylmethyl)-3,5-dimethoxyphenyl dimethylpyridinone ( 12 ) exhibited low nanomolar activity [ ic50 ( 12 , brd9 ) = 9 nm ] . binding of the structurally closely related compound 11 was confirmed by x - ray co - crystal structure determination ( figure 2a and supporting information figures 9 and 10 and table 2 ) . the anchor region binding is similar to the one observed with fragment 4 : the pyridinone core binds through its carbonyl to asn100 and to the conserved water molecule bound to tyr57 , a methyl group occupies the small lipophilic pocket , and the pyridinone makes a -stacking interaction with tyr106 . in the za channel , the dimethoxyphenyl linker adopts a conformation that permits optimal t - stacking with phe44 , with the distance between the aromatic core and phe44 now being the ideal 3.9 , while keeping the c h -interaction with ile53 , with the distance between aromatic core and ile53 being 3.6 . the azedin-3-ol forms an h - bond to his42 ; additionally , for this molecule , we observe an induced fit with phe47 closing onto the molecule due to a c h -interaction with the methoxy group of compound 11 . ( a c ) binding mode of brd9 bd inhibitors ( a ) 11 , ( b ) 1 , and ( c ) 2 [ compound 11 : pdb code 5f1l ; compound 1 : pdb code 5eu1 ; compound 2 : pdb code 5f1h ] . ( d ) x - ray co - crystal structure of 1 in brd9 ( not shown ) aligned with brd4-bd1 ( compound 1 : pdb code 5eu1 ) . ( a ) binding mode of compound 11 in brd9 bd : h - bond to asn100 , water - bridged interaction with tyr57 , -stacking with tyr106 , c h -interaction with ile53 , t - stacking with phe44 , h - bond to his42 , and induced fit phe47/c ( b ) binding mode of compound 1 in brd9 bd : two h - bonds to asn100 , water - mediated hydrogen bond with tyr57 , -stacking with tyr106 , c h -interaction with ile53 , and t - stacking with phe44 . ( c ) binding mode of compound 2 in brd9 bd : two h - bonds to asn100 , water - bridged interaction with tyr57 , -stacking with tyr106 , c h -interaction with ile53 , t - stacking with phe44,and induced fit phe47/c h -interaction . ( d ) clash between 1 and brd4-bd1 bd amino acids ( trp81 , gln85 , and leu92 ) is shown ( brown circles ) . while improving the potency toward brd9 , we also observed an enhancement of the selectivity against the first bd of brd4 ( brd4-bd1 ) compared to that of starting hit 4 , which showed higher potency for the off - target brd4-bd1 than for brd9 . encouraged by these results , we investigated modifications in the anchor region in order to further improve selectivity . as stated previously , we considered it to be essential that our final brd9 chemical probes were inactive against brd4-bd1 and other bet family members to avoid any misinterpretation of biological results [ i.e. , ic50 ( bet family members ) > 100 m ] . after comparing the surface of brd9 and brd4-bd1 , we hypothesized that enhanced selectivity could be achieved by forcing a clash with key amino acids in the anchor region or the za channel of brd4-bd1 . this could be done by introducing substituents at the 4 or 6 position on the pyridine-2-one core of our inhibitors to force a change in the torsion angle between the anchor binder ring and the za channel linker ring ( table 2 ) . a methyl group at position 4 ( or 6 ) forced a twist between the 2 aryl moieties , ( measured torsion angle approximately 60 vs 27 to 44 when hydrogen is present ) , and indeed translated to an improvement of selectivity against the bet family [ compounds 13 ( bi-7189 ) and 14 , table 2 ] . on the basis of the same principle , an aromatic ring merged to the pyridinone scaffold gave an improved selectivity against the bet family and concomitantly improved the -stacking interaction with tyr106 in the brd9 anchor region [ compounds 15 ( bi-7271 ) to 19 , table 2 ) . the most efficient inhibitor was 2-methyl-2,7-naphthyridin-1-one compound 1 with a 3-fold increase in affinity for brd9 and 50-fold increase in selectivity against brd4-bd1 over compound 7 ( selectivity brd9 vs brd4-bd1 : 100-fold for compound 7 and > 5200-fold for 1 ) . 1 forms an additional positive interaction with the carbonyl of asn100 in brd9 ; indeed , the presence of a nitrogen atom at position 7 on the naphthyridinone ring acidifies the ch bond at position 8 , permitting an interaction with the carbonyl side chain of asn100 ( figure 2b and supporting information figures 11 and 12 and table 2 ) . 1 displays no measurable activity toward bet family bds up to a concentration of 100 m in our biochemical alpha assay , which can be explained by a potential clash of the anchor part of the molecule with leu92 in brd4-bd1 and a clash of the trisubstituted phenyl linker of the molecule with gln85 and trp81 in brd4-bd1 ( figure 2d , superimposition of 1 with the brd4-bd1 surface ) . * finally , while investigating modifications on the za linker part , we discovered that para - substituted dimethoxy groups resulted in an enhanced selectivity for brd9 over its closest homologue , brd7 ( table 3 ) . we believe that this selectivity might stem from differences in flexibility between the two proteins . on the basis of the protein melting properties shown in the thermal shift assay , we hypothesized that the brd7 bd was more flexible and dynamic in solution than brd9 . 2 was slightly less potent toward brd9 compared to that of 1 in the alpha assay , but it showed improved selectivity against brd7 ( 45-fold more potent for brd9 vs brd7 ) and , most importantly , remained inactive toward bet family members ( scheme 1 ) . the binding mode of 2 in brd9 bd was confirmed by x - ray co - crystal structure determination ( figure 2c and supporting information figures 13 and 14 and table 2 ) . 2 bound with an induced fit of phe47 in the za channel , similar to that for compound 11 . synthesis of para - substituted dimethoxy analogues of 2 , which could directly address the carbonyl of his42 with either a benzyl amine linker or sulphonamide linker at the 4 position of the za linker , yielded compounds with an improved brd9 potency and similar selectivity profile compared to those of 2 . however , these compounds later presented less attractive pharmacokinetic properties ( e.g. , low permeability or high efflux ratio ) , which made them unsuitable for in vivo testing . * alpha format , mean value ; number of measurements : 25 . reagents and conditions : ( a ) nah , mei , dmf , rt , 71% ; ( b ) hnme2hcl , naoac , acoh , nabh(oac)3 , dcm , rt , 89% ; ( c ) b2pin2 , koac , pd(dppf)cl2 , 1,4-dioxane , 90 c , 63% ; ( d ) pd(dppf)cl2dcm , na2co3(aq ) , dmf , 100 c , 17% finally , binding affinities of 1 and 2 toward brd9 were measured by isothermal titration calorimetry ( itc ) and bromokdelect ( figure 3 , table 4 , and supporting information figures 17 and 18 ) . high - affinity binding to brd9 bd was confirmed for both compounds [ kd ( 1 , itc ) = 15.4 nm and kd ( 2 , itc ) = 14.1 nm ] . 1 and 2 were selected as brd9 bd chemical probes for further in vitro and in vivo profiling . ( a ) compound 1 binds with a kd value of 15.4 nm ( h = 12.1 kcal / mol ) and ( b ) 2 binds with a kd value of 14.1 nm ( h = 11.2 kcal / mol ) . target engagement in the cell was demonstrated in a semiquantitative frap assay using a green fluorescent protein 2 showed inhibition of brd9 in cells at 100 nm , whereas 1 was active in the cell at 1 m ( with 1 m being the lowest concentration tested ) ( figure 4a no compound - related toxicity was observed in u2os cell lines after 24 h. frap assay using u2os cells transfected with gfp brd9 . ( a ) recovery half times of wild - type ( wt ) cells treated with dmso in the absence or presence of 2.5 m saha or treated with 1 at 1 m and saha as indicated . in addition , cells expressing gfp brd9 with a bd - inactivating mutation ( n100f ) were analyzed . significant differences relative to cells treated with saha ( p < 0.0001 ) are shown by * * * * . ( b ) time dependence of fluorescence recovery in the bleached area of cells expressing wt or mutant gfp ( c ) recovery half times of cells expressing wt gfp brd9 treated with various concentrations of dmso and 2 in the presence or absence of saha as indicated . significant differences relative to cells treated with saha ( p < 0.0001 ) are shown by * * * * . ( d ) time dependence of fluorescence recovery in the bleached area of cells expressing wt or mutant gfp brd9 with the corresponding treatments shown in ( c ) . 1 shows potency ( 100% inhibition ) at 1 m in the brd9 frap assay . 2 shows potency ( 90% inhibition ) at 0.1 m in the brd9 frap assay . both compounds showed no toxicity in u2os cells after 24 h. the n100f construct is a negative control brd9 mutant in which asn100 is replaced by phe100 and therefore acetylated histone can not bind because of the lack of interaction to the anchor asn and because of steric hindrance . saha is added to the mixture to increase the signal - to - noise ratio by inhibiting the deacetylation of histones . to assess selectivity , bd selectivity was checked by differential scanning fluorimetry for 48 bds followed by itc kd determination ( table 4 and supporting information figures 15 , 16 , 19 , and 20 ) . the same selectivity pattern was observed using the bromomax / bromokdmax technology ( 32 bds screened ; supporting information figures 17 and 18 ) . high selectivity against bet family members was confirmed , with kd values exceeding 10 m . aside from brd9 , highly homologous brd7 and cecr2 were the only two bds identified as additional targets ( table 4 and supporting information figures 19 and 20 ) . although 2 showed potency at 1 m in a brd7 frap assay ( table 4 and supporting information figure 22 ) , pleasingly , no cellular inhibition of the cecr2 bd was observed at this concentration ( table 4 and supporting information figure 23 ) . cecr2 has been described as being part of the cecr2-containing - remodeling factor ( cerf ) complex . aside from its bd , cecr2 contains an at hook motif , a dna - binding motif with a preference for a / t - rich regions , that might contribute to the reduced displacement from chromatin by 2 in the frap assay compared to brd9/7 , which do not possess any additional dna or chromatin binding domains . further profiling was conducted to assess the selectivity over a range of targets , particularly kinases and g - protein coupled receptors ( gpcrs ) . concentrations of compound 2 of less than 5 m showed no activity against 324 kinases , and at 10 m , an inhibition > 40% was observed for only 2 out of 55 gpcrs ( table 4 and supporting information figure 24 ) . the cellular response to brd9 inhibition was assessed in a broad cancer cell line panel . treatment of the panel with 2 resulted in selective growth inhibition of a significant proportion of aml cell lines tested ( supporting information figure 31 ) . this result was in agreement with decreased proliferation in the murine aml rn2 cell line following treatment with 1 . cecr2 mrna expression was hardly detectable in any of the eight sensitive cell lines , which , in addition to the lack of cellular activity of 2 in the cecr2 frap assay ( supporting information figure 23 ) , suggests that the antiproliferative effect was not due to the effects of the compound on this bd . the most sensitive cell line was human acute myeloid eosinophilic leukemia cell line eol-1 [ ec50 ( 2 , eol-1 ) = 800 nm ; ec50 ( 1 , eol-1 ) = 1400 nm ] ( figure 5 , supporting information figure 31 ) . as observed from the phenotype of murine cells exposed to 1 , brd9 inhibition translated into a potent but only partial inhibition of myc expression in aml cell lines ( supporting information figure 32a d ) . complete suppression of myc expression , observed at higher concentrations with some of the less selective compounds , was likely due to the effects of the compounds on bet family members [ e.g. , ic50 ( 13 , brd4-bd1 ) = 31 m , ic50 ( 15 , brd4-bd1 ) = 24 m ] ( supporting information figure 32a , b ) . brd9 can fulfill its cellular function in murine leukemia cells when its bd is exchanged with that of brd4 . these domain - swap experiments were used to demonstrate that the antiproliferative activities of 1 in this cell line are due to its effect on the brd9 bd . using 2 in such domain - swap experiments up to 5 m concentration , we could further confirm that brd9 bd is responsible for mediating the antiproliferative effects of 2 . however , we also noted a degree of off - target effects of 2 in this murine leukemia cell line when it was used at higher concentrations ( data not shown ) . nonetheless , these findings suggested that 2 is a suitable chemical probe for probing the in vitro and in vivo functions of brd9 . compounds 1 and 2 both showed attractive adme / pk profiles for in vivo proof - of - concept studies , namely , high solubility at ph 6.8 , moderate to high in vitro metabolic stability , low plasma protein binding , and no cytochrome p450 inhibition , together with moderate to high absorptive permeability and moderate in vivo plasma clearances upon i.v . dosing ( table 4 and supporting information figures 25 and 28 ) . despite elevated efflux ratios in the caco-2 transporter assay , both compounds displayed high oral bioavailability ( table 4 and supporting information figures 2530 ) . in order to explore the potential of 2 and 1 as in vivo chemical probes , female bomtac : nmri - foxn1nu mice were given two doses orally ( 20 and 180 mg / kg p.o . ) and the compound concentration in plasma over time was measured . dose - dependent but nonlinear aucs were obtained for both compounds , achieving exposures that were higher compared to the ec50 levels determined for both compounds in proliferation assays with eol-1 cells ( figure 5 ) . 2 presented an exposure over time that was twice that of 1 and a higher oral bioavailability ( table 4 ) . this justified in vivo efficacy experiments in mice at the highest dose of 2 ( 180 mg / kg p.o . ) in the disseminated eol-1 aml mouse model ( summary of properties : table 4 and supporting information figures 26 , 27 , 29 , and 30 ) . 1 and 2 block eol-1 cell proliferation with ec50 s of 1400 and 800 nm , respectively . prior to performing efficacy studies , 1 week tolerability studies were carried out using 180 mg / kg of 2 in non - tumor - bearing female ciea - nog mice . continuous daily dosing of the selected dose was well - tolerated , with a medium weight change on day 7 of 3.8% ( n = 4 ) . the human acute myeloid eosinophilic leukemia cell line eol-1 was chosen for in vivo experiments as it was the most sensitive cell line in vitro to brd9 inhibition . a disseminated model , which more closely reflects the clinical situation compared to that of a subcutaneous xenograft , was chosen to assess the efficacy of 2 . eol-1 cells ( 10 ) , stably transduced with a luciferase - expressing vector to allow continuous assessment of tumor load by bioluminescence , were injected in the tail vein of ciea - nog mice . oral treatment with 180 mg / kg of 2 was initiated on day 5 and applied daily ( q.d . ) with an interruption at days 18 and 19 . a significant ( p = 0.0086 ) reduction in tumor growth ( measured in average radiance [ p / s / cm / sr ] ) compared to that of controls was observed on day 18 , resulting in a median tumor growth inhibition ( tgi ) value of 52% ( figure 6a ) . imaging data on day 18 provided evidence of a significantly reduced disease burden ( figure 6b ) in mice treated with compound 2 . the animals were closely monitored for clinical signs and were sacrificed when the disease burden exceeded a prespecified grade as a surrogate end point for survival . an increase in tumor burden in the control mice resulted in body weight loss at the end of the study . on day 18 , dosing with 2 was interrupted for 2 days , as continuous body weight loss was observed , and a body loss of 15% was observed in one mouse . the body weight loss might be explainable by an increased tumor burden because on this day the control group showed a median body weight loss of 11 compared to 8 in the treated group ; however , tolerability issues with the compound can not be ruled out ( figure 6c ) . in the disseminated eol-1 mouse model , the median survival of the vehicle ( natrosol 0.5% ) treated control animals was 20 days . median survival of the animals treated with 180 mg / kg 2 resulted in a modest but significant additional survival benefit of 2 days compared to survival of the control group ( figure 6d ) . pharmacokinetic analysis of plasma samples taken on the last day of treatment showed that high systemic exposure had been reached , with an auc0last of 268 000 nmh , as expected from single - dose pk experiments in mice ( table 4 ) . the mean total plasma concentrations of compound 2 exceeded the ec50 of 800 nm from the eol-1 cellular proliferation assay for 20 h following dosing . while the antileukemia effects of 2 in this model are modest , these experiments demonstrate that 2 is a suitable probe to evaluate the effects of in vivo modulation of brd9 bd activity . efficacy and tolerability of brd9 inhibitor 2 in a xenograft model of human aml . data for panels a d was collected in the same in vivo experiment . ( a ) ciea - nog mice were injected intravenously with 10 eol-1 aml cells . starting on day 5 after cell injection , mice were orally treated with vehicle ( daily ) or with 180 mg / kg 2 ( qd517 and qd2022 ) . with the help of bioluminescence imaging , the tumor burden was assessed in each animal on days 5 , 8 , 11 , 14 , and 18 and calculated as median average radiance [ p / s / cm / sr ] . ( b ) bioluminescence imaging on day 18 : ( upper ) vehicle control and ( lower ) 180 mg / kg 2 . ( c ) average body weight changes as a percentage of the initial weight . meier curve showing prolonged survival of animals treated with 180 mg / kg 2 ( vehicle = black line ; 180 mg / kg 2 = blue line ) . epigenetic modifications have been linked to many diseases , in particular , cancer and immune / inflammatory disorders . the molecular machinery required to read and modify chromatin has been shown to comprise large protein complexes where the activities of multiple domains are coordinated . the need to identify the activities that drive disease pathology has spurred the development of high - quality inhibitors to probe the function of individual domains in the context of the native cellular complexes . in this article , we focused on the bd of brd9 , a subunit of the swi / snf chromatin remodeling complex . our study describes how a combination of fbs and virtual screening of proprietary libraries allowed us to identify a new scaffold class of brd9 bd inhibitors . among the current bet - sparing brd9 bd inhibitors , 2 and 1 displayed the greatest cellular potency this was principally achieved following a structure - guided chemical optimization that led to the introduction of 2-methyl-2,7-naphthyridin-1-one as an anchor region binder . optimization of the compounds focused on addressing all key interactions as efficiently as possible , keeping the overall size of the final molecule small and the ligand efficiency high [ mw ( 1 ) = mw ( 2 ) = 353.4 da ; ligand efficiency ( 1 ) = 0.41 and le ( 2 ) = 0.38 ; lipophilic ligand efficiency ( 1 ) = 5.7 and lle ( 2 ) = 5.7 ] . it is of note that cecr2 was the only in vitro off - target identified outside the brd9/brd7 subfamily ; however , at 1 m , no cellular inhibition of this bd was observed in the cecr2 frap assay . the overall adme properties of the two compounds permit them to be used in in vivo experiments . early work in a disseminated mouse model of aml showed efficacy for 2 ( at 180 mg / kg ) with a median tgi value of 52% on day 18 , which translated into an additional survival benefit compared to that of the control group . three structurally unrelated brd9 inhibitors have been recently published in the literature : lp99,i - brd9 , and ketone compound 28 ( supporting information table 3 ) . as selective , potent compounds with cellular activity , 1 and 2 will prove to be invaluable as tools to further explore brd9 biology ( table 4 ) . the adme parameters of these two inhibitors will , in addition , allow the scientific community to elucidate the role of brd9 , either as a single agent or in combination with other inhibitors , in both in vitro and in vivo settings . compound 2 is available to the scientific community via the sgc consortium as a brd9/brd7 potent , selective , cell - permeable , and noncytotoxic probe compound ( http://www.thesgc.org/chemical-probes/bi-9564 ) . we believe that this molecular probe will provide a useful tool to broaden the study of chromatin regulators not only in oncology but also potentially in additional therapeutic areas such as neurology , immunology , and inflammation . unless otherwise indicated , all reactions were carried out in standard commercially available glassware using standard synthetic chemistry methods . air- and moisture - sensitive reactions were performed under an atmosphere of dry nitrogen or argon with dried glassware . solvents used for reactions were of commercial dry , extra - dry , or analytical grade . preparative rp - hplc was carried out on an agilent or gilson system using columns from waters ( sunfire c18 obd , 5 or 10 m , 20 50 mm , 30 50 mm , or 50 150 mm ; x - bridge c18 obd , 5 or 10 m , 20 50 , 30 50 , or 50 150 mm ) or ymc ( triart c18 , 5 , or 10 m , 20 50 or 30 50 mm ) . unless otherwise indicated , compounds were eluted with mecn / water gradients using either acidic ( 0.2% hcooh or tfa ) or basic water ( 5 ml 2 m nh4hco3 + 2 ml of nh3 ( 32% ) brought up to 1 l with water ) . nmr experiments were recorded on bruker avance 400 and 500 mhz spectrometers at 298 k. samples were dissolved in 600 l of dmso - d6 or cdcl3 , and tms was added as an internal standard . one - dimensional h spectra were acquired with 30 excitation pulses and an interpulse delay of 4.2 s with 64k data points and 20 ppm sweep width . one - dimensional c spectra were acquired with broadband composite pulse decoupling ( waltz16 ) and an interpulse delay of 3.3 s with 64k data points and a sweep width of 240 ppm . processing and analysis of 1d spectra were performed with bruker topspin 2.0 software . no zero filling was performed , and spectra were manually integrated after automatic baseline correction . analytical lc / ms data [ lc / ms(bas1 ) ] were measured on an agilent hplc 1100 series with an agilent lc / msd sl detector using a waters x - bridge ( c18 , 2.5 m , 2.1 20 mm ) column ( part no . 186003201 ) and solvents a [ 20 mm aqueous nh4hco3/nh3 ( ph 9 ) ] and b [ acetonitrile hplc grade ] as eluent ( additional settings : flow , 1 ml / min ; injection volume , 5 l ; column temp , 60 c ) . standard gradient was as follows : 0.00 min , 10% b ; 0.001.50 min , 10% 95% b ; 1.502.00 min , 95% b ; and 2.002.10 min , 95% 10% b. for some intermediates , analytical lc / ms data was measured using different methods : lc / ms(int1 ) was measured on a shamadzu hplc lc-20ab , spd - m20a 190370 nm system using a luna c18(2 ) ( 5 m , 50 2 mm ) column and solvents a [ h2o containing 0.0375% tfa ] and b [ acetonitrile hplc grade containing 0.018% tfa ] as eluent ( additional settings : flow , 0.8 ml / min ; column temp , 40 c ) . standard gradient was as follows : 0.00 min , 10% b ; 0.004.00 min , 10% 80% b ; 4.004.90 min , 80% b ; 4.904.92 min , 80% b 10% b ; and 4.925.50 min , 10% b. lc / ms(int2 ) was measured on an agilent hplc 1200 series ( dad 200400 nm ) with an agilent 6120 ms system using a luna c18(2 ) ( 3 m , 30 2 mm ) column and solvents a [ h2o containing 0.0375% tfa ] and b [ acetonitrile hplc grade containing 0.018% tfa ] as eluent ( additional settings : flow , 1.0 ml / min ; column temp , 50 c ) . standard gradient was as follows : 0.00 min , 10% b ; 0.001.15 min , 10% 80% b ; 1.151.55 min , 80% b ; 1.551.56 min , 80% b 10% b ; and 1.562.99 min , 10% b. hrms data were recorded using a thermo scientific orbitrap elite hybrid ion trap / orbitrap spectrometer system with an ultimate 3000 series lpg-3400xrs pump system . mass calibration was performed using the pierce ltq velos esi positive ion calibration solution from thermo scientific ( lot pf200011 , product no . 88323 ) the purity of the biologically evaluated compounds was determined by lc / ms ( for all compounds ) and q - nmr ( for compounds 1 and 2 ) to be above > 95% . sodium hydride ( 3.41 g , 142 mmol ) was added slowly to a cooled solution ( 0 c ) of 4-bromo-1,2-dihydro-2,7-naphthyridin-1-one ( 23 ) ( 16.0 g , 71.1 mmol ; commercial from activate ) in dmf ( 300 ml ) , and the resulting mixture was stirred for 0.5 h. methyl iodide ( 40.4 g , 285 mmol ) was added slowly , and stirring was continued for 2 h. the reaction mixture was quenched with ice water , whereupon the product precipitates . the solid was collected by filtration , washed , and dried in vacuo to give pure 4-bromo-2-methyl-1,2-dihydro-2,7-naphthyridin-1-one ( 24 ) ( 12.0 g , 50.2 mmol , 71% ) . h nmr ( 400 mhz , dmso - d6 ) 9.36 ( s , 1h ) , 8.87 ( d , j = 5.6 hz , 1h ) , 8.26 ( s , 1h ) , 7.62 ( d , j = 5.6 hz , 1h ) , 3.53 ( s , 3h ) ; lc / ms ( bas1 ) : [ m + h ] = 239/241 ; tr = 0.92 . a solution of naoac ( 6.10 g , 44.9 mmol ) , acoh ( 2.45 g , 40.8 mmol ) , and dimethylamine hydrochloride ( 6.98 g , 85.7 mmol ) in dcm ( 160 ml ) 4-bromo-2,5-dimethoxybenzaldehyde ( 25 ) ( 10.0 g , 40.8 mmol ) was added , and stirring was continued . after 30 min , sodium triacetoxyborohydride ( 17.2 g , 81.6 mmol ) was added in one portion , and the reaction mixture was stirred at rt for 16 h. a saturated nahco3 solution was added , and the layers were separated . the combined organic layer was dried over mgso4 , filtered , and evaporated to give [ ( 4-bromo-2,5-dimethoxyphenyl)-methyl]dimethylamine ( 26 ) ( 10.0 g , 36.5 mmol , 89% ) . h nmr ( 500 mhz , dmso - d6 ) 7.17 ( d , j = 1.4 hz , 1h ) , 7.06 ( s , 1h ) , 3.78 ( d , j = 1.5 hz , 3h ) , 3.74 ( d , j = 1.4 hz , 3h ) , 3.35 ( s , 2h ) , 2.16 ( s , 6h ) ; lc / ms ( bas1 ) : [ m + h]+ = 274/276 ; tr = 1.11 min . [ ( 4-bromo-2,6-dimethoxyphenyl)methyl]dimethylamine ( 26 ) ( 7.80 g , 28.5 mmol ) and bis(pinacolato)diboron ( 21.7 g , 85.5 mol ) were dissolved / suspended in 1,4-dioxane ( 150 ml ) under n2 . potassium acetate ( 8.43 g , 49.6 mmol ) and pd(dppf)cl2 ( 1.00 g , 1.37 mmol ) were added , and the mixture was stirred at 90 c for 8 h. after cooling to rt , the mixture was concentrated and the residue was taken up in dcm . water was added , the layers were separated , and the aqueous phase was extracted three times with dcm . the crude product was purified by preparative rp - hplc using a mecn / water ( 0.2% tfa added to the water ) gradient as eluent to give the tfa salt of { [ 2,6-dimethoxy-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl}-dimethylamine ( 27 ) , which was transferred into the corresponding hydrochloride by dissolving and stirring in hcl / meoh for 30 min ( 5.80 g , 18.1 mmol , 63% ) . h nmr ( 500 mhz , dmso - d6 ) 10.15 ( s , 1h , prot . amine ) , 7.24 ( s , 1h ) , 7.16 ( s , 1h ) , 4.24 ( d , j = 5.4 hz , 2h ) , 3.80 ( s , 3h ) , 3.74 ( s , 3h ) , 2.70 ( d , j = 4.9 hz , 6h ) , 1.29 ( s , 12h ) ; lc / ms ( bas1 ) : [ m + h ] = 240 ( ester cleaved ) ; tr = 0.70 min . 4-bromo-2-methyl-1,2-dihydro-2,7-naphthyridin-1-one ( 24 ) ( 7.44 g , 31.1 mmol ) , 27 ( 10.0 g , 31.1 mmol ) , and pd(dppf)cl2dcm ( 2.54 g , 3.11 mmol ) were suspended in dmf ( 100 ml ) under argon . a degassed na2co3 solution ( 2 n , 38.9 ml , 77.8 mmol ) was subsequently added , and the resulting mixture was heated at 100 c for 2 h. after cooling to rt , dmf was evaporated and a mixture of meoh / dcm was added . all solids were filtered off , and the filtrate was evaporated again to give the crude material , which was purified by flash chromatography on sio2 using a meoh / dcm gradient as eluent to give 4-{4-[(dimethylamino)methyl]-2,5-dimethoxy - phenyl}-2-methyl-1,2-dihydro-2,7-naphthyridin-1-one ( 2 ) ( 3.40 g , 9.62 mmol , 31% ) . further purification was achieved by preparative rp - hplc ( x - bridge c18 50 100 mm , 10 m ) using a mecn / water gradient as eluent to give highly pure material ( 1.90 g , 5.38 mmol , 17% ) . h nmr ( 500 mhz , dmso - d6 ) 9.41 ( s , 1h ) , 8.64 ( d , j = 5.6 hz , 1h ) , 7.74 ( s , 1h ) , 7.12 ( s , 1h ) , 7.03 ( d , j = 5.6 hz , 1h ) , 6.93 ( s , 1h ) , 3.75 ( s , 3h ) , 3.63 ( s , 3h ) , 3.58 ( s , 3h ) , 3.483.44 ( m , 2h ) , 2.22 ( s , 6h ) ; c nmr ( 125 mhz , dmso - d6 ) 161.1 , 151.7 , 151.4 , 150.8 , 150.6 , 142.0 , 138.5 , 128.4 , 122.2 , 120.0 , 118.6 , 115.1 , 113.6 , 113.5 , 57.1 , 56.5 , 56.2 , 45.8 ( 2c ) , 36.8 . hrms ( ci+ ) : calcd for c20h24n3o3 ( mh+ ) , 354.18122 ; found , 354.18091 ; 0.88 ppm ; lc / ms ( bas1 ) : [ m + h ] = 354 ; tr = 0.91 min .

components of the chromatin remodelling switch / sucrose nonfermentable ( swi / snf ) complex are recurrently mutated in tumors , suggesting that altering the activity of the complex plays a role in oncogenesis . however , the role that the individual subunits play in this process is not clear . we set out to develop an inhibitor compound targeting the bromodomain of brd9 in order to evaluate its function within the swi / snf complex . here , we present the discovery and development of a potent and selective brd9 bromodomain inhibitor series based on a new pyridinone - like scaffold . crystallographic information on the inhibitors bound to brd9 guided their development with respect to potency for brd9 and selectivity against brd4 . these compounds modulate brd9 bromodomain cellular function and display antitumor activity in an aml xenograft model . two chemical probes , bi-7273 ( 1 ) and bi-9564 ( 2 ) , were identified that should prove to be useful in further exploring brd9 bromodomain biology in both in vitro and in vivo settings .

Introduction Results Discussion Experimental Section

the mammalian switch / sucrose nonfermentable ( swi / snf ) complex is one of four mammalian chromatin remodelling complexes . despite its known roles in tumor suppression , the mammalian swi / snf complex this stems from the recognition that residual swi / snf complexes are critical for the growth of genetically defined cancers , including swi / snf mutant and max mutant tumors as well as acute leukemias . in acute leukemias , it was found that the swi / snf complex supports an oncogenic transcriptional program . in the absence of the swi / snf atpase a recent study highlighted a role of another swi / snf subunit , brd9 , in leukemia growth . the brd9 bromodomain ( bd ) was shown to be required for the proliferation of acute myeloid leukemia ( aml ) cells . we set out to develop a probe compound targeting the bd of brd9 in order to evaluate the function of this domain within the swi / snf complex . the acetyl - lysine binding pockets of these domains have been shown to be amenable to inhibition by drug - like small molecules , and the activity of several inhibitors directed against bromodomain and extra - terminal motif ( bet ) containing proteins ( brd2 , brd3 , brd4 , and brd - t ) is being clinically assessed in cancer , including hematopoietic malignancies , and atherosclerosis ( http://www.resverlogix.com/blog/tag/atherosclerosis/ ) . lp99 is the first published potent and selective inhibitor of brd9 and brd7 [ kd ( brd9 , itc ) = 99 nm vs kd ( brd7 , itc ) = 909 nm ] ; its structure is based on a methylquinolinone scaffold . i - brd9 , which is derived from a thienopyridone scaffold , is a potent cell - active selective binder to brd9 [ kd ( brd9 , discoverx ) = 1.9 nm vs kd ( brd7 , discoverx ) = 380 nm ; ic50 ( brd9-h3.3 , nanobret ) = 158 nm ] , but it presents some residual affinity toward other bet family members [ kd ( brd4-bd1 , discoverx ) = 1400 nm ] . finally , ketone compound 28 was developed starting from a keto - indolizine baz2a / b chemical probe ; the compound is potent and selective toward brd9 and brd7 [ kd ( brd9 , itc ) = 68 nm vs kd ( brd7 , itc ) = 368 nm ] and shows cellular activity at 1 m in a fluorescence recovery after photobleaching ( frap ) assay . in this article , we describe the discovery and development of a potent and selective brd9 bd inhibitor series based on a new scaffold arising from two parallel screening approaches consisting of fragment - based screening and virtual screening of proprietary libraries . in particular , we report the structure - based design of brd9 inhibitor 1 ( bi-7273 ) , which was previously demonstrated to mimic genetic perturbation of brd9.1 also targets brd7 bd , which is a bd protein that has been found in a subclass of swi / snf remodelling complexes ( pbaf ) and shares high sequence homology with brd9 . we further describe a second brd9 inhibitor , 2 ( bi-9564 ) , which displays enhanced selectivity against the brd7 bd as well as improved pharmacokinetic properties when compared to those of 1 . these two chemical probes , 1 and 2 , should prove to be useful in further probing brd9 bd biology in both in vitro and in vivo settings . three parallel biophysical assays , a differential scanning fluorimetry ( dsf ) assay , a surface plasmon resonance ( spr ) assay , and a microscale thermophoresis ( mst ) assay , were used to screen our proprietary fragment library of around 1700 compounds against the brd9 bd ( supporting information figures 1 and 2 and table 1 ) . seventy - seven hits showed significant cross peak shifts in the 2d h/ n hsqc nmr spectra , and 55 of these compounds were successfully soaked into the crystals of the brd9 bd . however , despite the higher primary hit rate , mst screening yielded 38 hits validated by hsqc nmr , from which 29 were identified solely by this technology . additionally , a proprietary high - concentration screening ( hicos ) library of 74 500 compounds was screened by glide docking , followed by brd9 bd pharmacophore mapping and finally filtering based on molecular weight ( mw < 280 ) and lipophilicity ( clogp < 2 ) ( supporting information figure 4 ) . the binding affinity ( kd ) of the hits was quantified using spr ( supporting information figure 1 ) , leading to the discovery of 23 additional candidates , of which 11 compounds had their binding mode elucidated by x - ray co - crystal structure determination . structure - based medicinal chemistry was then initiated using the x - ray co - crystal structures of the fragments obtained in the brd9 bd ( supporting information figures 58 and table 2 ) . finally , in the specific case of compound 4 , we observe a c h -interaction between ile53 and the phenyl moiety ( distance ch3phenyl = 3.6 ) , which could be beneficial for improving potency toward brd9 ( figure 1c ) . in parallel to the biophysical spr assay , we developed a biochemical assay measuring the inhibitory effect of our compounds on the binding between acetylated histone h3 and brd9 bd . as the aim of the project was to develop a potent and selective inhibitor , we developed in vitro peptide displacement assays to routinely monitor the inhibitory effect of our compounds on the binding between brd9 , brd7 , brd4-bd1 , brd4-bd2 , or brd2-bd1 bd and acetylated histone h3 or h4 . considering that this binding mode is conserved among all bds , we decided to concentrate on improving potency by growing the initial fragment toward the less conserved za channel area ( figure 1b ) , as we anticipated that this approach would give us a greater chance of successfully achieving selectivity against bet family members . in order to optimize the za channel linker , we used the dimethylpyridinone scaffold as an anchor binder . following this principle , we replaced the amide functional group by a methylene dimethylamine ( compound 5 , table 1 ) , which led to an 8-fold increase in potency . binding of the structurally closely related compound 11 was confirmed by x - ray co - crystal structure determination ( figure 2a and supporting information figures 9 and 10 and table 2 ) . while improving the potency toward brd9 , we also observed an enhancement of the selectivity against the first bd of brd4 ( brd4-bd1 ) compared to that of starting hit 4 , which showed higher potency for the off - target brd4-bd1 than for brd9 . encouraged by these results , we investigated modifications in the anchor region in order to further improve selectivity . as stated previously , we considered it to be essential that our final brd9 chemical probes were inactive against brd4-bd1 and other bet family members to avoid any misinterpretation of biological results [ i.e. after comparing the surface of brd9 and brd4-bd1 , we hypothesized that enhanced selectivity could be achieved by forcing a clash with key amino acids in the anchor region or the za channel of brd4-bd1 . this could be done by introducing substituents at the 4 or 6 position on the pyridine-2-one core of our inhibitors to force a change in the torsion angle between the anchor binder ring and the za channel linker ring ( table 2 ) . a methyl group at position 4 ( or 6 ) forced a twist between the 2 aryl moieties , ( measured torsion angle approximately 60 vs 27 to 44 when hydrogen is present ) , and indeed translated to an improvement of selectivity against the bet family [ compounds 13 ( bi-7189 ) and 14 , table 2 ] . on the basis of the same principle , an aromatic ring merged to the pyridinone scaffold gave an improved selectivity against the bet family and concomitantly improved the -stacking interaction with tyr106 in the brd9 anchor region [ compounds 15 ( bi-7271 ) to 19 , table 2 ) . the most efficient inhibitor was 2-methyl-2,7-naphthyridin-1-one compound 1 with a 3-fold increase in affinity for brd9 and 50-fold increase in selectivity against brd4-bd1 over compound 7 ( selectivity brd9 vs brd4-bd1 : 100-fold for compound 7 and > 5200-fold for 1 ) . 1 forms an additional positive interaction with the carbonyl of asn100 in brd9 ; indeed , the presence of a nitrogen atom at position 7 on the naphthyridinone ring acidifies the ch bond at position 8 , permitting an interaction with the carbonyl side chain of asn100 ( figure 2b and supporting information figures 11 and 12 and table 2 ) . * finally , while investigating modifications on the za linker part , we discovered that para - substituted dimethoxy groups resulted in an enhanced selectivity for brd9 over its closest homologue , brd7 ( table 3 ) . on the basis of the protein melting properties shown in the thermal shift assay , we hypothesized that the brd7 bd was more flexible and dynamic in solution than brd9 . 2 was slightly less potent toward brd9 compared to that of 1 in the alpha assay , but it showed improved selectivity against brd7 ( 45-fold more potent for brd9 vs brd7 ) and , most importantly , remained inactive toward bet family members ( scheme 1 ) . however , these compounds later presented less attractive pharmacokinetic properties ( e.g. , low permeability or high efflux ratio ) , which made them unsuitable for in vivo testing . high - affinity binding to brd9 bd was confirmed for both compounds [ kd ( 1 , itc ) = 15.4 nm and kd ( 2 , itc ) = 14.1 nm ] . 1 and 2 were selected as brd9 bd chemical probes for further in vitro and in vivo profiling . target engagement in the cell was demonstrated in a semiquantitative frap assay using a green fluorescent protein 2 showed inhibition of brd9 in cells at 100 nm , whereas 1 was active in the cell at 1 m ( with 1 m being the lowest concentration tested ) ( figure 4a no compound - related toxicity was observed in u2os cell lines after 24 h. frap assay using u2os cells transfected with gfp brd9 . although 2 showed potency at 1 m in a brd7 frap assay ( table 4 and supporting information figure 22 ) , pleasingly , no cellular inhibition of the cecr2 bd was observed at this concentration ( table 4 and supporting information figure 23 ) . cecr2 has been described as being part of the cecr2-containing - remodeling factor ( cerf ) complex . cecr2 mrna expression was hardly detectable in any of the eight sensitive cell lines , which , in addition to the lack of cellular activity of 2 in the cecr2 frap assay ( supporting information figure 23 ) , suggests that the antiproliferative effect was not due to the effects of the compound on this bd . the most sensitive cell line was human acute myeloid eosinophilic leukemia cell line eol-1 [ ec50 ( 2 , eol-1 ) = 800 nm ; ec50 ( 1 , eol-1 ) = 1400 nm ] ( figure 5 , supporting information figure 31 ) . these domain - swap experiments were used to demonstrate that the antiproliferative activities of 1 in this cell line are due to its effect on the brd9 bd . however , we also noted a degree of off - target effects of 2 in this murine leukemia cell line when it was used at higher concentrations ( data not shown ) . nonetheless , these findings suggested that 2 is a suitable chemical probe for probing the in vitro and in vivo functions of brd9 . compounds 1 and 2 both showed attractive adme / pk profiles for in vivo proof - of - concept studies , namely , high solubility at ph 6.8 , moderate to high in vitro metabolic stability , low plasma protein binding , and no cytochrome p450 inhibition , together with moderate to high absorptive permeability and moderate in vivo plasma clearances upon i.v . in order to explore the potential of 2 and 1 as in vivo chemical probes , female bomtac : nmri - foxn1nu mice were given two doses orally ( 20 and 180 mg / kg p.o . ) the human acute myeloid eosinophilic leukemia cell line eol-1 was chosen for in vivo experiments as it was the most sensitive cell line in vitro to brd9 inhibition . eol-1 cells ( 10 ) , stably transduced with a luciferase - expressing vector to allow continuous assessment of tumor load by bioluminescence , were injected in the tail vein of ciea - nog mice . while the antileukemia effects of 2 in this model are modest , these experiments demonstrate that 2 is a suitable probe to evaluate the effects of in vivo modulation of brd9 bd activity . efficacy and tolerability of brd9 inhibitor 2 in a xenograft model of human aml . the need to identify the activities that drive disease pathology has spurred the development of high - quality inhibitors to probe the function of individual domains in the context of the native cellular complexes . in this article , we focused on the bd of brd9 , a subunit of the swi / snf chromatin remodeling complex . our study describes how a combination of fbs and virtual screening of proprietary libraries allowed us to identify a new scaffold class of brd9 bd inhibitors . optimization of the compounds focused on addressing all key interactions as efficiently as possible , keeping the overall size of the final molecule small and the ligand efficiency high [ mw ( 1 ) = mw ( 2 ) = 353.4 da ; ligand efficiency ( 1 ) = 0.41 and le ( 2 ) = 0.38 ; lipophilic ligand efficiency ( 1 ) = 5.7 and lle ( 2 ) = 5.7 ] . it is of note that cecr2 was the only in vitro off - target identified outside the brd9/brd7 subfamily ; however , at 1 m , no cellular inhibition of this bd was observed in the cecr2 frap assay . the overall adme properties of the two compounds permit them to be used in in vivo experiments . as selective , potent compounds with cellular activity , 1 and 2 will prove to be invaluable as tools to further explore brd9 biology ( table 4 ) . the adme parameters of these two inhibitors will , in addition , allow the scientific community to elucidate the role of brd9 , either as a single agent or in combination with other inhibitors , in both in vitro and in vivo settings . 88323 ) the purity of the biologically evaluated compounds was determined by lc / ms ( for all compounds ) and q - nmr ( for compounds 1 and 2 ) to be above > 95% . a solution of naoac ( 6.10 g , 44.9 mmol ) , acoh ( 2.45 g , 40.8 mmol ) , and dimethylamine hydrochloride ( 6.98 g , 85.7 mmol ) in dcm ( 160 ml ) 4-bromo-2,5-dimethoxybenzaldehyde ( 25 ) ( 10.0 g , 40.8 mmol ) was added , and stirring was continued . potassium acetate ( 8.43 g , 49.6 mmol ) and pd(dppf)cl2 ( 1.00 g , 1.37 mmol ) were added , and the mixture was stirred at 90 c for 8 h. after cooling to rt , the mixture was concentrated and the residue was taken up in dcm . 4-bromo-2-methyl-1,2-dihydro-2,7-naphthyridin-1-one ( 24 ) ( 7.44 g , 31.1 mmol ) , 27 ( 10.0 g , 31.1 mmol ) , and pd(dppf)cl2dcm ( 2.54 g , 3.11 mmol ) were suspended in dmf ( 100 ml ) under argon . all solids were filtered off , and the filtrate was evaporated again to give the crude material , which was purified by flash chromatography on sio2 using a meoh / dcm gradient as eluent to give 4-{4-[(dimethylamino)methyl]-2,5-dimethoxy - phenyl}-2-methyl-1,2-dihydro-2,7-naphthyridin-1-one ( 2 ) ( 3.40 g , 9.62 mmol , 31% ) .

the dermatophytes include closely related keratinophilic fungi in the genera trichophyton , microsporum and epidermophyton that degrade the skin , nail and hair of mammalian and cause a cutaneous infection known as recent epidemiological studies have indicated that dermatophytosis has remained as a public health problem in the majority of communities , even in developed countries ( 35 ) . epidemiology of dermatophytosis and dermatophytes is not static and may alter due to changes in factors such as socioeconomic , environmental and climate conditions , occupation , lifestyle , immigration - movements , travel activities and cultural practices ( 3 , 6 , 7 ) . large scale epidemiological data about diversity of pathogenic dermatophytes in different regions of iran have rarely been documented , however dermatophytosis has increasingly been reported as the dominant superficial mycosis in the country ( 6 , 812 ) . apart from the fact that previous studies used morphological - based criteria which could not reflect the entire spectrum of dermatophytes species , these studies were mainly focused on tehran , capital of iran ( 6,16 ) and other geographical area were subjected to narrow consideration . recent advances in nucleotides sequencing and other dna - based methods have promoted precise insight into the identification and epidemiology of dermatophytes ( 6 , 1720 ) . the current study aimed to describe the spectrum of dermatophytoses and dermatophytes in some parts of iran , with special emphasis on the species delineation of isolates implicated in infection using a newly described pcr - rflp on -tubulin gene ( 21 ) . during june 2011 to june 2012 , among outpatients referred to medical mycology laboratories in four provinces of iran i.e. tehran , isfahan , mazandaran and guilan , the dermatophyte strains from 500 culture - proven cases of dermatophytosis were included in the study . direct microscopic examination of skin scrapings , nails and hair shafts with20% potassium hydroxide ( koh ) , as a routine screening method was used for verifying the presence of fungal elements . a portion of the specimens was inoculated onto mycobiotic agar ( difco , usa ) and the cultures were maintained up to 3 weeks at 28 c . the cultivated dermatophytes were subjected to gross and microscopic assessment of colonies for preliminary identification and for further molecular analysis . the genomic dna of all strains was extracted according to a previously described method ( 17 ) . in brief , a piece of fresh mycelium was scraped from the surface of the plate and crushed with a conical grinder in an 1.5 ml eppendorf tube containing 300 l of lysis buffer ( 200 mm tris - hcl ( ph 7.5 ) , 25 mm edta , 0.5% w / v sds , 250 mm nacl ) . the suspension was mixed with phenol chloroform , vortexed shortly and centrifuged at 10,000 g for 10 min , mixed with chloroform and centrifuged again . dna was precipitated with an equal volume of isopropanol and 0.1 volume of 3.0 m sodium acetate ( ph 5.2 ) and the dna pellet was washed with 70% ethanol , dried in air , suspended in 50 l of double distilled water and kept at -20c till use . beta tubulin ( bt2 ) region was partially amplified in a 25 l reaction mixture containing 2.5 l of 10x pcr buffer , 10 pmol of each forward t1 ( 22 ) and reverse bt2b ( 23 ) primers , 1 l of genomic dna , 1.5 m mgcl2 , 400 m deoxynucleotide triphosphates , 1.25 u of taq dna polymerase and enough sterile deionized water . the following conditions were set up for amplification : initial denaturation phase at 94c for 6 min , 35 cycles of 94c for 30 sec , 58c for 30 sec and 72c for 1 min , followed by an ultimate extension step at 72c for 10 min . species identification was performed in a two - step rflp assay , preliminary by using the restriction enzyme fati , ( new england biolabs ltd , nhitchin , uk ) and subsequently by mwoi and hpych4v ( new england biolabs ltd , nhitchin , uk ) , and alw21i ( fermentas , vilnius , lithuania ) according to the manufacturer s instructions . all digestion reactions were performed in a 15 l mixture containing 1.5 l of 10 buffer , 0.5 l of each enzyme ( 5 u/l ) , 8l of bt2 amplicon and enough ultrapure water up to the final volume . dna cleavage was performed at 37 c for 2 h. to confirm the production of amplified and digested dnas of the expected size , 10 l of each amplicons and digested fractions were respectively electrophoresed on 1.5 and 2 % gel agarose , stained with ethidium bromide ( 0.5 g / ml ) , visualized under uv illumination and the banding patterns were recorded by photography . identification of all isolates was carried out through comparing the obtained banding profiles summarized in fig . 1 , with those deduced from the previous study ( 21 ) . fig . 1flowchart of molecular identification steps for identification of dermatophytes in the study based on beta - tubulin rflp . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) flowchart of molecular identification steps for identification of dermatophytes in the study based on beta - tubulin rflp . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) during june 2011 to june 2012 , among outpatients referred to medical mycology laboratories in four provinces of iran i.e. tehran , isfahan , mazandaran and guilan , the dermatophyte strains from 500 culture - proven cases of dermatophytosis were included in the study . direct microscopic examination of skin scrapings , nails and hair shafts with20% potassium hydroxide ( koh ) , as a routine screening method was used for verifying the presence of fungal elements . a portion of the specimens was inoculated onto mycobiotic agar ( difco , usa ) and the cultures were maintained up to 3 weeks at 28 c . the cultivated dermatophytes were subjected to gross and microscopic assessment of colonies for preliminary identification and for further molecular analysis . the genomic dna of all strains was extracted according to a previously described method ( 17 ) . in brief , a piece of fresh mycelium was scraped from the surface of the plate and crushed with a conical grinder in an 1.5 ml eppendorf tube containing 300 l of lysis buffer ( 200 mm tris - hcl ( ph 7.5 ) , 25 mm edta , 0.5% w / v sds , 250 mm nacl ) . the suspension was mixed with phenol chloroform , vortexed shortly and centrifuged at 10,000 g for 10 min , mixed with chloroform and centrifuged again . dna was precipitated with an equal volume of isopropanol and 0.1 volume of 3.0 m sodium acetate ( ph 5.2 ) and the dna pellet was washed with 70% ethanol , dried in air , suspended in 50 l of double distilled water and kept at -20c till use . beta tubulin ( bt2 ) region was partially amplified in a 25 l reaction mixture containing 2.5 l of 10x pcr buffer , 10 pmol of each forward t1 ( 22 ) and reverse bt2b ( 23 ) primers , 1 l of genomic dna , 1.5 m mgcl2 , 400 m deoxynucleotide triphosphates , 1.25 u of taq dna polymerase and enough sterile deionized water . the following conditions were set up for amplification : initial denaturation phase at 94c for 6 min , 35 cycles of 94c for 30 sec , 58c for 30 sec and 72c for 1 min , followed by an ultimate extension step at 72c for 10 min . species identification was performed in a two - step rflp assay , preliminary by using the restriction enzyme fati , ( new england biolabs ltd , nhitchin , uk ) and subsequently by mwoi and hpych4v ( new england biolabs ltd , nhitchin , uk ) , and alw21i ( fermentas , vilnius , lithuania ) according to the manufacturer s instructions . all digestion reactions were performed in a 15 l mixture containing 1.5 l of 10 buffer , 0.5 l of each enzyme ( 5 u/l ) , 8l of bt2 amplicon and enough ultrapure water up to the final volume . dna cleavage was performed at 37 c for 2 h. to confirm the production of amplified and digested dnas of the expected size , 10 l of each amplicons and digested fractions were respectively electrophoresed on 1.5 and 2 % gel agarose , stained with ethidium bromide ( 0.5 g / ml ) , visualized under uv illumination and the banding patterns were recorded by photography . identification of all isolates was carried out through comparing the obtained banding profiles summarized in fig . 1 , with those deduced from the previous study ( 21 ) . fig . 1flowchart of molecular identification steps for identification of dermatophytes in the study based on beta - tubulin rflp . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) flowchart of molecular identification steps for identification of dermatophytes in the study based on beta - tubulin rflp . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) totally , 603 dermatophytes strains were isolated from patients of which 394 , 41 and 38 isolates were originated from three northern provinces i.e. tehran , guilan and mazandaran , respectively and 130 from isfahan province , in the center of iran . the patients from whom the strains were isolated included 245 ( 40.80% ) females and 358 ( 59.20% ) males ( table 1 ) and age range of 2 - 87 years ( table 2 ) . frequency of all infection forms was more in males than females except tinea corporis . in 93 patients who had lesions in more than one anatomical site ( combined dermatophytosis ) , amplification of the bt2 region in all strains yielded single band of approximately 770 to 800 base pair ( bp ) , depending on the species . in the two - step bt2-rflp by fati , hpych4v , mwoi and alw21i , 599 strains created banding patterns matched with expected species - specific profile , leading to the delineation of t. interdigitale as the highest in terms of frequency , followed by t. rubrum , epidermophyton floccosum , m. canis , t. tonsurans , t. violaceum , t. schoenleinii and m. gypseum . four remaining strains which created restriction fragments for the arthroderma benhamiae clade , were subjected to sequencing and identified as t. erinacei ( n=3 ) and t. anamorph of a. benhamiae ( n=1 ) . except in tinea cruris and tinea barbae , t. interdigitale was the dominant species isolated in all clinical conditions and all provinces . clinical presentation of the infection revealed that feet involvement ( tinea pedis ) was the predominant clinical condition ( 42.4% ) followed by tinea cruris ( 24.2% ) , tinea unguium ( 12.3% ) , tinea corporis ( 10.8% ) , tinea faciei ( 4% ) , tinea manuum ( 3.14% ) , tinea capitis ( 3% ) and tinea barbae ( 0.16% ) , respectively . regarding to the age distribution , the infection was manifested more in three age groups of 21 - 30,31 - 40 and 41 - 50 , respectively . anamorph of a. benhamiaetotal distribution of isolates in relation to sex(m / f)*tinea pedis16969115110000256 ( 165/91)tinea cruris6339394010000146(81/65)tinea unguium40284100100074 ( 50/24)tinea corporis241614910100065 ( 28/37)tinea faciei950360100024 ( 12/12)tinea barbae01000000001(1/0)tinea manuum761301000119 ( 11/8)tinea capitis611440011018 ( 10/8)total ( % ) and geographical distribution of isolates in the study294 ( 48.75)165 ( 27.36)94 ( 15.58)29 ( 4.8)12 ( 1.99)3 ( 0.49)3 ( 0.49)1 ( 0.16)1 ( 0.16)1 ( 0.16)maz:21maz : 6maz : 7maz : 1maz : 1maz : 1maz : 1maz : 0maz : 0maz : 0gui : 15gui : 14gui : 9gui : 1gui : 2gui : 0gui : 0gui : 0gui : 0gui : 0603 ( 358/245)isf : 66isf : 25isf : 24isf : 6isf : 2isf : 2isf : 2isf : 1isf : 1isf : 1teh : 192teh : 120teh : 54teh : 21teh : 7teh : 0teh : 0teh : 0teh : 0teh : 0 * m / f : male/ female maz : mazandaran , gui : guilan , isf : isfahan , teh : tehran table.2distribution of dermatophytosis among patients of the study in regard to the age groupsage group clinical form1 - 10 ( % ) 11 - 20 ( % ) 21 - 30 ( % ) 31 - 40 ( % ) 41 - 50 ( % ) 51 - 60 ( % ) 61 - 70 ( % ) 71 - 80 ( % ) 80 < ( % ) total ( % ) tinea pedis2 ( 0.33\)10 ( 1.65)31 ( 5.14)54 ( 8.95)61 ( 10.11)57 ( 9.45)37 ( 6.13)2 ( 0.33)2 ( 0.33)256 ( 42.45)tinea cruris1 ( 0.16\)13 ( 2.15)55 ( 9.12)33 ( 5.47)23 ( 3.81)8 ( 1.32)10 ( 1.65)1 ( 0.16)2 ( 0.33)146 ( 24.21)tinea unguium2 ( 0.33)15 ( 2.48)10 ( 1.65)6 ( 0.99)22 ( 3.64)16 ( 2.65)3 ( 0.49)74 ( 12.27)tinea manuum2 ( 0.33)9 ( 1.49)3 ( 0.49)4 ( 0.66)1 ( 0.16)19 ( 3.15)tinea corporis11 ( 1.82)29 ( 4.80)2 ( 0.33)13 ( 2.15)5 ( 0.82)4 ( 0.66)1 ( 0.16)65 ( 10.77)tinea capitis12 ( 1.99)6 ( 0.99)18 ( 2.98)tinea faciei4 ( 0.66)8 ( 1.32)6 ( 0.99)1 ( 0.16)1 ( 0.16)4 ( 0.66)1 ( 0.16)24 ( 4)tinea barbae11 ( 0.16)total ( % ) 19 ( 3.15)52 ( 8.62)145 ( 24.04)103 ( 17.08)103 ( 17.08)97 ( 16.08)71 ( 11.77)8 ( 1.32)5 ( 0.82%)603 ( 100 ) frequency of dermatophytoses regarding to the dermatophyte species and patients gender distribution of dermatophytosis among patients of the study in regard to the age groups dermatophytosis annually impacts millions of individuals and imposes a substantial cost to the societies for treatment ( 2 , 24 ) . the distribution pattern and causative agents of infection continuously change , therefore it is essential to update our knowledge in regard to the epidemiology of dermatophytes . to clarify the accurate epidemiology and to resolve the problems attributed to the morphology - based identification of dermatophytes , molecular biology methods focusing on genetic markers have met with significant successes over the recent years ( 1821 ) . in the current study , dermatophytes causing different clinical forms of infection were identified at the species level based on a set of bt2-rflp profiles , which already was developed in our laboratory ( 21 ) . thus , this is the first application of pcr - rflp on -tubulin in a molecular epidemiological monitoring of dermat - ophytes worldwide . the distribution of various clinical manifestations and frequencies of isolates in four geographical areas of iran was also det - ermined . the frequency arrangement of causative species in the present study was similar to the recent molecular epidemiology report from tehran ( 6 ) , in which t. interdigitale , t. rubrum , e. floccosum , m. canis and t. tonsurans were the main agents of infection in decreasing order . however , currently t. rubrum is the most common species observed throughout the world ( 2 , 4 , 25 ) . the incidence of infection by t. interdigitale ( 48.75% ) as the most common species increased in this survey , but not significantly compared with that described by rezaei - matehkolaei et al.(6 ) . tinea pedis , the main clinical manifestation , was accounted for 42.4% of all infections followed by tinea cruris and tinea corporis and this finding was concordant with some reports from iran ( 6 , 16 ) . rubrum and e. floccosum were the most frequent , whereas currently t. rubrum is the main species implicated in feet infection worldwide ( 2 , 3 , 7 ) . our study highlighted this fact that dermatophytosis of the feet is more common in adult - males and infrequent in females and children ( 16 , 26 , 27 ) , because the infection more occurred in age groups between 31 - 60 years , was nearly twice as frequent in men than women ( 64.4% vs 35.5% ) and only 0.33% of the cases involved prepubescent patients . it was noteworthy that we found a case of foot infection by t. erinacei among our samples , which was the first proven report of tinea pedis by this species from iran . heretofore , this zoophilic less - common dermatophyte was isolated in tehran from two cases of tinea cruris and tinea manuum using dna - based methods ( 6 ) . similar to two recent studies conducted in iran ( 6 , 16 ) involvement of groin , perianal and perineal ranked second in prevalence and e. floccosum was the most common agent which was in accordance with the reports from the past till now in iran ( 6 , 8 , 10 , 12 , 13 , 16 ) . while t. rubrum still remains the most frequent cause worldwide ( 24 ) , e. floccosum seems to become more prominent as the main agent of tinea cruris in iran . tinea unguium , an old unsolved problem , ranked third in frequency and accounted for 12.27% of all clinical conditions seen during the study , which was nearly equal to those observed in qazvin and ahvaz ( 10 , 12 ) . overall , nail infection due to dermatophytes is known to predominantly occurs in males and older adults ( 25 ) and reports which found infection more prevalent in women are few ( 10 ) . in agreement with this point , in current study it remarkably more affected men and the age group of 51 - 60 years . at present , t. rubrum is the most common dermatophyte causing onychomycosis throughout the world ( 24 ) and it also was addressed as the predominant agent of nail infection in some reports from tehran , qazvin and babol ( 6 , 10 , 16 , 28 ) . however , in our study t. interdigitale was the main agent implicated and this result was in keeping with other investigations from iran ( 8 , 12 , 2930 ) . as a whole , these three dermatophytes are the principal species responsible for dermatophytic nail infection in the world , though their frequency pattern varies geographically . the causative agents of tinea corporis , the fourth prevalent infection in the study , are extensively various in different areas and a wide range of species were reported from inside of the country and overseas . in previous reports from ahvaz , hamadan , qazvin , isfahan and tehran , t. interdigitale ( 12 ) , e. floccosum ( 13 ) , t. tonsurans ( 16 ) and t. verrucosum ( 810 ) were enumerated as the main agents involved in ringworm of the body . however , in agreement with the latest molecular epidemiological survey , performed in tehran ( 6 ) the infection predominantly caused by t. interdigitale and t. rubrum in this study . it should also be noted that like the study of rezaei - matehkolaei et al . ( 6 ) , among the strains of m. canis the most cases of isolation ( 31% ) were correlated to tinea corporis . this zoophilic dermatophyte was cited as the dominant agent of tinea corporis in some european countries ( 3 , 31 ) and currently is the most common microsporum species causing dermatophytosis in iran ( 6 , 8 , 9 , 10 , 12 , 13 , 16 ) . irrespective of all these , most cases of tinea corporis are currently associated with t. rubrum worldwide ( 3 , 4 , 25 ) . dermatophytosis of glabrous skin of the face has less extensively been studied , because it often not regarded as an independent entity , but as part of tinea corporis ( 2 , 16 , 32 ) or tinea gladiatorum ( 11 ) . the infection is more frequently diagnosed in children due to contact with pets such as cat , dog and rabbit , and in adults as a result of contact with livestock or physical activities typical of this age group ( 32 , 33 ) . different studies showed that the predominant species responsible for tinea faciei varies regarding to the geographic location and potential animal reservoir ( 32 , 33 ) . studies performed in ahvaz , tehran , qazvin and hamadan showed that the infection was more common in males than females and the related frequent species were t. interdigitale ( 12 ) , m. gypseum and t. rubrum ( 13 , 14 ) and t. verrucosum(9 , 10 ) . in the studies carried out in greece and italy m. canis was mentioned as the prevailing agent of tinea faciei(25 , 31 , 32 ) . in our study the infection was manifested as the same ratio in males and females and t. interdigitale , t. tonsurans , t. rubrum and m. canis were respectively the main corresponding species , which partially differed from the recent report from tehran ( 6 ) that found t. tonsurans and m.canis as the dominant agents . ringworm of the hand accounted for 3.14% of all infections and like the study performed in tehran t. interdigitale and t. rubrum comprised the most frequent causes , respectively . similarly , these two species were reported as the prevalent causes of tinea manuum in the majority of reports from iran and europe ( 10,12 , 13 , 25 , 31 , 34 ) . one of the remarkable aspects of the current survey was the first confirmed report of tinea manuum in an iranian 60-year - old female due to t.anamorph of a. benhamiae . this trichophyton sp . and t. erinacei are two zoophilic dermatophytes that formerly known as t. mentagrophytes var . grannulosum , respectively , however currently both fungi have species status and are considered as the anamorphic stages of the teleomorph a. benhamiae ( 35 ) . ecologically , both species adapted with house pets like hedgehogs , rabbits , guinea pigs , dogs and cats as the main hosts and the possible sources of human infections ( 35 , 36 ) . since , pets are not well - liked in iran ( 6 , 16 ) , incidence of infection by a. benhamiae is low and the sources of infection for such species are likely domestic or farm animals . generally , the data pertaining to the epidemiology of taxa in a. benhamiae complex are limited , nevertheless in some recent reports the species was considered as an emerging pathogen of dermatophytosis in human and animals ( 36 , 37 ) . a long time ago , infection of the scalp skin and hair shafts , which predominantly is common among preschool going and preadolescent populations ( 4 ) was the main form of dermatophytosis in iran ( 8 , 9 ) . in contrary , recent studies showed a gradual decrease in the frequency of infection ( 6,10 , 12 ) and it also ranked the least in our study ( 2.8% of all infections ) , after tinea barbae . on the other hand , congruent with the observations made in iran it was limited to the lower age groups of 1 - 10 and 11 - 20 year ( 6 , 8 , 9 , 12 , 15 ) . generally , the high prevalence rate of tinea capitis in some areas of africa , especially in warm countries of the tropics was attributed to the inter - human transmission of infection among the school children and low level of lifestyle , however in asiatic countries the prevalence of infection decreased due to improvement in socioeconomic and sanitary condition ( 4 , 38 , 39 ) . epidemiologically , different geographic regions maintain distinct species of dermatophytes as agents of tinea capitis . while , t. tonsurans represents most cases of infection in uk and north america , m. canis is the predominant species in most parts of continental europe and australia , and t. violaceum in south asia and north africa ( 4 , 40 ) . in studies performed in tehran and ahvaz , t. violaceum and m. canis were cited as the most common etiologic agents of tinea capitis ( 6 , 12 , 13 , 15 ) , but comparatively , in our study t. interdigitale surpassed over other species . a likely explanation for this transition may be that the distribution profile of dermatophytes involved in distinct type of dermatophytosis varies geographically , since the current survey was extended to four different provinces . another salient finding , like the recent study from tehran , was that no case of tinea capitis by t. violaceum was encountered . from the available data , it seems that dermatophytoses due to t. violaceum in general , and scalp infection in particular are eradicating in iran . the most interesting finding in our survey might be an episode of tinea capitis ( ectothrix type ) by e. floccosum in a6-year - old girl . epidermophyton floccosum is an anthropophilic species with worldwide distribution ( 1,2 , 5 ) , however reports in relation to tinea capitis by this species are very rare . to the extent of our knowledge , there are sporadic reports from palestine ( 41 ) , nigeria ( 42 ) , italy ( 43 , 44 ) and canada ( 45 ) regarding the isolation of e. floccosum from scalp lesions and our case is the sixth worldwide . albeit rarely , this finding makes emphatic that this species has the potential to parasitize and infect the hair . in recent years , incidence of scalp lesions by trichophyton schoenleinii ( tinea favosa ) has decreased worldwide ( 39 ) and there are evidences for eradication of this dermatophyte in developed nations ( 5 , 7 ) . in line with this fact , the isolation rate of this fungus in the present study and some recent surveys from iran substantially decreased ( 6 , 13 , 14 ) . like the study of aghamirian and ghiasian(10 ) and maraki et al.(25 ) a significant number of our patients ( n=93 ) had mixed infections , consisting of concomitant tinea unguium / tinea pedis , tinea cruris / tinea pedis and tinea cruris / tinea corporis . obviously , in these patients the same dermatophyte species was isolated from both skin and nail samples and the responsible agents , in order of frequency were t. interdigitale , t. rubrum and e. floccosum . in the few studies in greece and spain , which paid attention to this subject it was also found that t. rubrum and t. interdigitale tend to establish co - infection in multiple organs ( 25 , 27 ) . generally , derma - tophytosis simultaneouslyin more than one anatomical site is expected and frequently occurs due to scratching of the lesions by infected patient and mechanical spreading ( 7 , 25 ) . recently it was shown that infection of the beard and mustache took a decreasing trend in iran since at the moment men pay more attention to self - care ( 6 , 10 ) . interestingly , the only observed case of tinea barbae was due to the anthropophilic species t. rubrum , while in many reports from iran the dermatophytes other than t. rubrum , especially zoophilic species were listed as the infectious agents ( 812 ) . from the etiological point of view , while t. verrucosum constituted a notable portion of isolates in earlier studies in iran , exceptionally no case of infection by this species were recognized in this investigation and the recent study conducted in tehran ( 6 ) . in other words , all referred strains from four provinces in which morphologically identified as t. verrucosum , in bt2-rflp analysis were known as t. interdigitale . species - to - species transition in the dermatophytes taxonomy meaning that most of the strains which morphologically identified as t. verrucosum in many parts of the world ( including iran ) , are unambiguously the former t. verrucosum var . autotrophicum ( an invalid name ) , which recently regarded to be conspecific with t. interdigitale based on new molecular analyses ( 35 , 46 ) . t. interdigitale was the predominant agent of most infections in this study and given to the increase in occurrence of tinea pedis and tinea unguium , two old unsolved problems , it is likely to remain as the main pathogenic species in iran . although infection with t. verrucosum and some other species was not detected in recent molecular epidemiology surveys , even so this does not mean that the country is free of these species . additional investigations , with emphasis to transition from phenotypic methods towards molecular approaches relying on new perspective to concept of species , are required to elucidate the full spectra of dermatophytes specie in iran , since the regional and sectional studies may not necessarily be the representative of overall pattern in the country . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

abstractbackgroundsurveillance of dermatophytosis is essential to determine the likely changes in etiological trends and distribution profile of this infection . in this study beta tubulin gene ( bt2 ) , was used as the first time in a pcr - rflp format to clarify the distribution of dermatophytosis agents in some parts of iran.methodsa total of 603 clinical isolates was obtained from 500 patients in tehran , isfahan , mazandaran and guilan provinces . the isolates were identified using macro / micro - morphological criteria and electrophoretic patterns of pcr amplicons of bt2after digestion with each of the restriction enzymes fati , hpych4v , mwoi and alw21i.resultsamong the patients , 59.2% were male and 40.8% female . the most prevalent clinical form was tinea pedis ( 42.4% ) , followed by tinea cruris ( 24.2% ) , tinea unguium ( 12.3% ) , tinea corporis ( 10.8% ) , tinea faciei ( 4% ) , tinea manuum ( 3.14% ) , tinea capitis ( 3% ) and tinea barbae ( 0.16% ) , respectively . trichophyton interdigitale ranked the first , followed by t. rubrum , epidermophyton floccosum , microsporum canis , t. tonsurans , t. erinacei and t. violaceum ( each 0.49% ) and the less frequent species were t. schoenleinii , m. gypseum and t.anamorph of arthroderma benhamiae ( each 0.16% ) . a case of scalp infection by e. floccosum was an exceptional event in the study . no case of t. verrucosum was found.conclusiontrichophyton species and e. floccosum are yet the predominant agents of infection in iran , while microsporum species are decreasing . t. interdigitale and tinea pedis remain as the most causal agent and clinical form of dermatophytosis , respectively . it seems that bt2 can be a useful genetic marker for epidemiological survey of common pathogenic dermatophytes .

Introduction Materials and Methods Isolates Molecular identification Results Discussion Conclusion Ethical considerations

large scale epidemiological data about diversity of pathogenic dermatophytes in different regions of iran have rarely been documented , however dermatophytosis has increasingly been reported as the dominant superficial mycosis in the country ( 6 , 812 ) . the current study aimed to describe the spectrum of dermatophytoses and dermatophytes in some parts of iran , with special emphasis on the species delineation of isolates implicated in infection using a newly described pcr - rflp on -tubulin gene ( 21 ) . tehran , isfahan , mazandaran and guilan , the dermatophyte strains from 500 culture - proven cases of dermatophytosis were included in the study . beta tubulin ( bt2 ) region was partially amplified in a 25 l reaction mixture containing 2.5 l of 10x pcr buffer , 10 pmol of each forward t1 ( 22 ) and reverse bt2b ( 23 ) primers , 1 l of genomic dna , 1.5 m mgcl2 , 400 m deoxynucleotide triphosphates , 1.25 u of taq dna polymerase and enough sterile deionized water . species identification was performed in a two - step rflp assay , preliminary by using the restriction enzyme fati , ( new england biolabs ltd , nhitchin , uk ) and subsequently by mwoi and hpych4v ( new england biolabs ltd , nhitchin , uk ) , and alw21i ( fermentas , vilnius , lithuania ) according to the manufacturer s instructions . dna cleavage was performed at 37 c for 2 h. to confirm the production of amplified and digested dnas of the expected size , 10 l of each amplicons and digested fractions were respectively electrophoresed on 1.5 and 2 % gel agarose , stained with ethidium bromide ( 0.5 g / ml ) , visualized under uv illumination and the banding patterns were recorded by photography . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) flowchart of molecular identification steps for identification of dermatophytes in the study based on beta - tubulin rflp . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) during june 2011 to june 2012 , among outpatients referred to medical mycology laboratories in four provinces of iran i.e. tehran , isfahan , mazandaran and guilan , the dermatophyte strains from 500 culture - proven cases of dermatophytosis were included in the study . beta tubulin ( bt2 ) region was partially amplified in a 25 l reaction mixture containing 2.5 l of 10x pcr buffer , 10 pmol of each forward t1 ( 22 ) and reverse bt2b ( 23 ) primers , 1 l of genomic dna , 1.5 m mgcl2 , 400 m deoxynucleotide triphosphates , 1.25 u of taq dna polymerase and enough sterile deionized water . species identification was performed in a two - step rflp assay , preliminary by using the restriction enzyme fati , ( new england biolabs ltd , nhitchin , uk ) and subsequently by mwoi and hpych4v ( new england biolabs ltd , nhitchin , uk ) , and alw21i ( fermentas , vilnius , lithuania ) according to the manufacturer s instructions . dna cleavage was performed at 37 c for 2 h. to confirm the production of amplified and digested dnas of the expected size , 10 l of each amplicons and digested fractions were respectively electrophoresed on 1.5 and 2 % gel agarose , stained with ethidium bromide ( 0.5 g / ml ) , visualized under uv illumination and the banding patterns were recorded by photography . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) flowchart of molecular identification steps for identification of dermatophytes in the study based on beta - tubulin rflp . ( the numbers in parentheses are size of the fragments after digestion with each relevant restriction endonuclease ) totally , 603 dermatophytes strains were isolated from patients of which 394 , 41 and 38 isolates were originated from three northern provinces i.e. tehran , guilan and mazandaran , respectively and 130 from isfahan province , in the center of iran . in the two - step bt2-rflp by fati , hpych4v , mwoi and alw21i , 599 strains created banding patterns matched with expected species - specific profile , leading to the delineation of t. interdigitale as the highest in terms of frequency , followed by t. rubrum , epidermophyton floccosum , m. canis , t. tonsurans , t. violaceum , t. schoenleinii and m. gypseum . four remaining strains which created restriction fragments for the arthroderma benhamiae clade , were subjected to sequencing and identified as t. erinacei ( n=3 ) and t. anamorph of a. benhamiae ( n=1 ) . except in tinea cruris and tinea barbae , t. interdigitale was the dominant species isolated in all clinical conditions and all provinces . clinical presentation of the infection revealed that feet involvement ( tinea pedis ) was the predominant clinical condition ( 42.4% ) followed by tinea cruris ( 24.2% ) , tinea unguium ( 12.3% ) , tinea corporis ( 10.8% ) , tinea faciei ( 4% ) , tinea manuum ( 3.14% ) , tinea capitis ( 3% ) and tinea barbae ( 0.16% ) , respectively . anamorph of a. benhamiaetotal distribution of isolates in relation to sex(m / f)*tinea pedis16969115110000256 ( 165/91)tinea cruris6339394010000146(81/65)tinea unguium40284100100074 ( 50/24)tinea corporis241614910100065 ( 28/37)tinea faciei950360100024 ( 12/12)tinea barbae01000000001(1/0)tinea manuum761301000119 ( 11/8)tinea capitis611440011018 ( 10/8)total ( % ) and geographical distribution of isolates in the study294 ( 48.75)165 ( 27.36)94 ( 15.58)29 ( 4.8)12 ( 1.99)3 ( 0.49)3 ( 0.49)1 ( 0.16)1 ( 0.16)1 ( 0.16)maz:21maz : 6maz : 7maz : 1maz : 1maz : 1maz : 1maz : 0maz : 0maz : 0gui : 15gui : 14gui : 9gui : 1gui : 2gui : 0gui : 0gui : 0gui : 0gui : 0603 ( 358/245)isf : 66isf : 25isf : 24isf : 6isf : 2isf : 2isf : 2isf : 1isf : 1isf : 1teh : 192teh : 120teh : 54teh : 21teh : 7teh : 0teh : 0teh : 0teh : 0teh : 0 * m / f : male/ female maz : mazandaran , gui : guilan , isf : isfahan , teh : tehran table.2distribution of dermatophytosis among patients of the study in regard to the age groupsage group clinical form1 - 10 ( % ) 11 - 20 ( % ) 21 - 30 ( % ) 31 - 40 ( % ) 41 - 50 ( % ) 51 - 60 ( % ) 61 - 70 ( % ) 71 - 80 ( % ) 80 < ( % ) total ( % ) tinea pedis2 ( 0.33\)10 ( 1.65)31 ( 5.14)54 ( 8.95)61 ( 10.11)57 ( 9.45)37 ( 6.13)2 ( 0.33)2 ( 0.33)256 ( 42.45)tinea cruris1 ( 0.16\)13 ( 2.15)55 ( 9.12)33 ( 5.47)23 ( 3.81)8 ( 1.32)10 ( 1.65)1 ( 0.16)2 ( 0.33)146 ( 24.21)tinea unguium2 ( 0.33)15 ( 2.48)10 ( 1.65)6 ( 0.99)22 ( 3.64)16 ( 2.65)3 ( 0.49)74 ( 12.27)tinea manuum2 ( 0.33)9 ( 1.49)3 ( 0.49)4 ( 0.66)1 ( 0.16)19 ( 3.15)tinea corporis11 ( 1.82)29 ( 4.80)2 ( 0.33)13 ( 2.15)5 ( 0.82)4 ( 0.66)1 ( 0.16)65 ( 10.77)tinea capitis12 ( 1.99)6 ( 0.99)18 ( 2.98)tinea faciei4 ( 0.66)8 ( 1.32)6 ( 0.99)1 ( 0.16)1 ( 0.16)4 ( 0.66)1 ( 0.16)24 ( 4)tinea barbae11 ( 0.16)total ( % ) 19 ( 3.15)52 ( 8.62)145 ( 24.04)103 ( 17.08)103 ( 17.08)97 ( 16.08)71 ( 11.77)8 ( 1.32)5 ( 0.82%)603 ( 100 ) frequency of dermatophytoses regarding to the dermatophyte species and patients gender distribution of dermatophytosis among patients of the study in regard to the age groups dermatophytosis annually impacts millions of individuals and imposes a substantial cost to the societies for treatment ( 2 , 24 ) . the distribution pattern and causative agents of infection continuously change , therefore it is essential to update our knowledge in regard to the epidemiology of dermatophytes . thus , this is the first application of pcr - rflp on -tubulin in a molecular epidemiological monitoring of dermat - ophytes worldwide . the frequency arrangement of causative species in the present study was similar to the recent molecular epidemiology report from tehran ( 6 ) , in which t. interdigitale , t. rubrum , e. floccosum , m. canis and t. tonsurans were the main agents of infection in decreasing order . the incidence of infection by t. interdigitale ( 48.75% ) as the most common species increased in this survey , but not significantly compared with that described by rezaei - matehkolaei et al. tinea pedis , the main clinical manifestation , was accounted for 42.4% of all infections followed by tinea cruris and tinea corporis and this finding was concordant with some reports from iran ( 6 , 16 ) . rubrum and e. floccosum were the most frequent , whereas currently t. rubrum is the main species implicated in feet infection worldwide ( 2 , 3 , 7 ) . our study highlighted this fact that dermatophytosis of the feet is more common in adult - males and infrequent in females and children ( 16 , 26 , 27 ) , because the infection more occurred in age groups between 31 - 60 years , was nearly twice as frequent in men than women ( 64.4% vs 35.5% ) and only 0.33% of the cases involved prepubescent patients . it was noteworthy that we found a case of foot infection by t. erinacei among our samples , which was the first proven report of tinea pedis by this species from iran . similar to two recent studies conducted in iran ( 6 , 16 ) involvement of groin , perianal and perineal ranked second in prevalence and e. floccosum was the most common agent which was in accordance with the reports from the past till now in iran ( 6 , 8 , 10 , 12 , 13 , 16 ) . while t. rubrum still remains the most frequent cause worldwide ( 24 ) , e. floccosum seems to become more prominent as the main agent of tinea cruris in iran . at present , t. rubrum is the most common dermatophyte causing onychomycosis throughout the world ( 24 ) and it also was addressed as the predominant agent of nail infection in some reports from tehran , qazvin and babol ( 6 , 10 , 16 , 28 ) . the causative agents of tinea corporis , the fourth prevalent infection in the study , are extensively various in different areas and a wide range of species were reported from inside of the country and overseas . in previous reports from ahvaz , hamadan , qazvin , isfahan and tehran , t. interdigitale ( 12 ) , e. floccosum ( 13 ) , t. tonsurans ( 16 ) and t. verrucosum ( 810 ) were enumerated as the main agents involved in ringworm of the body . however , in agreement with the latest molecular epidemiological survey , performed in tehran ( 6 ) the infection predominantly caused by t. interdigitale and t. rubrum in this study . this zoophilic dermatophyte was cited as the dominant agent of tinea corporis in some european countries ( 3 , 31 ) and currently is the most common microsporum species causing dermatophytosis in iran ( 6 , 8 , 9 , 10 , 12 , 13 , 16 ) . studies performed in ahvaz , tehran , qazvin and hamadan showed that the infection was more common in males than females and the related frequent species were t. interdigitale ( 12 ) , m. gypseum and t. rubrum ( 13 , 14 ) and t. verrucosum(9 , 10 ) . in our study the infection was manifested as the same ratio in males and females and t. interdigitale , t. tonsurans , t. rubrum and m. canis were respectively the main corresponding species , which partially differed from the recent report from tehran ( 6 ) that found t. tonsurans and m.canis as the dominant agents . ringworm of the hand accounted for 3.14% of all infections and like the study performed in tehran t. interdigitale and t. rubrum comprised the most frequent causes , respectively . one of the remarkable aspects of the current survey was the first confirmed report of tinea manuum in an iranian 60-year - old female due to t.anamorph of a. benhamiae . grannulosum , respectively , however currently both fungi have species status and are considered as the anamorphic stages of the teleomorph a. benhamiae ( 35 ) . since , pets are not well - liked in iran ( 6 , 16 ) , incidence of infection by a. benhamiae is low and the sources of infection for such species are likely domestic or farm animals . in contrary , recent studies showed a gradual decrease in the frequency of infection ( 6,10 , 12 ) and it also ranked the least in our study ( 2.8% of all infections ) , after tinea barbae . generally , the high prevalence rate of tinea capitis in some areas of africa , especially in warm countries of the tropics was attributed to the inter - human transmission of infection among the school children and low level of lifestyle , however in asiatic countries the prevalence of infection decreased due to improvement in socioeconomic and sanitary condition ( 4 , 38 , 39 ) . while , t. tonsurans represents most cases of infection in uk and north america , m. canis is the predominant species in most parts of continental europe and australia , and t. violaceum in south asia and north africa ( 4 , 40 ) . in studies performed in tehran and ahvaz , t. violaceum and m. canis were cited as the most common etiologic agents of tinea capitis ( 6 , 12 , 13 , 15 ) , but comparatively , in our study t. interdigitale surpassed over other species . another salient finding , like the recent study from tehran , was that no case of tinea capitis by t. violaceum was encountered . from the available data , it seems that dermatophytoses due to t. violaceum in general , and scalp infection in particular are eradicating in iran . the most interesting finding in our survey might be an episode of tinea capitis ( ectothrix type ) by e. floccosum in a6-year - old girl . epidermophyton floccosum is an anthropophilic species with worldwide distribution ( 1,2 , 5 ) , however reports in relation to tinea capitis by this species are very rare . to the extent of our knowledge , there are sporadic reports from palestine ( 41 ) , nigeria ( 42 ) , italy ( 43 , 44 ) and canada ( 45 ) regarding the isolation of e. floccosum from scalp lesions and our case is the sixth worldwide . (25 ) a significant number of our patients ( n=93 ) had mixed infections , consisting of concomitant tinea unguium / tinea pedis , tinea cruris / tinea pedis and tinea cruris / tinea corporis . obviously , in these patients the same dermatophyte species was isolated from both skin and nail samples and the responsible agents , in order of frequency were t. interdigitale , t. rubrum and e. floccosum . in the few studies in greece and spain , which paid attention to this subject it was also found that t. rubrum and t. interdigitale tend to establish co - infection in multiple organs ( 25 , 27 ) . interestingly , the only observed case of tinea barbae was due to the anthropophilic species t. rubrum , while in many reports from iran the dermatophytes other than t. rubrum , especially zoophilic species were listed as the infectious agents ( 812 ) . from the etiological point of view , while t. verrucosum constituted a notable portion of isolates in earlier studies in iran , exceptionally no case of infection by this species were recognized in this investigation and the recent study conducted in tehran ( 6 ) . species - to - species transition in the dermatophytes taxonomy meaning that most of the strains which morphologically identified as t. verrucosum in many parts of the world ( including iran ) , are unambiguously the former t. verrucosum var . t. interdigitale was the predominant agent of most infections in this study and given to the increase in occurrence of tinea pedis and tinea unguium , two old unsolved problems , it is likely to remain as the main pathogenic species in iran .

although extracellular , axenic development of the erythrocyte cycle of p. falciparum has been obtained [ 2 - 4 ] , the number of merozoites completing the cycle is not sufficient to permit continuous extracellular culture . under the best conditions , only 1% of the merozoites further develops into trophozoites therefore , the isolation of trophozoites still relies on their liberation from the host cells . several methods for the isolation of trophozoites have been developed including : agglutination and lysis by passage through a series of filters , glycerol - enhanced haemolysis and sorbitol lysis . however , to prepare intact trophozoites in high yield and of high purity remains technically difficult . these methods either affect the integrity of the parasites or fail to remove host materials from the parasites . the most widely used method for freeing parasites from their host cells is saponin lysis , but electron microscopy studies have shown that these parasites are still trapped within the erythrocyte plasma membrane . released parasites need to be further cleansed of host cell materials , in particular erythrocytes and their ghosts , as well as unlysed infected erythrocytes . while methods to isolate host cell ghosts from parasitised erythrocytes have been developed , there is no published method to isolate the parasite plasma membrane . the isolation of the parasite plasma membrane presents technical difficulties due to the presence of three closely related membranes ( 1 ) the erythrocyte plasma membrane ( 2 ) the parasitophorous vacuolar membrane and ( 3 ) the parasite plasma membrane , as well as the food vacuole membrane . unfortunately these preparations were found to contain membrane elements of both parasites and host cells . although the p. falciparum vacuolar atpase activity have been characterised , the atpase activity of the parasite plasma membrane has not yet been investigated . to date , investigation of the atpase activity of the plasma membrane has been hampered by the presence of contaminants in the membrane preparations . preparation of uncontaminated parasite plasma membranes would represent a major experimental advance for approaching this problem . for this reason , a method was developed to prepare pure and intact parasite plasma membranes from p. falciparum infected - erythrocytes . the dependence of p. falciparum plasma membrane atpase activity on atp and other nucleotides , divalent cations , time , atp and mg concentrations was also evaluated . , polystyrene beads coated with anti - erythrocyte antibodies were incubated with the trophozoite preparation . trophozoites were then biotinylated with nhs - ss - biotin prior to nitrogen cavitation in the presence of protease inhibitors and dnasei . the resulting biotinylated membranes bound to streptavidin - magnetic beads and were separated from the lysate using a magnet ( figure 1 ) . the integrity of purified trophozoites was monitored by measuring the uptake of the trypan blue dye into the parasite cytoplasm . it was shown that 98% of the trophozoites released by saponin lysis did not concentrate the dye , indicating an intact plasma membrane . purified p. falciparum plasma membranes examined using transmission electron microscopy were observed to form intact vesicles , although some open membranes could be seen . no visible contamination by other membranes or organelles was observed , although some liberated hemozoin crystal could be seen ( results not shown ) . the purity of the parasite plasma membrane preparations was further assayed by the use of enzyme markers of p. falciparum cytosol , the parasite lactate dehydrogenase ( pldh ) and of human erythrocyte membranes , the acetylcholine esterase ( ache ) . there were no detectable ache or pldh activities ( less than 0.01 mol / min / mg protein ) in the parasite plasma membrane preparations ( table 1 ) . by comparison , the specific activity of ache was 2.30 0.15 and 0.05 0.03 mol / min / mg protein in erythrocyte ghosts and isolated trophozoites ( n = 3 ) respectively , while the specific pldh activity in isolated trophozoites was 0.14 0.01 mol / min / mg protein ( n = 3 ) . acetylcholine esterase ( ache ) and parasite lactate dehydrogenase ( pldh ) activities of isolated trophozoites , parasite plasma membranes and/or erythrocyte ghosts were determined as described under materials and methods section . values are means standard deviation of three separate experiments from separate parasite plasma isolations , trophozoite isolations or erythrocyte membrane preparations . time dependence of the atpase activity of plasma membranes isolated from the p. falciparum strain d10 was evaluated over a time period of 45 minutes . a linear relationship of time versus atpase activity was found with a correlation coefficient of 0.717 ( n = 3 ) ( figure 2 ) . atpase in plasma membrane preparations required the presence of divalent cations to maintain its activity . atpase activity was maximal with 2 mm mg in the presence of 2 mm atp ( figures 3,4 ) . the ability of 2 mm concentration of mg and the other divalent cations mn and ca to support the atpase activity of purified plasma membranes isolated from the p. falciparum strain d10 was evaluated , along with no divalent cation addition as a control ( table 2 ) . a significant activity ( 88 33% ) was measured when mg was replaced with mn . when ca was substituted for mg , only 21 19% of the atpase activity was retained . plasma membranes were also capable of hydrolysing other nucleoside triphosphates as effectively as atp ( table 3 ) . purified parasite plasma membranes isolated from the sensitive strains d10 displayed an atpase activity of 3.2 1.7 nmol pi / min / mg protein . atpase activity ( nmol pi / mg protein ) of d10 plasma membranes was determined over a time period of 45 minutes and the activity of the membranes tested without atp was subtracted . error bars represent standard deviations from means of three separate experiments , each performed in quadruplicate . atpase activity of freshly purified d10 plasma membranes expressed as nmol pi / min / mg protein was measured over mg concentrations ranging from 0 to 5 mm . atpase activity of parasite plasma membranes tested without atp and absorbance due to atp hydrolysis was subtracted . error bars represent standard deviations from means of three separate experiments , each performed in quadruplicate . atpase activity of freshly purified d10 plasma membranes expressed as nmol pi / min / mg protein was measured over atp concentrations ranging from 0.5 to 5 mm . atpase activity of parasite plasma membranes tested without atp and absorbance due to atp hydrolysis were subtracted . error bars represent standard deviations from means of three separate experiments , each performed in quadruplicate . atpase activity was determined using 2 mm concentration of mg , mn and ca and the atpase activity in the absence of divalent cation was subtracted . values are means of three separate experiments , each performed in quadruplicate standard deviations and are expressed as % of control in presence of mg . atpase activity of isolated d10 plasma membranes tested without nucleotide and absorbance due to the nucleotide hydrolysis was subtracted . values are means of three separate experiments , each performed in quadruplicate standard deviations and are expressed as % of control in presence of atp . n.d . a procedure for isolating parasite plasma membrane from p. falciparum - infected erythrocytes was developed . in this method , trophozoites were released from the erythrocytes by saponin lysis , the remaining erythrocytes membranes being removed by immunoaffinity using anti - erythrocyte antibodies . previously , anti - erythrocyte monoclonal antibodies have been used to remove unlysed erythrocytes and ghost membranes after sorbitol treatment . however , in order to remove most of the erythrocyte ghosts by centrifugation , all the erythrocytes must be lysed by the detergent , thus allowing isolation of the trophozoites on a larger scale . saponin , on the other hand lysed uninfected and infected erythrocytes . for this reason , sorbitol lysis of the trophozoites was not retained in this study . however , because saponin - treatment of parasitized erythrocytes has been shown to permeabilise both the erythrocyte plasma membrane and the parasitophorous membrane the exposure of the trophozoites to saponin was very brief to minimize membrane damage . contamination of the plasma membrane vesicle preparations by the parasitophorous membrane , however , could not be discounted since , to our knowledge , there is no specific marker for this membrane . a number of criteria have to be met when isolating subcellular fractions for biochemical experiments . the subcellular constituents have to be pure , intact , isolated in high yield and retain their normal physiological capabilities . evidence for the membrane integrity of saponin - freed parasites came from studies measuring the rate of incorporation of [ c]-isoleucine into protein and the rate of phosphorylation of the pantothenic acid . in this study , experiments with the trypan blue showed that 98% of the trophozoites released by saponin lysis were capable of maintaining an intact plasma membrane . nitrogen decompression has been previously shown to be effective for the disruption of cells and was used in this study to disrupt the parasite plasma membranes . a cocktail of protease inhibitors ( aprotinin , leupeptin and phenylmethylsulfonyl fluoride ) was used to prevent the degradation of membrane proteins by the proteases released during the nitrogen cavitation step . the presence of dna presents an additional problem , as the parasites adhere to the sticky dna liberated during parasite lysis . dnase i was , therefore , included in the isolation process to eliminate the dna liberated during the nitrogen cavitation step . the recovery yield of the parasite plasma membranes , 8.2% of the total isolated - trophozoite protein , is consistent with the percent protein yield ( 810% ) obtained for membrane vesicles prepared from cancer cells . purified p. falciparum examined using transmission electron microscopy were observed to be intact and free of contaminating membranes and organelles , even though a small amount of liberated haemozoin could occasionally be seen in certain fields . contamination of the plasma membrane vesicles with fragments of food vacuole membrane could not be ruled out since nitrogen cavitation is likely to disrupt the food vacuole membrane as well as the plasma membrane . it would therefore had been interesting to investigate whether the parasite plasma membrane preparations were contaminated by food vacuole membrane components using antibodies to pfcrt . the absence of parasite and erythrocyte protein in the parasite plasma membrane preparation was confirmed by non - detectable parasite lactate dehydrogenase and erythrocyte acetylcholine esterase activities . the fact that the membranes were bound to a support facilitated their purification from contaminants and their recovery . the atpase activity of parasite plasma membranes ( 3.2 1.7 nmol pi / min / mg protein ) obtained in this study was found to be of similar magnitude to that of digestive vacuoles isolated from p. falciparum . for comparison , the specific atpase activity of plasma membranes prepared from multidrug - resistant cancer cells is 1.15 mol / min / mg membrane protein . conditions required for optimal p. falciparum plasma membrane atpase activity were determined using the chloroquine - sensitive strain d10 . parasite plasma membranes showed optimal atpase activity at 2 mm mg and 2 mm atp . other divalent cations can be substituted for mg and maintain the function of a variety of different atpases . in this study , it was found that ca could not be substituted for mg to sustain atpase activity in parasite plasma membranes , whereas activity was preserved when mg was replaced by mn , indicating that the parasite plasma membrane atpase activity was similar to the vacuolar membrane atpase activity . however , this contradicts the choi and mego study which showed that ca could support the p. falciparum vacuolar atpase activity in the absence of mg . however , the nucleoside monophosphate amp was not hydrolysed , suggesting that the parasite plasma membrane atpase activity is distinguishable from that of membrane - associated alkaline and acid phosphatases . since the parasite plasma membranes were isolated in the form of intact vesicles the right - side - out , the atp hydrolysing regions of the atpase proteins should be enclosed within the vesicles . this raises the question of whether all of the compounds tested are gaining access to the relevant part of the protein , and whether the differences between the atpase activities measured in the presence of the different agents might reflect , at least to some extent , the different ability of these agents to gain access to the interior of the vesicles . therefore , it could not be excluded that the saturable dependence of atpase activity on the concentration of atp in the medium could reflect the saturable transport of atp into the vesicles , rather than the saturation of the atpase itself . similarly , the differential ability of divalent cations to support atpase activity could be the result of a differential transport of these cations into the vesicles . in conclusion , a method for the isolation and purification of the p. falciparum plasma membrane was developed and optimal conditions for p. falciparum plasma membrane atpase activity were determined . these purified membranes were intact and isolated in a high enough yield to enable a characterisation of their atpase activity . thus , plasma membranes cleansed of p. falciparum can be used as a model to establish the role played by atpases in ion transport . the availability of purified p. falciparum plasma membrane will be of value not only to investigate processes such as nutrient uptake / efflux , ph regulation and ion balance but also in the understanding of drug transport in this organelle . parasites were maintained in continuous culture at a 5% haematocrit in rpmi-1640 with glutamine ( sigma ) supplemented with hypoxanthine ( 44 mg / l ) , hepes ( 6 g / l ) , gentamycin ( 50 mg / l ) , and 10% a human serum ( western province blood transfusion service and haematology department , groote schuur hospital , cape town , south africa ) . parasites were grown in o - positive red cells and the parasitemia was kept between 1015 % . parasite cultures were incubated at 37c in dessicator cabinets under an atmosphere of 93% nitrogen , 4% carbon dioxide , 3% oxygen . infected erythrocytes were washed twice in 10 volumes of phosphate buffered saline ( pbs ) . the pellet was suspended in 10 volumes of pbs containing 0.05 % ( w / v ) saponin in order to lyse the erythrocytes . it was then incubated for 23 min at room temperature and centrifuged at 1,500 g for 10 min . the resulting pellet was washed twice in pbs and resuspended in 5 volumes of pbs . the protease inhibitors aprotinin , leupeptin and phenylmethylsulfonylfluoride ( boehringer mannheim ) were added to final concentration of 10 g / ml , 10 g / ml and 0.1 mm , respectively . unlysed erythrocytes and erythrocyte membranes contained in the trophozoite preparation were adsorbed by immuno - affinity . anti - human erythrocytes ( polyvalent immunoglobulin ( g , a , m ) rabbit , sigma ) were diluted 1 : 1000 in pbs . 5 g of polystyrene beads ( pierce ) were incubated in 20 ml of the above solution for two hours at room temperature with gentle agitation . the beads were washed in pbs and further incubated for one hour with 20 ml 1 % bovine serum albumin in pbs . the beads were then washed again in pbs and incubated with the trophozoite preparation for 30 min . the ability of purified trophozoites to exclude trypan blue was monitored by adding 10 l of trypan blue ( 10 mg / ml in distilled water ) to 100 l of trophozoite suspension in pbs ( phosphate buffered saline ) . after five minutes incubation at room temperature , the proportion of parasites showing trypan blue accumulation was evaluated using a phase contrast microscope . the biotylination reaction was performed by incubating 250 l of 1 mg / ml nhs - ss - biotin ( sulfosuccinimidyl 2-[biotin - amido ] ethyl-1,3-dithiopropionate ) ( pierce ) with 500 l of trophozoites ( 2 mg of protein ) for 30 min at room temperature . the resulting biotinylated trophozoites were washed once in pbs , then twice in vesicle buffer i ( 0.25 m sucrose , 1 mm e.d.t.a . vesicles were prepared by nitrogen cavitation according to the method of lever et al .. the cells were resuspended in 500 l vesicle buffer i containing aprotinin , leupeptin and phenylmethylsulfonylfluoride ( 10 g / ml , 10 g / ml and 0.1 mm , respectively ) and equilibrated at 4c under nitrogen pressure at 800 psi for 30 min . the homogenate was then incubated for five min at 37c with dnase i ( 50 g / ml ) . the biotinylated vesicles were incubated with 100 l of streptavidin immobilized on iron oxide ( sigma ) previously washed with pbs for 15 min at room temperature and then separated using a magnet ( magnetic cell sorting , myltenyi biotec ) . the magnetic particles were bound to the side of the tube and washed with 1 ml of 0.25 m sucrose , 10 mm tris - hcl ph 7.4 . parasite plasma membrane vesicles were fixed with glutaraldehyde ( 0.8%)/formaldehyde ( 8% ) in pbs for one hour at room temperature and washed in pbs , followed by addition of 50 mm nh4cl for 30 minutes and two washes in pbs . samples were dehydrated in 70% ethanol and embedded in lr white resin ( polyscience inc . ) sections were mounted on copper grids and stained with 2% uranyl acetate and lead acetate . the acetylcholine esterase ( ache ) activity was determined by a modification of the method of ellman et al . . enzyme assays were carried out with 200 l of freshly prepared 0.1 m sodium phosphate ph 7.5 , 0.5 mm 2,2'-dinitro-5,5'-dithiobenzoic acid , 0.6 mm s - acetylthiocholine iodide to which was added 50 l of a suspension of erythrocyte ghost , trophozoites or parasite plasma membrane , in microtitre 96-well plates . 10 g of erythrocyte ghost protein , 35 g of trophozoites protein or 20 g of parasite plasma membrane protein were used to measure an initial rate . wells containing 200 l h2o and 50 l of erythrocyte ghosts/ trophozoites/ vesicles suspensions were included to subtract any absorbance due to the cells . changes in absorbance at 405 nm were measured for at least 20 min , using a 7520 microplate reader ( cambridge technology , inc ) . measurements of parasite lactate dehydrogenase ( pldh ) activity were carried out essentially as described by makler and hinrichs . for these measurements , 5 g of trophozoite protein or membrane vesicle protein suspended in 20 l pbs or vesicle buffer were added to 100 l malstat reagent ( flow , inc . ) and 25 l 0.24 mm phenazine ethosulfate ( pes)/1.96 mm nitro blue tetrazolium ( nbt ) in microtitre 96-well plates . 20 wells containing 20 l trophozoites/ membrane vesicles and 125 l h2o were included to subtract any absorbance due to the cells . changes in absorbance at 620 nm were measured for at least 20 min , using a 7520 microplate reader ( cambridge technology , inc ) . the atpase activity was determined using a colorimetric assay adapted from those of chifflet et al . and doige et al .. enzyme assays were carried out with 50 g of freshly isolated p. falciparum plasma membranes suspended in 450 l of reaction buffer ( 50 mm tris - hcl , 0.15 m nh4cl , 2 mm mgcl2 , 0.02% nan3 , ph 7.4 ) . to initiate the reaction 50 l of atp or the appropriate nucleotide tested in the reaction buffer was added , giving a final concentration of 2 mm atp or the appropriate nucleotide . a control containing 50 g of parasite plasma membranes suspended in 500 l of reaction buffer was included to subtract any absorbance due to the membranes . another control consisting of 500 l reaction buffer containing 2 mm atp was added to evaluate the atp hydrolysis . , the suspensions were centrifuged at 13,000 rpm for three minutes to pellet the membranes . aliquots of 100 l of the resulting supernatant were transferred to the wells of a 96-well microtitre plate . the reaction was stopped by addition of 100 l of fresly prepared solution a ( 6% sds , 3% l - ascorbic acid , 0.5% ammonium molybdate in 0.25 m sulfuric acid ) . after 15 minutes , the phosphoammoniummolybdate complex formed was stabilised by the addition of 100 l of solution b ( 2% sodium citrate , 2% sodium arsenite , 2% acetic acid ) . after 15 minutes , the absorbance at 710 nm was measured using a 7520 microplate reader ( cambridge technology , inc ) . parasites were maintained in continuous culture at a 5% haematocrit in rpmi-1640 with glutamine ( sigma ) supplemented with hypoxanthine ( 44 mg / l ) , hepes ( 6 g / l ) , gentamycin ( 50 mg / l ) , and 10% a human serum ( western province blood transfusion service and haematology department , groote schuur hospital , cape town , south africa ) . parasites were grown in o - positive red cells and the parasitemia was kept between 1015 % . parasite cultures were incubated at 37c in dessicator cabinets under an atmosphere of 93% nitrogen , 4% carbon dioxide , 3% oxygen . infected erythrocytes were washed twice in 10 volumes of phosphate buffered saline ( pbs ) . the pellet was suspended in 10 volumes of pbs containing 0.05 % ( w / v ) saponin in order to lyse the erythrocytes . it was then incubated for 23 min at room temperature and centrifuged at 1,500 g for 10 min . the resulting pellet was washed twice in pbs and resuspended in 5 volumes of pbs . the protease inhibitors aprotinin , leupeptin and phenylmethylsulfonylfluoride ( boehringer mannheim ) were added to final concentration of 10 g / ml , 10 g / ml and 0.1 mm , respectively . unlysed erythrocytes and erythrocyte membranes contained in the trophozoite preparation were adsorbed by immuno - affinity . anti - human erythrocytes ( polyvalent immunoglobulin ( g , a , m ) rabbit , sigma ) were diluted 1 : 1000 in pbs . 5 g of polystyrene beads ( pierce ) were incubated in 20 ml of the above solution for two hours at room temperature with gentle agitation . the beads were washed in pbs and further incubated for one hour with 20 ml 1 % bovine serum albumin in pbs . the beads were then washed again in pbs and incubated with the trophozoite preparation for 30 min . the ability of purified trophozoites to exclude trypan blue was monitored by adding 10 l of trypan blue ( 10 mg / ml in distilled water ) to 100 l of trophozoite suspension in pbs ( phosphate buffered saline ) . after five minutes incubation at room temperature , the proportion of parasites showing trypan blue accumulation was evaluated using a phase contrast microscope . the biotylination reaction was performed by incubating 250 l of 1 mg / ml nhs - ss - biotin ( sulfosuccinimidyl 2-[biotin - amido ] ethyl-1,3-dithiopropionate ) ( pierce ) with 500 l of trophozoites ( 2 mg of protein ) for 30 min at room temperature . the resulting biotinylated trophozoites were washed once in pbs , then twice in vesicle buffer i ( 0.25 m sucrose , 1 mm e.d.t.a . , 10 mm tris - hcl ph 7.4 ) . vesicles were prepared by nitrogen cavitation according to the method of lever et al .. the cells were resuspended in 500 l vesicle buffer i containing aprotinin , leupeptin and phenylmethylsulfonylfluoride ( 10 g / ml , 10 g / ml and 0.1 mm , respectively ) and equilibrated at 4c under nitrogen pressure at 800 psi for 30 min . the homogenate was then incubated for five min at 37c with dnase i ( 50 g / ml ) . the biotinylated vesicles were incubated with 100 l of streptavidin immobilized on iron oxide ( sigma ) previously washed with pbs for 15 min at room temperature and then separated using a magnet ( magnetic cell sorting , myltenyi biotec ) . the magnetic particles were bound to the side of the tube and washed with 1 ml of 0.25 m sucrose , 10 mm tris - hcl ph 7.4 . parasite plasma membrane vesicles were fixed with glutaraldehyde ( 0.8%)/formaldehyde ( 8% ) in pbs for one hour at room temperature and washed in pbs , followed by addition of 50 mm nh4cl for 30 minutes and two washes in pbs . samples were dehydrated in 70% ethanol and embedded in lr white resin ( polyscience inc . ) sections were mounted on copper grids and stained with 2% uranyl acetate and lead acetate . the acetylcholine esterase ( ache ) activity was determined by a modification of the method of ellman et al . . enzyme assays were carried out with 200 l of freshly prepared 0.1 m sodium phosphate ph 7.5 , 0.5 mm 2,2'-dinitro-5,5'-dithiobenzoic acid , 0.6 mm s - acetylthiocholine iodide to which was added 50 l of a suspension of erythrocyte ghost , trophozoites or parasite plasma membrane , in microtitre 96-well plates . 10 g of erythrocyte ghost protein , 35 g of trophozoites protein or 20 g of parasite plasma membrane protein were used to measure an initial rate . wells containing 200 l h2o and 50 l of erythrocyte ghosts/ trophozoites/ vesicles suspensions were included to subtract any absorbance due to the cells . changes in absorbance at 405 nm were measured for at least 20 min , using a 7520 microplate reader ( cambridge technology , inc ) . measurements of parasite lactate dehydrogenase ( pldh ) activity were carried out essentially as described by makler and hinrichs . for these measurements , 5 g of trophozoite protein or membrane vesicle protein suspended in 20 l pbs or vesicle buffer were added to 100 l malstat reagent ( flow , inc . ) and 25 l 0.24 mm phenazine ethosulfate ( pes)/1.96 mm nitro blue tetrazolium ( nbt ) in microtitre 96-well plates . wells containing 20 l trophozoites/ membrane vesicles and 125 l h2o were included to subtract any absorbance due to the cells . changes in absorbance at 620 nm were measured for at least 20 min , using a 7520 microplate reader ( cambridge technology , inc ) . the atpase activity was determined using a colorimetric assay adapted from those of chifflet et al . and doige et al .. enzyme assays were carried out with 50 g of freshly isolated p. falciparum plasma membranes suspended in 450 l of reaction buffer ( 50 mm tris - hcl , 0.15 m nh4cl , 2 mm mgcl2 , 0.02% nan3 , ph 7.4 ) . to initiate the reaction 50 l of atp or the appropriate nucleotide tested in the reaction buffer was added , giving a final concentration of 2 mm atp or the appropriate nucleotide . a control containing 50 g of parasite plasma membranes suspended in 500 l of reaction buffer was included to subtract any absorbance due to the membranes . another control consisting of 500 l reaction buffer containing 2 mm atp was added to evaluate the atp hydrolysis . , the suspensions were centrifuged at 13,000 rpm for three minutes to pellet the membranes . aliquots of 100 l of the resulting supernatant were transferred to the wells of a 96-well microtitre plate . the reaction was stopped by addition of 100 l of fresly prepared solution a ( 6% sds , 3% l - ascorbic acid , 0.5% ammonium molybdate in 0.25 m sulfuric acid ) . after 15 minutes , the phosphoammoniummolybdate complex formed was stabilised by the addition of 100 l of solution b ( 2% sodium citrate , 2% sodium arsenite , 2% acetic acid ) . after 15 minutes , the absorbance at 710 nm was measured using a 7520 microplate reader ( cambridge technology , inc ) .

backgroundin host erythrocytes , the malaria parasite must contend with ion and drug transport across three membranes ; its own plasma membrane , the parasitophorous membrane and the host plasma membrane . isolation of pure and intact plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport.resultsthis study describes a procedure for isolating parasite plasma membrane from p. falciparum - infected erythrocytes . with this method , the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti - erythrocyte antibodies fixed to polystyrene beads . these trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation . this process allows the membranes to reform into vesicles . the magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet . these vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol . the parasite plasma membrane showed optimal atpase activity at 2 mm atp and 2 mm mg2 + . it was also found that ca2 + could not substitute for mg2 + atpase activity in parasite plasma membranes whereas activity was completely preserved when mn2 + was used instead of mg2 + . other nucleoside triphosphates tested were hydrolysed as efficiently as atp , while the nucleoside monophosphate amp was not.conclusionswe have described the successful isolation of intact p. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum atpase activity .

Background Results Discussion Conclusions Materials and Methods Cell cultures Trophozoite isolation Trypan blue uptake Plasma membrane isolation Electron microscopy analysis Acetylcholine esterase activity Parasite lactate dehydrogenase activity Protein determinations ATPase activity Acknowledgements

although extracellular , axenic development of the erythrocyte cycle of p. falciparum has been obtained [ 2 - 4 ] , the number of merozoites completing the cycle is not sufficient to permit continuous extracellular culture . under the best conditions , only 1% of the merozoites further develops into trophozoites therefore , the isolation of trophozoites still relies on their liberation from the host cells . released parasites need to be further cleansed of host cell materials , in particular erythrocytes and their ghosts , as well as unlysed infected erythrocytes . while methods to isolate host cell ghosts from parasitised erythrocytes have been developed , there is no published method to isolate the parasite plasma membrane . the isolation of the parasite plasma membrane presents technical difficulties due to the presence of three closely related membranes ( 1 ) the erythrocyte plasma membrane ( 2 ) the parasitophorous vacuolar membrane and ( 3 ) the parasite plasma membrane , as well as the food vacuole membrane . although the p. falciparum vacuolar atpase activity have been characterised , the atpase activity of the parasite plasma membrane has not yet been investigated . to date , investigation of the atpase activity of the plasma membrane has been hampered by the presence of contaminants in the membrane preparations . preparation of uncontaminated parasite plasma membranes would represent a major experimental advance for approaching this problem . for this reason , a method was developed to prepare pure and intact parasite plasma membranes from p. falciparum infected - erythrocytes . the dependence of p. falciparum plasma membrane atpase activity on atp and other nucleotides , divalent cations , time , atp and mg concentrations was also evaluated . , polystyrene beads coated with anti - erythrocyte antibodies were incubated with the trophozoite preparation . trophozoites were then biotinylated with nhs - ss - biotin prior to nitrogen cavitation in the presence of protease inhibitors and dnasei . the resulting biotinylated membranes bound to streptavidin - magnetic beads and were separated from the lysate using a magnet ( figure 1 ) . it was shown that 98% of the trophozoites released by saponin lysis did not concentrate the dye , indicating an intact plasma membrane . purified p. falciparum plasma membranes examined using transmission electron microscopy were observed to form intact vesicles , although some open membranes could be seen . the purity of the parasite plasma membrane preparations was further assayed by the use of enzyme markers of p. falciparum cytosol , the parasite lactate dehydrogenase ( pldh ) and of human erythrocyte membranes , the acetylcholine esterase ( ache ) . there were no detectable ache or pldh activities ( less than 0.01 mol / min / mg protein ) in the parasite plasma membrane preparations ( table 1 ) . by comparison , the specific activity of ache was 2.30 0.15 and 0.05 0.03 mol / min / mg protein in erythrocyte ghosts and isolated trophozoites ( n = 3 ) respectively , while the specific pldh activity in isolated trophozoites was 0.14 0.01 mol / min / mg protein ( n = 3 ) . acetylcholine esterase ( ache ) and parasite lactate dehydrogenase ( pldh ) activities of isolated trophozoites , parasite plasma membranes and/or erythrocyte ghosts were determined as described under materials and methods section . values are means standard deviation of three separate experiments from separate parasite plasma isolations , trophozoite isolations or erythrocyte membrane preparations . time dependence of the atpase activity of plasma membranes isolated from the p. falciparum strain d10 was evaluated over a time period of 45 minutes . a linear relationship of time versus atpase activity was found with a correlation coefficient of 0.717 ( n = 3 ) ( figure 2 ) . atpase activity was maximal with 2 mm mg in the presence of 2 mm atp ( figures 3,4 ) . the ability of 2 mm concentration of mg and the other divalent cations mn and ca to support the atpase activity of purified plasma membranes isolated from the p. falciparum strain d10 was evaluated , along with no divalent cation addition as a control ( table 2 ) . when ca was substituted for mg , only 21 19% of the atpase activity was retained . plasma membranes were also capable of hydrolysing other nucleoside triphosphates as effectively as atp ( table 3 ) . purified parasite plasma membranes isolated from the sensitive strains d10 displayed an atpase activity of 3.2 1.7 nmol pi / min / mg protein . atpase activity ( nmol pi / mg protein ) of d10 plasma membranes was determined over a time period of 45 minutes and the activity of the membranes tested without atp was subtracted . atpase activity of freshly purified d10 plasma membranes expressed as nmol pi / min / mg protein was measured over mg concentrations ranging from 0 to 5 mm . atpase activity of parasite plasma membranes tested without atp and absorbance due to atp hydrolysis was subtracted . atpase activity of freshly purified d10 plasma membranes expressed as nmol pi / min / mg protein was measured over atp concentrations ranging from 0.5 to 5 mm . atpase activity of parasite plasma membranes tested without atp and absorbance due to atp hydrolysis were subtracted . atpase activity was determined using 2 mm concentration of mg , mn and ca and the atpase activity in the absence of divalent cation was subtracted . atpase activity of isolated d10 plasma membranes tested without nucleotide and absorbance due to the nucleotide hydrolysis was subtracted . a procedure for isolating parasite plasma membrane from p. falciparum - infected erythrocytes was developed . in this method , trophozoites were released from the erythrocytes by saponin lysis , the remaining erythrocytes membranes being removed by immunoaffinity using anti - erythrocyte antibodies . previously , anti - erythrocyte monoclonal antibodies have been used to remove unlysed erythrocytes and ghost membranes after sorbitol treatment . however , in order to remove most of the erythrocyte ghosts by centrifugation , all the erythrocytes must be lysed by the detergent , thus allowing isolation of the trophozoites on a larger scale . for this reason , sorbitol lysis of the trophozoites was not retained in this study . however , because saponin - treatment of parasitized erythrocytes has been shown to permeabilise both the erythrocyte plasma membrane and the parasitophorous membrane the exposure of the trophozoites to saponin was very brief to minimize membrane damage . contamination of the plasma membrane vesicle preparations by the parasitophorous membrane , however , could not be discounted since , to our knowledge , there is no specific marker for this membrane . in this study , experiments with the trypan blue showed that 98% of the trophozoites released by saponin lysis were capable of maintaining an intact plasma membrane . nitrogen decompression has been previously shown to be effective for the disruption of cells and was used in this study to disrupt the parasite plasma membranes . a cocktail of protease inhibitors ( aprotinin , leupeptin and phenylmethylsulfonyl fluoride ) was used to prevent the degradation of membrane proteins by the proteases released during the nitrogen cavitation step . dnase i was , therefore , included in the isolation process to eliminate the dna liberated during the nitrogen cavitation step . the recovery yield of the parasite plasma membranes , 8.2% of the total isolated - trophozoite protein , is consistent with the percent protein yield ( 810% ) obtained for membrane vesicles prepared from cancer cells . purified p. falciparum examined using transmission electron microscopy were observed to be intact and free of contaminating membranes and organelles , even though a small amount of liberated haemozoin could occasionally be seen in certain fields . contamination of the plasma membrane vesicles with fragments of food vacuole membrane could not be ruled out since nitrogen cavitation is likely to disrupt the food vacuole membrane as well as the plasma membrane . it would therefore had been interesting to investigate whether the parasite plasma membrane preparations were contaminated by food vacuole membrane components using antibodies to pfcrt . the absence of parasite and erythrocyte protein in the parasite plasma membrane preparation was confirmed by non - detectable parasite lactate dehydrogenase and erythrocyte acetylcholine esterase activities . the fact that the membranes were bound to a support facilitated their purification from contaminants and their recovery . the atpase activity of parasite plasma membranes ( 3.2 1.7 nmol pi / min / mg protein ) obtained in this study was found to be of similar magnitude to that of digestive vacuoles isolated from p. falciparum . for comparison , the specific atpase activity of plasma membranes prepared from multidrug - resistant cancer cells is 1.15 mol / min / mg membrane protein . conditions required for optimal p. falciparum plasma membrane atpase activity were determined using the chloroquine - sensitive strain d10 . parasite plasma membranes showed optimal atpase activity at 2 mm mg and 2 mm atp . in this study , it was found that ca could not be substituted for mg to sustain atpase activity in parasite plasma membranes , whereas activity was preserved when mg was replaced by mn , indicating that the parasite plasma membrane atpase activity was similar to the vacuolar membrane atpase activity . however , this contradicts the choi and mego study which showed that ca could support the p. falciparum vacuolar atpase activity in the absence of mg . however , the nucleoside monophosphate amp was not hydrolysed , suggesting that the parasite plasma membrane atpase activity is distinguishable from that of membrane - associated alkaline and acid phosphatases . since the parasite plasma membranes were isolated in the form of intact vesicles the right - side - out , the atp hydrolysing regions of the atpase proteins should be enclosed within the vesicles . this raises the question of whether all of the compounds tested are gaining access to the relevant part of the protein , and whether the differences between the atpase activities measured in the presence of the different agents might reflect , at least to some extent , the different ability of these agents to gain access to the interior of the vesicles . therefore , it could not be excluded that the saturable dependence of atpase activity on the concentration of atp in the medium could reflect the saturable transport of atp into the vesicles , rather than the saturation of the atpase itself . similarly , the differential ability of divalent cations to support atpase activity could be the result of a differential transport of these cations into the vesicles . in conclusion , a method for the isolation and purification of the p. falciparum plasma membrane was developed and optimal conditions for p. falciparum plasma membrane atpase activity were determined . these purified membranes were intact and isolated in a high enough yield to enable a characterisation of their atpase activity . thus , plasma membranes cleansed of p. falciparum can be used as a model to establish the role played by atpases in ion transport . the availability of purified p. falciparum plasma membrane will be of value not only to investigate processes such as nutrient uptake / efflux , ph regulation and ion balance but also in the understanding of drug transport in this organelle . anti - human erythrocytes ( polyvalent immunoglobulin ( g , a , m ) rabbit , sigma ) were diluted 1 : 1000 in pbs . after five minutes incubation at room temperature , the proportion of parasites showing trypan blue accumulation was evaluated using a phase contrast microscope . vesicles were prepared by nitrogen cavitation according to the method of lever et al .. the cells were resuspended in 500 l vesicle buffer i containing aprotinin , leupeptin and phenylmethylsulfonylfluoride ( 10 g / ml , 10 g / ml and 0.1 mm , respectively ) and equilibrated at 4c under nitrogen pressure at 800 psi for 30 min . the biotinylated vesicles were incubated with 100 l of streptavidin immobilized on iron oxide ( sigma ) previously washed with pbs for 15 min at room temperature and then separated using a magnet ( magnetic cell sorting , myltenyi biotec ) . the magnetic particles were bound to the side of the tube and washed with 1 ml of 0.25 m sucrose , 10 mm tris - hcl ph 7.4 . parasite plasma membrane vesicles were fixed with glutaraldehyde ( 0.8%)/formaldehyde ( 8% ) in pbs for one hour at room temperature and washed in pbs , followed by addition of 50 mm nh4cl for 30 minutes and two washes in pbs . enzyme assays were carried out with 200 l of freshly prepared 0.1 m sodium phosphate ph 7.5 , 0.5 mm 2,2'-dinitro-5,5'-dithiobenzoic acid , 0.6 mm s - acetylthiocholine iodide to which was added 50 l of a suspension of erythrocyte ghost , trophozoites or parasite plasma membrane , in microtitre 96-well plates . 10 g of erythrocyte ghost protein , 35 g of trophozoites protein or 20 g of parasite plasma membrane protein were used to measure an initial rate . wells containing 200 l h2o and 50 l of erythrocyte ghosts/ trophozoites/ vesicles suspensions were included to subtract any absorbance due to the cells . 20 wells containing 20 l trophozoites/ membrane vesicles and 125 l h2o were included to subtract any absorbance due to the cells . the atpase activity was determined using a colorimetric assay adapted from those of chifflet et al . and doige et al .. enzyme assays were carried out with 50 g of freshly isolated p. falciparum plasma membranes suspended in 450 l of reaction buffer ( 50 mm tris - hcl , 0.15 m nh4cl , 2 mm mgcl2 , 0.02% nan3 , ph 7.4 ) . to initiate the reaction 50 l of atp or the appropriate nucleotide tested in the reaction buffer was added , giving a final concentration of 2 mm atp or the appropriate nucleotide . a control containing 50 g of parasite plasma membranes suspended in 500 l of reaction buffer was included to subtract any absorbance due to the membranes . another control consisting of 500 l reaction buffer containing 2 mm atp was added to evaluate the atp hydrolysis . , the suspensions were centrifuged at 13,000 rpm for three minutes to pellet the membranes . after 15 minutes , the phosphoammoniummolybdate complex formed was stabilised by the addition of 100 l of solution b ( 2% sodium citrate , 2% sodium arsenite , 2% acetic acid ) . after 15 minutes , the absorbance at 710 nm was measured using a 7520 microplate reader ( cambridge technology , inc ) . it was then incubated for 23 min at room temperature and centrifuged at 1,500 g for 10 min . vesicles were prepared by nitrogen cavitation according to the method of lever et al .. the cells were resuspended in 500 l vesicle buffer i containing aprotinin , leupeptin and phenylmethylsulfonylfluoride ( 10 g / ml , 10 g / ml and 0.1 mm , respectively ) and equilibrated at 4c under nitrogen pressure at 800 psi for 30 min . the biotinylated vesicles were incubated with 100 l of streptavidin immobilized on iron oxide ( sigma ) previously washed with pbs for 15 min at room temperature and then separated using a magnet ( magnetic cell sorting , myltenyi biotec ) . the magnetic particles were bound to the side of the tube and washed with 1 ml of 0.25 m sucrose , 10 mm tris - hcl ph 7.4 . parasite plasma membrane vesicles were fixed with glutaraldehyde ( 0.8%)/formaldehyde ( 8% ) in pbs for one hour at room temperature and washed in pbs , followed by addition of 50 mm nh4cl for 30 minutes and two washes in pbs . enzyme assays were carried out with 200 l of freshly prepared 0.1 m sodium phosphate ph 7.5 , 0.5 mm 2,2'-dinitro-5,5'-dithiobenzoic acid , 0.6 mm s - acetylthiocholine iodide to which was added 50 l of a suspension of erythrocyte ghost , trophozoites or parasite plasma membrane , in microtitre 96-well plates . 10 g of erythrocyte ghost protein , 35 g of trophozoites protein or 20 g of parasite plasma membrane protein were used to measure an initial rate . wells containing 200 l h2o and 50 l of erythrocyte ghosts/ trophozoites/ vesicles suspensions were included to subtract any absorbance due to the cells . wells containing 20 l trophozoites/ membrane vesicles and 125 l h2o were included to subtract any absorbance due to the cells . the atpase activity was determined using a colorimetric assay adapted from those of chifflet et al . and doige et al .. enzyme assays were carried out with 50 g of freshly isolated p. falciparum plasma membranes suspended in 450 l of reaction buffer ( 50 mm tris - hcl , 0.15 m nh4cl , 2 mm mgcl2 , 0.02% nan3 , ph 7.4 ) . to initiate the reaction 50 l of atp or the appropriate nucleotide tested in the reaction buffer was added , giving a final concentration of 2 mm atp or the appropriate nucleotide . a control containing 50 g of parasite plasma membranes suspended in 500 l of reaction buffer was included to subtract any absorbance due to the membranes . another control consisting of 500 l reaction buffer containing 2 mm atp was added to evaluate the atp hydrolysis . , the suspensions were centrifuged at 13,000 rpm for three minutes to pellet the membranes . after 15 minutes , the phosphoammoniummolybdate complex formed was stabilised by the addition of 100 l of solution b ( 2% sodium citrate , 2% sodium arsenite , 2% acetic acid ) .

hcc is characterized by multiple causes , multiple stages and multifocal process of tumorigenesis related intimately to the overexpression / suppression of many cellular factors . in general , chemical - induced liver cancer ( hepatocarcinogenesis ) animal models have been widely used for investigating therapeutic and other combating strategies in vivo against hcc . one of which is insulin - like growth factor - ii ( igf - ii ) . igf - ii , a ligand of the insulin - like growth factor - i receptor ( igf - ir ) , is believed to exert its effect during cellular proliferation and widely distributed in various fetal and neonatal human and rat tissues , including liver and during hcc . a relationship exists between the igf / igf - ir systems in development of various types of cancer , including hcc . report suggests that igf - ii promotes hepatocyte proliferation through a paracrine mechanism in the pre - cancerous stage and when hepatocytes are transformed into malignant cells , they secrete igf - ii and promote malignant cell proliferation by an autocrine mechanism . thus , an appropriate silencing of igf - ii gene overexpression by synthetic antisense oligonucleotides / oligomers ( asos ) may be useful in the treatment of liver tumors in which igf - ii has a predominant role , as asos are believed to possess great specificity to an intended molecular target . in the present study , we have investigated the effectiveness of phosphorothioate asos targeted against igf - ii messenger ribonucleic acids ( mrnas ) of three coding exons ( exon-1 , exon-2 and exon-3 ) in controlling the progress of rat hepatocarcinogenesis . the 30-base sequence 5-tccatggttacc cctagggtcaccccttca-3 targeted against exon-1 partially ( aso-1 ) , 24-base sequence 5-cgtccggaagttcggcacggttgg-3 targeted against exon-2 partially ( aso-2 ) and 32-base sequence 5-tgctgaaggggtctatggggcacccg ttcaag-3 targeted against exon-3 partially ( aso-3 ) synthesized on a 0.05 mol scale , hplc purification and phosphorothioate backbone modification ( synthesized by eurofins genomics india pvt . ltd . , a set of them also contained fluorescein isothiocyanate ( fitc)-labeled igf - ii asos used for in vivo localization of igf - ii asos . male sprague - dawley rats ( procured from the indian institute of chemical biology , kolkata , india ) with body weights approximately 130 - 150 g were maintained in polypropylene cages and housed at a temperature ( 25c 1c ) and a relative humidity 55% 5% with a normal day and night photoperiod , in the university animal house . all the animal experiments were conducted upon receiving the approval of the animal ethics committee , jadavpur university . rats were divided into eight groups [ figure 1 ] - ( groups a - h ) . group - a ( normal control rats ) and group - b ( carcinogen control rats ) , group - c ( carcinogen treated rats received aso-1 ) , group - d ( carcinogen treated rats received aso-2 ) , group - e ( carcinogen treated rats received aso-3 ) , group - f ( normal rats received aso-1 ) , group - g ( normal rats received aso-2 ) and group - h ( normal rats received aso-3 ) . aso : antisense oligomer , 2aaf : 2-acetylaminfluorine , dena : diethylnitrosamine , ip : intraperitoneal in groups b - e animals , carcinogenesis was initiated in liver by diethylnitrosamine ( sigma aldrich , bangalore , india ) at a dose of 200 mg / kg body weight once intraperitoneally at the start of the experiment , i.e. , day 0 . the animals were then treated with 0.5% w / w of 2-acetylaminofluorene ( sigma aldrich , bangalore , india ) in the diet to promote carcinogenesis . carcinogenesis was promoted from the beginning of 3 week to the end of 20 week , i.e. , for a period of 18 weeks . injection of aso-1/aso-2/aso-3 in normal saline ( 5 mg / kg body weight ) thrice a week , respectively [ figure 1 ] . igf - ii aso treatment was started from 23 week and continued up to 31 week in groups c - e rats . groups f - h received the treatment of aso-1/aso-2/aso-3 , respectively . at the 36 week , the animals were sacrificed and further experiments were conducted . the livers were removed , sliced ( 5 - 10 mm thick ) and were snap - frozen in liquid nitrogen . the tissues were stored at 80c in ultra - low temperature freezer ( so - low , usa ) until further use . liver tissues were subjected to macroscopic examination on the surfaces for visible hyperplastic nodules ( hns ) . the nodules were identified and differentiated from the reddish brown non - nodular surrounding liver by their grayish - white and darkish red color with clear demarcation . the liver tissue samples were undergone serial sections of hepatic tissues ( 6 m thick ) , which were further processed histochemically by periodic acid - schiff reaction , toluidine blue and hematoxylin - eosin for investigating tissue architecture and detecting different hepatic altered foci ( haf ) . liver lesions were identified as haf and were classified as clear endoplasmic reticulum rich clear cell foci ( ground glass appearance ) , mixed cell foci and ribosome rich basophilic foci . numbers and area of lesions were determined by zeiss light microscope and axio vision software 4.7.1 . microsomal cytochrome p-450 ( cyt.p-450 ) , glutathione s - transferase ( gst ) activity and uridine diphosphate glucuronyl transferase ( udpgt ) activity were determined using the referred methods . catalase activity using catalase activity assay kit ( biovision , milpitas , usa ) , superoxide dismutase ( sod ) activity using sod activity assay kit ( biovision , mountain view , usa ) and protein using modified lowry protein assay kit ( thermo scientific , rockford , usa ) were determined . in situ hybridization using igf - ii sense and antisense digoxigenin - labeled mrna obtained by in vitro transcription was conducted on 6 m cryosections of the liver samples to localize igf - ii gene expression in the cancerous and non - cancerous rat hepatic tissues . morphology of liver tissue from different experimental groups of rats was performed with the help of fesem ( jsm electron microscope , jeol , tokyo , japan ) . the platinum coating was applied on samples using an ion sputtering device , dried in a vacuum and examined using fesem . intravenous injection of fitc labeled igf - ii asos was given in normal rats . rats were dissected at different time points ( after injection ) 2 h , 4 h , 8 h and 12 h. liver samples were collected , washed and fixed with formalin solution and embedded in paraffin blocks by the conventional methods . the unstained tissue sections were observed at 400 magnification under a confocal laser scanning microscope ( lsm 510 ; carl zeiss , jena , germany ) at an excitation wavelength of 494 nm and emission wavelength of 521 nm to observe the distribution of igf - ii asos in liver tissue . fitc - labeled oligomers in liver were quantified using a spectrofluorimeter ( perkin - elmer ls 55 , walthan , usa ) with excitation and emission wavelength at 494 nm and 521 nm respectively , for fitc activity . the data were analyzed either by one - way analysis of variance or by dunnett 's t - test to determine statistical level of significance with a p < 0.05 . the 30-base sequence 5-tccatggttacc cctagggtcaccccttca-3 targeted against exon-1 partially ( aso-1 ) , 24-base sequence 5-cgtccggaagttcggcacggttgg-3 targeted against exon-2 partially ( aso-2 ) and 32-base sequence 5-tgctgaaggggtctatggggcacccg ttcaag-3 targeted against exon-3 partially ( aso-3 ) synthesized on a 0.05 mol scale , hplc purification and phosphorothioate backbone modification ( synthesized by eurofins genomics india pvt . ltd . , a set of them also contained fluorescein isothiocyanate ( fitc)-labeled igf - ii asos used for in vivo localization of igf - ii asos . male sprague - dawley rats ( procured from the indian institute of chemical biology , kolkata , india ) with body weights approximately 130 - 150 g were maintained in polypropylene cages and housed at a temperature ( 25c 1c ) and a relative humidity 55% 5% with a normal day and night photoperiod , in the university animal house . all the animal experiments were conducted upon receiving the approval of the animal ethics committee , jadavpur university . rats were divided into eight groups [ figure 1 ] - ( groups a - h ) . group - a ( normal control rats ) and group - b ( carcinogen control rats ) , group - c ( carcinogen treated rats received aso-1 ) , group - d ( carcinogen treated rats received aso-2 ) , group - e ( carcinogen treated rats received aso-3 ) , group - f ( normal rats received aso-1 ) , group - g ( normal rats received aso-2 ) and group - h ( normal rats received aso-3 ) . each group contained 10 rats . experimental regimen . aso : antisense oligomer , 2aaf : 2-acetylaminfluorine , dena : diethylnitrosamine , ip : intraperitoneal in groups b - e animals , carcinogenesis was initiated in liver by diethylnitrosamine ( sigma aldrich , bangalore , india ) at a dose of 200 mg / kg body weight once intraperitoneally at the start of the experiment , i.e. , day 0 . the animals were then treated with 0.5% w / w of 2-acetylaminofluorene ( sigma aldrich , bangalore , india ) in the diet to promote carcinogenesis . carcinogenesis was promoted from the beginning of 3 week to the end of 20 week , i.e. , for a period of 18 weeks . injection of aso-1/aso-2/aso-3 in normal saline ( 5 mg / kg body weight ) thrice a week , respectively [ figure 1 ] . igf - ii aso treatment was started from 23 week and continued up to 31 week in groups c - e rats . groups f - h received the treatment of aso-1/aso-2/aso-3 , respectively . at the 36 week , the animals were sacrificed and further experiments were conducted . the livers were removed , sliced ( 5 - 10 mm thick ) and were snap - frozen in liquid nitrogen . the tissues were stored at 80c in ultra - low temperature freezer ( so - low , usa ) until further use . liver tissues were subjected to macroscopic examination on the surfaces for visible hyperplastic nodules ( hns ) . the nodules were identified and differentiated from the reddish brown non - nodular surrounding liver by their grayish - white and darkish red color with clear demarcation . the liver tissue samples were undergone serial sections of hepatic tissues ( 6 m thick ) , which were further processed histochemically by periodic acid - schiff reaction , toluidine blue and hematoxylin - eosin for investigating tissue architecture and detecting different hepatic altered foci ( haf ) . liver lesions were identified as haf and were classified as clear endoplasmic reticulum rich clear cell foci ( ground glass appearance ) , mixed cell foci and ribosome rich basophilic foci . numbers and area of lesions were determined by zeiss light microscope and axio vision software 4.7.1 . microsomal cytochrome p-450 ( cyt.p-450 ) , glutathione s - transferase ( gst ) activity and uridine diphosphate glucuronyl transferase ( udpgt ) activity were determined using the referred methods . catalase activity using catalase activity assay kit ( biovision , milpitas , usa ) , superoxide dismutase ( sod ) activity using sod activity assay kit ( biovision , mountain view , usa ) and protein using modified lowry protein assay kit ( thermo scientific , rockford , usa ) were determined . in situ hybridization using igf - ii sense and antisense digoxigenin - labeled mrna obtained by in vitro transcription was conducted on 6 m cryosections of the liver samples to localize igf - ii gene expression in the cancerous and non - cancerous rat hepatic tissues . morphology of liver tissue from different experimental groups of rats was performed with the help of fesem ( jsm electron microscope , jeol , tokyo , japan ) . the platinum coating was applied on samples using an ion sputtering device , dried in a vacuum and examined using fesem . intravenous injection of fitc labeled igf - ii asos was given in normal rats . rats were dissected at different time points ( after injection ) 2 h , 4 h , 8 h and 12 h. liver samples were collected , washed and fixed with formalin solution and embedded in paraffin blocks by the conventional methods . the unstained tissue sections were observed at 400 magnification under a confocal laser scanning microscope ( lsm 510 ; carl zeiss , jena , germany ) at an excitation wavelength of 494 nm and emission wavelength of 521 nm to observe the distribution of igf - ii asos in liver tissue . fitc - labeled oligomers in liver were quantified using a spectrofluorimeter ( perkin - elmer ls 55 , walthan , usa ) with excitation and emission wavelength at 494 nm and 521 nm respectively , for fitc activity . statistical calculations were performed with graphpad instat version 3.0 ( graphpad software , inc . , san diego california ) . the data were analyzed either by one - way analysis of variance or by dunnett 's t - test to determine statistical level of significance with a p < 0.05 . cellular uptake of all the three asos ( aso-1,-2 and-3 ) along with their sense controls were studied in vitro in rat hepatoma h4iiec3 cells ( b.d biosciences , mumbai , india ) . all the sense and antisense oligomers were found to be internalized by the cells as detected by using fitc labeled oligomers with the help of confocal microscopy and all the antisense oligomers ( aso-1,-2 and -3 ) were found to control cellular proliferation investigated by thymidine incorporated deoxyribonucleic acid ( dna ) synthesis in cells ( data not shown ) , although aso-2 had about 30% less activity as compared to aso-1 and -3 in this regard . all the three sense oligomers had a similar level of dna synthesis as detected for untreated rat hepatoma h4iiec3 cells . antisense oligomers therefore were administered in carcinogen treated rats and in normal rats to investigate their efficacy in vivo . . this may be due to the absence of igf - ii expression in normal rats . carcinogen control rats ( group - b ) had maximum numbers and size of macroscopic hepatic hns [ figure 2a ] in liver . appearance of hns varied predominantly [ figure 2b ] in carcinogen treated rats treated with or without igf - ii asos . carcinogen treated rats received aso-1 ( group - c ) had minimum number of hns on the liver surfaces and the carcinogen treated rats received aso-2 ( group - d ) had the maximum number of hns and tumor in liver , among the carcinogen with aso treated rats . tumor incidences were not seen in group - c and group - e rats . hepatic hns , preneoplastic and neoplastic lesions and tumors were not seen in case of normal control ( group - a ) rats and rats treated with aso-1/aso-2/aso-3 ( groups f - h , respectively ) . group - b and group - d rats showed 100% incidence of liver lesions and hepatic tumors . however , 40% of carcinogen treated rats which received aso-1 or aso-3 did not show any hepatic lesions , hns or tumors [ table 1 ] . morphology , histology , insulin - like growth factor - ii ( igf - ii ) gene expression in experimental rats . ( a and b ) external morphology of liver showing multiple hyperplastic nodules ( white arrow ) on liver surface of group - b and group - c rats , respectively . ( c ) section of hepatic tumor ( shown by black arrow ) in hepatocellular carcinoma ( group - b ) ; using periodic acid - schiff reaction 100 . ( d ) a prominent hepatic mixed cell lesion ( shown by white arrow ) in group - b rats , using toluidine blue 100 . ( e ) highly differentiated hcc composed of a mixed population of clear ( glycogenetic ) cells , acidophilic cells ( ground - glass hepatocytes ) and some glycogen - poor , basophilic cells with pas in group - b rats 100 . ( f ) section of experimental rat hepatic tissue showing spongiosis hepatis ( white arrow ) with nucleus ( black arrow ) with pas in group - b rats 400 . ( g and h ) igf - ii overexpressed lesions in rat liver shown by white arrow and yellow arrow of group - b and group - d rats respectively 400 . ( i ) shows igf - ii expression in scattered hepatocytes in group - c rats , shown by yellow arrow 400 effect of antisense oligomers on number of haf lesions and area of lesions in group - b and groups c - e rats , numbers of haf were counted / unit area ( /cm ) of hepatic tissue [ table 1 ] . maximum count for haf was observed in group - b rats , followed by group - d rats . however , 60% of group - c and group - e rats showed lesion incidence and had about 59% and 55% reduction in the numbers of haf , respectively . reductions in the total lesion area compared to group - b rats were 64% and 53% , respectively for group - c and group - e rats . group - d rats had total lesion area even slightly more ( statistically non - significant ) compared to group - b rats . clear cell lesions ( ground glass appearance ) , basophilic lesions and mixed cell lesions [ figure 2d and e ] were predominantly scattered in hepatic tissues . those lesions were significantly ( p < 0.05 ) less in numbers , size and area in group - c and group - e animals [ table 1 ] . group - b and group - d rats had some spongioblast cells [ figure 2f ] . igf - ii overexpression as focal lesions and in individual hepatocytes was observed in group - b and group - d rats [ figure 2 g and h ] . no predominant igf - ii overexpression was observed in rats treated with aso-1/-3 ( for group - c / group - e rats ) . however , scattered igf - ii expression in few hepatocytes was seen in some of those rats [ figure 2i ] . we further investigated whether the experimental igf - ii asos ( fitc - labeled ) reached liver after their intravenous administration . livers were eventually rich in fitc fluorescence with the increasing period of time ( until 12 h in this case ) [ figure 3a ] . fitc labeled igf - ii aso was about 0.25 nm / g of hepatic tissue after 12 h of administration . the value was about 13 times more than that at 2 h , with an increasing trend of values through 4 - 8 h of assessment [ figure 3b ] . hepatic localization of fluorescein isothiocyanate - labeled insulin - like growth factor - ii ( igf - ii ) antisense oligomers and quantification of fitc - labeled oligomers in experimental rats . ( a ) confocal microscopic image of liver section of rats treated with fitc - labeled igf - ii antisense oligomer 400 . a : tissue alone , b : fitc - oligomer alone , c : fitc - oligomer in liver tissue . ( b ) tissue content of fitc - labeled oligomer in liver tissue of experimental animals another interesting study with the experimental livers using a simple fesem technique provides some significant findings related to the structure of the hepatocytes and the tissues . in many cases , adjacent cell walls were not connected in group - a [ figure 4a and b ] and groups f - h rats ( data not shown ) . the cellular structures appear well preserved with nuclei and on occasions , the spaces between hepatocytes were occupied by non - cellular materials [ figure 4b ] . carcinogen control livers had cellular structures without much space between the hepatocytes compared with group - a and groups f - h rat livers . nuclei were comparatively positioned centrally in the cells and more number of binucleated cells often existed in group - b rats [ figure 4c and d ] . less fine granular cytoplasm with less structural compactness was seen in group - b rats when compared to group - a and groups f - h rats . treatment of igf - ii aso in carcinogen treated rats ( group - c ) show better hepatocellular integrity [ figure 4e and f ] compared with that of group - b rats . group - d rats had hepatocellular architecture similar ( data not shown ) to that of group - b rats . ( a and b ) normal control rat liver tissues ( group - a ) . ( c and d ) carcinogen control rat liver tissue ( group - b ) . ( e and f ) carcinogen treated rats received antisense oligomer-1 ( group - c ) rat liver tissue . white arrows indicate space between the hepatocytes and black arrows indicate nucleus of the cell variation in activities or levels of some important marker isoenzyme / enzymes known for their role in hepatocellular cancer development , were observed in the livers of the experimental rats . in group - b rats , the content of cyt.p-450 was reduced compared to normal control animals ( group - a ) [ figure 5a ] . upon comparison of cyt.p-450 content of group - b animals with those values of groups c - e rats , cyt.p-450 contents were found to increase significantly ( p < 0.05 ) in group - c and group - e rats , whereas in group - d rats the isoenzyme content was almost similar to that of group - b rats . groups f - h and group - a rats had significantly ( p < 0.05 ) higher values of cyt.p-450 content than that of group - b rats . cyt.p-450 content was about double ( p < 0.05 ) in group - f rats when compared to group - a rats . various enzyme activities , iso - enzyme level in hepatic tissues of the experimental rats , ( a ) cytochrome p-450 content , ( b ) glutathione s - transferase activity , ( c ) uridine diphosphate glucuronyl transferase activity , ( d ) superoxide dismutase activity , ( e ) catalase activity . values represent mean standard deviation ( n = 10 ) , * p < 0.05 , a s tatistical l evel o f s ignificance ( using d unnett 's t - test ) when all groups were compared with group - a , b statistical level of significance ( using dunnett 's t - test ) when group - c , group - d , and group - e were compared with group - b in group - b animals , the activities of phase - ii biotransforming enzymes such as gst and udpgt were significantly ( p < 0.05 ) higher [ figure 5b and c compared to group - a rats . when compared to group - b rats , group - c and group - e rats had significantly decreased ( p < 0.05 ) gst and udpgt activities . in group - d animals , when groups f - h rats were compared with group - a rats , there was no significant change of gst activity in group - f and group - g rats . however administration of asos in group - h animals enhanced gst activity twice more than that value in group - a rats . udpgt activity was decreased to near normal values in groups c - e rats when compared to group - b rats which had about 2 times higher udpgt activity than that in group - a rats . injection of asos in group - f and - h rats caused enhancement of udpgt activity compared with group - a rats , although the data were not statistically significant . hepatic sod markedly decreased ( about 2.5 times ) in group - b rats compared to group - a animals [ figure 5d ] . sod activity was significantly ( p < 0.05 ) higher in groups c - e rats when compared to group - b rats , upon asos treatment . however , except for group - d animals , the values were less than those in group - a rats . rats belonged to groups f - h did not alter sod activities significantly as compared to group - a rats . similar trend was found in case of catalase activity in carcinogen treated rats which received asos [ figure 5e ] , except in group - d rats . catalase activity was found to decrease significantly ( p < 0.05 ) in group - b rats compared to group - a rats . upon asos administration ( except aso-2 which had even less activity of catalase than the carcinogen control animals ) in groups - c and - e rats , the activity was found to enhance toward the normal value . no predominant statistically significant difference in the activity of catalase was observed in groups f - h rats compared to group - a rats . all the carcinogen control animals showed 100% incidences of haf and tumors along with hns scattered predominantly on liver surface . aso-1/aso-3 treatment in carcinogen treated rats showed no haf and tumor in 40% of the rats and remaining 60% of the rats of those groups showed haf without any tumor incidence and reddish hns much smaller in size and less in number compared to grey - white , larger hns of group - b rats . this might be by modulating some genetic pathways in which igf - ii gene has a predominant role in cell signaling for hcc development . application of aso-2 in carcinogen - treated rats was found to be ineffective to control hcc development as the group of rats had 100% incidence of haf and tumors in liver . overexpression of igf - ii has been found in hcc and in hepatic lesions . in group - b and group - d rats , overexpression of igf - ii gene was observed in focal lesions and in some scattered hepatocytes . rats of group - c and group - e ( the rats of those two groups in which haf were seen ) had very few scattered igf - ii gene - expressed hepatocytes , suggesting silencing of igf - ii gene by aso-1/aso-3 in those rats to a great extent . further confocal microscopic images of the liver sections showed that asos reached the hepatocytes within 2 h and the quantity of oligomers increased with time ( at least up to 12 h ) . fesem data depict that there was a loss of structural integrity in the livers of carcinogen control rats . more dividing cells were observed . unlike normal control rat livers , cytoplasm had more space with less finely granular appearance . aso-1/aso-3 treatment in carcinogen treated rats show hepatocytes with more compact cytoplasm with finer granular structure . nuclei were positioned more centrally in the hepatocytes as in carcinogen control animals , but space between the hepatocytes was more similar to those in normal control rats . thus , aso-1/-3 treatment might partially control the structural changes of hepatocytes during the transformation of normal cells to neoplastic cells . various enzyme activities and isoenzyme level have long been considered as the hepatic preneoplastic and/or neoplastic markers and many studies have attested to this claim by the findings . cyt.p-450 is an isoenzyme of a collection of cyt.p-450 monooxygenases , which catalyze different types of the oxidation reaction . this enzyme family plays an important role in phase - i metabolism of many drugs . the enzymes are present in many tissues but the highest levels are found in liver . our data suggest that oxidizing activities through cyt.p-450 enzymes might be suppressed due to the presence of carcinogen and unlike aso-1/-3 treatment , aso-2 administration had no marked effect to improve cyt.p-450 enzyme activity in carcinogen treated rats . a significant 100% enhancement of cyt.p-450 content in group - f animals ( i.e. , aso-1 control rats ) might be due to the use of cyt.p-450 enzymes much more in livers of those rats during the degradation of the phosphorothioate oligomer or its products compared with the rats of groups g and h which showed no predominant change in cyt.p-450 contents . gst and udpgt ( two important phase ii enzymes ) are known to play a key role in detoxification of both xenobiotics and endogenous compounds and in reduction of reactive oxygen species ( ros ) and dna adduct formation . gst and udpgt are known to be important preneoplastic and neoplastic determinants both in the susceptibility to mutagenic effects of chemical carcinogenesis and in the response of liver tumors to chemotherapy . an increase in gst activity is therefore an elevation in the mechanism that protects against the noxious effects of xenobiotics , including carcinogens . unlike carcinogen control rats , the rats treated with aso-1/aso-3 decreased gst activity significantly toward the normal value . however , carcinogen treated animals received aso-2 ( group - d ) had gst activity similar to that of carcinogen control animals , indicating gst activity was not altered by the administration of aso-2 . aso-3 treatment in normal rats enhanced gst activity by almost 2.5 times of that of normal control rats . gst might have a direct role to degrade aso-3 or its products by transferase activity or aso-3 has a direct role to induce gst activity . the synthesis of glucuronides by liver microsomal udpgt is a major pathway by which the body inactivates both endogenous and foreign compounds . a significantly ( p < 0.05 ) higher udpgt activity in carcinogen control rats than normal control rats was reduced to near normal value upon application of aso-1/-3 , suggesting that aso-1/-3 influence the enzyme to inactivate xenobiotics in carcinogen treated rats . carcinogen administration produces ros , which can induce several kinds of dna damage , activation of transcription factors and proto - oncogenes , genomic instability , invasion or metastasis etc . many antioxidant enzymes such as sod , catalase , glutathione peroxidase and reductase etc . , prevent or repair the damage caused by ros . it is reported that during hepatocarcinogenesis both sod and catalase levels decrease , except some cancer cells which process high level of sod expression and activity . catalase activity was not significantly different between normal control rats and igf - ii aso control rats . in carcinogen control animals , carcinogen treated animals received aso-1/aso-3 enhanced catalase activities toward the normal control value compared to carcinogen control animals . however , the application of aso-2 in carcinogen treated animals did not show any enhancement , rather reduced the catalase activity in carcinogen treated rats . results indicate that treatment of aso-1/aso-3 significantly improved the antioxidant defense enzyme system , in particular catalase activity . the differential toxicity due to ros generated by carcinogen in hepatocytes as compared to normal animals may be brought about by the lack of sod in preneoplastic and neoplastic cells , as loss of sod ( or increased ros ) occurs in all cancerous cells . thus loss of sod from the normal cells causes the cells to appear transformed in some ways or makes the cells more susceptible to transformation . sod activity did not vary significantly in aso treated rats ( aso control ) compared to normal animals , indicating that in normal rats aso-1/-2/-3 had no effect on sod . the findings suggest that administration of aso-1/-2/-3 had induced sod activity directly or indirectly by triggering some other enzyme systems in carcinogen treated rats . out of the three asos used here to block igf - ii gene overexpression , aso-1 was found to control most significantly the development and/or progress of hepatic cancer . function of antisense oligomers in vivo depends on many facts such as mrna - antisense oligomer duplex formation , rate of binding of oligomers with mrna , stability of the formed duplex in vivo etc . in the present study , production of mrna in vivo might be much faster and distinctly more in an amount than the mrna - aso-2 duplex formation . this has been supported by the findings of overexpression of igf ii in aso-2 administered carcinogen treated rat livers . occurrence of quicker degradation and/or elimination of mrna - aso-2 duplex in vivo should not be ignored , too . aso-2 could show non - antisense effects as a report suggests that the presence of certain oligonucleotide sequences , such as cg ( which were maximum in numbers in aso-2 ) , have sequence - dependent , non - antisense effects . thus , aso-2 found to be ineffective in vivo may be because of any one of the mentioned reasons or a combine effect of some of the mentioned reasons or due to a cumulative effect of them . miltu kumar ghosh , department of pharmaceutical technology , jadavpur university , kolkata - 700 032 , west bengal , india and georg - august - universitt gttingen , department of human genetics , heinrich - dker - weg 12 , 37073 gttingen , germany . falguni patra , department of pharmaceutical technology , jadavpur university , kolkata - 700 032 , west bengal , india . dr . shampa ghosh , department of pharmaceutical technology , jadavpur university , kolkata - 700 032 , west bengal , india . chowdhury mobaswar hossain , department of pharmaceutical technology , jadavpur university , kolkata - 700 032 , west bengal , india . biswajit mukherjee , department of pharmaceutical technology , jadavpur university , kolkata - 700 032 , west bengal , india .

background : hepatocellular carcinoma ( hcc ) is a multistep complex process , caused by many of genetic alteration . insulin - like growth factors and their receptor have been widely implicated to hcc . insulin - like growth factor - ii ( igf - ii ) is a mitogenic polypeptide , found in various fetal and neonatal tissues of humans and rats and expresses in hcc . here we investigated anticancer potential of phosphorothioate antisense oligonucleotides ( asos ) against three coding exons ( exon-1/exon-2/exon-3 ) of igf - ii messenger ribonucleic acid in rat hepatocarcinogenesis model.materials and methods : during diethylnitrosamine and 2-acetylaminofluorene induced hepatocarcinogenesis , rats were treated with asos . various biochemical and histological studies were conducted.results:about 40% of carcinogen treated rats , which received two oligomers ( against exon-1 or-3 ) did not show any hepatic lesion , hyperplastic nodule or tumor and remaining 60% of those rats showed lesion incidence and had about 59% and 55% reductions in the numbers of hepatic altered foci , respectively . reductions in the total lesion - area when compared with carcinogen control rats were 64% and 53% , respectively for the animals treated with carcinogen and received the asos against exon-1/-3 . fluorescein isothiocyanate - labeled aso reached in the hepatocytes in 2 h. no predominant igf - ii overexpression was observed in case of rats treated with the two asos . treatment of the antisense igf - ii oligomers in carcinogen treated rats show better hepatocellular integrity along with several preneoplastic / neoplastic marker isoenzyme / enzyme modulations.conclusions:two of the three antisense oligomer - types effectively controlled igf - ii overexpression , causing the delay of the development and/or progress of hepatic cancer in rats .

INTRODUCTION MATERIALS AND METHODS Oligonucleotides Rat cancer model Liver morphology study Histopathological study Marker enzymes study Detection of IGF-II mRNA by Field emission scanning electron microscopy of liver tissue None None Statistical calculations RESULTS DISCUSSION CONCLUSIONS AUTHOR'S PROFILE

in general , chemical - induced liver cancer ( hepatocarcinogenesis ) animal models have been widely used for investigating therapeutic and other combating strategies in vivo against hcc . one of which is insulin - like growth factor - ii ( igf - ii ) . igf - ii , a ligand of the insulin - like growth factor - i receptor ( igf - ir ) , is believed to exert its effect during cellular proliferation and widely distributed in various fetal and neonatal human and rat tissues , including liver and during hcc . report suggests that igf - ii promotes hepatocyte proliferation through a paracrine mechanism in the pre - cancerous stage and when hepatocytes are transformed into malignant cells , they secrete igf - ii and promote malignant cell proliferation by an autocrine mechanism . thus , an appropriate silencing of igf - ii gene overexpression by synthetic antisense oligonucleotides / oligomers ( asos ) may be useful in the treatment of liver tumors in which igf - ii has a predominant role , as asos are believed to possess great specificity to an intended molecular target . in the present study , we have investigated the effectiveness of phosphorothioate asos targeted against igf - ii messenger ribonucleic acids ( mrnas ) of three coding exons ( exon-1 , exon-2 and exon-3 ) in controlling the progress of rat hepatocarcinogenesis . , a set of them also contained fluorescein isothiocyanate ( fitc)-labeled igf - ii asos used for in vivo localization of igf - ii asos . all the animal experiments were conducted upon receiving the approval of the animal ethics committee , jadavpur university . group - a ( normal control rats ) and group - b ( carcinogen control rats ) , group - c ( carcinogen treated rats received aso-1 ) , group - d ( carcinogen treated rats received aso-2 ) , group - e ( carcinogen treated rats received aso-3 ) , group - f ( normal rats received aso-1 ) , group - g ( normal rats received aso-2 ) and group - h ( normal rats received aso-3 ) . aso : antisense oligomer , 2aaf : 2-acetylaminfluorine , dena : diethylnitrosamine , ip : intraperitoneal in groups b - e animals , carcinogenesis was initiated in liver by diethylnitrosamine ( sigma aldrich , bangalore , india ) at a dose of 200 mg / kg body weight once intraperitoneally at the start of the experiment , i.e. the animals were then treated with 0.5% w / w of 2-acetylaminofluorene ( sigma aldrich , bangalore , india ) in the diet to promote carcinogenesis . igf - ii aso treatment was started from 23 week and continued up to 31 week in groups c - e rats . groups f - h received the treatment of aso-1/aso-2/aso-3 , respectively . at the 36 week , the animals were sacrificed and further experiments were conducted . the liver tissue samples were undergone serial sections of hepatic tissues ( 6 m thick ) , which were further processed histochemically by periodic acid - schiff reaction , toluidine blue and hematoxylin - eosin for investigating tissue architecture and detecting different hepatic altered foci ( haf ) . in situ hybridization using igf - ii sense and antisense digoxigenin - labeled mrna obtained by in vitro transcription was conducted on 6 m cryosections of the liver samples to localize igf - ii gene expression in the cancerous and non - cancerous rat hepatic tissues . morphology of liver tissue from different experimental groups of rats was performed with the help of fesem ( jsm electron microscope , jeol , tokyo , japan ) . intravenous injection of fitc labeled igf - ii asos was given in normal rats . rats were dissected at different time points ( after injection ) 2 h , 4 h , 8 h and 12 h. liver samples were collected , washed and fixed with formalin solution and embedded in paraffin blocks by the conventional methods . the unstained tissue sections were observed at 400 magnification under a confocal laser scanning microscope ( lsm 510 ; carl zeiss , jena , germany ) at an excitation wavelength of 494 nm and emission wavelength of 521 nm to observe the distribution of igf - ii asos in liver tissue . fitc - labeled oligomers in liver were quantified using a spectrofluorimeter ( perkin - elmer ls 55 , walthan , usa ) with excitation and emission wavelength at 494 nm and 521 nm respectively , for fitc activity . , a set of them also contained fluorescein isothiocyanate ( fitc)-labeled igf - ii asos used for in vivo localization of igf - ii asos . group - a ( normal control rats ) and group - b ( carcinogen control rats ) , group - c ( carcinogen treated rats received aso-1 ) , group - d ( carcinogen treated rats received aso-2 ) , group - e ( carcinogen treated rats received aso-3 ) , group - f ( normal rats received aso-1 ) , group - g ( normal rats received aso-2 ) and group - h ( normal rats received aso-3 ) . aso : antisense oligomer , 2aaf : 2-acetylaminfluorine , dena : diethylnitrosamine , ip : intraperitoneal in groups b - e animals , carcinogenesis was initiated in liver by diethylnitrosamine ( sigma aldrich , bangalore , india ) at a dose of 200 mg / kg body weight once intraperitoneally at the start of the experiment , i.e. the animals were then treated with 0.5% w / w of 2-acetylaminofluorene ( sigma aldrich , bangalore , india ) in the diet to promote carcinogenesis . igf - ii aso treatment was started from 23 week and continued up to 31 week in groups c - e rats . groups f - h received the treatment of aso-1/aso-2/aso-3 , respectively . at the 36 week , the animals were sacrificed and further experiments were conducted . the liver tissue samples were undergone serial sections of hepatic tissues ( 6 m thick ) , which were further processed histochemically by periodic acid - schiff reaction , toluidine blue and hematoxylin - eosin for investigating tissue architecture and detecting different hepatic altered foci ( haf ) . in situ hybridization using igf - ii sense and antisense digoxigenin - labeled mrna obtained by in vitro transcription was conducted on 6 m cryosections of the liver samples to localize igf - ii gene expression in the cancerous and non - cancerous rat hepatic tissues . morphology of liver tissue from different experimental groups of rats was performed with the help of fesem ( jsm electron microscope , jeol , tokyo , japan ) . rats were dissected at different time points ( after injection ) 2 h , 4 h , 8 h and 12 h. liver samples were collected , washed and fixed with formalin solution and embedded in paraffin blocks by the conventional methods . the unstained tissue sections were observed at 400 magnification under a confocal laser scanning microscope ( lsm 510 ; carl zeiss , jena , germany ) at an excitation wavelength of 494 nm and emission wavelength of 521 nm to observe the distribution of igf - ii asos in liver tissue . fitc - labeled oligomers in liver were quantified using a spectrofluorimeter ( perkin - elmer ls 55 , walthan , usa ) with excitation and emission wavelength at 494 nm and 521 nm respectively , for fitc activity . cellular uptake of all the three asos ( aso-1,-2 and-3 ) along with their sense controls were studied in vitro in rat hepatoma h4iiec3 cells ( b.d biosciences , mumbai , india ) . all the sense and antisense oligomers were found to be internalized by the cells as detected by using fitc labeled oligomers with the help of confocal microscopy and all the antisense oligomers ( aso-1,-2 and -3 ) were found to control cellular proliferation investigated by thymidine incorporated deoxyribonucleic acid ( dna ) synthesis in cells ( data not shown ) , although aso-2 had about 30% less activity as compared to aso-1 and -3 in this regard . antisense oligomers therefore were administered in carcinogen treated rats and in normal rats to investigate their efficacy in vivo . this may be due to the absence of igf - ii expression in normal rats . appearance of hns varied predominantly [ figure 2b ] in carcinogen treated rats treated with or without igf - ii asos . carcinogen treated rats received aso-1 ( group - c ) had minimum number of hns on the liver surfaces and the carcinogen treated rats received aso-2 ( group - d ) had the maximum number of hns and tumor in liver , among the carcinogen with aso treated rats . hepatic hns , preneoplastic and neoplastic lesions and tumors were not seen in case of normal control ( group - a ) rats and rats treated with aso-1/aso-2/aso-3 ( groups f - h , respectively ) . however , 40% of carcinogen treated rats which received aso-1 or aso-3 did not show any hepatic lesions , hns or tumors [ table 1 ] . morphology , histology , insulin - like growth factor - ii ( igf - ii ) gene expression in experimental rats . ( c ) section of hepatic tumor ( shown by black arrow ) in hepatocellular carcinoma ( group - b ) ; using periodic acid - schiff reaction 100 . ( g and h ) igf - ii overexpressed lesions in rat liver shown by white arrow and yellow arrow of group - b and group - d rats respectively 400 . ( i ) shows igf - ii expression in scattered hepatocytes in group - c rats , shown by yellow arrow 400 effect of antisense oligomers on number of haf lesions and area of lesions in group - b and groups c - e rats , numbers of haf were counted / unit area ( /cm ) of hepatic tissue [ table 1 ] . maximum count for haf was observed in group - b rats , followed by group - d rats . however , 60% of group - c and group - e rats showed lesion incidence and had about 59% and 55% reduction in the numbers of haf , respectively . reductions in the total lesion area compared to group - b rats were 64% and 53% , respectively for group - c and group - e rats . igf - ii overexpression as focal lesions and in individual hepatocytes was observed in group - b and group - d rats [ figure 2 g and h ] . no predominant igf - ii overexpression was observed in rats treated with aso-1/-3 ( for group - c / group - e rats ) . however , scattered igf - ii expression in few hepatocytes was seen in some of those rats [ figure 2i ] . we further investigated whether the experimental igf - ii asos ( fitc - labeled ) reached liver after their intravenous administration . fitc labeled igf - ii aso was about 0.25 nm / g of hepatic tissue after 12 h of administration . hepatic localization of fluorescein isothiocyanate - labeled insulin - like growth factor - ii ( igf - ii ) antisense oligomers and quantification of fitc - labeled oligomers in experimental rats . ( a ) confocal microscopic image of liver section of rats treated with fitc - labeled igf - ii antisense oligomer 400 . ( b ) tissue content of fitc - labeled oligomer in liver tissue of experimental animals another interesting study with the experimental livers using a simple fesem technique provides some significant findings related to the structure of the hepatocytes and the tissues . carcinogen control livers had cellular structures without much space between the hepatocytes compared with group - a and groups f - h rat livers . treatment of igf - ii aso in carcinogen treated rats ( group - c ) show better hepatocellular integrity [ figure 4e and f ] compared with that of group - b rats . white arrows indicate space between the hepatocytes and black arrows indicate nucleus of the cell variation in activities or levels of some important marker isoenzyme / enzymes known for their role in hepatocellular cancer development , were observed in the livers of the experimental rats . various enzyme activities , iso - enzyme level in hepatic tissues of the experimental rats , ( a ) cytochrome p-450 content , ( b ) glutathione s - transferase activity , ( c ) uridine diphosphate glucuronyl transferase activity , ( d ) superoxide dismutase activity , ( e ) catalase activity . values represent mean standard deviation ( n = 10 ) , * p < 0.05 , a s tatistical l evel o f s ignificance ( using d unnett 's t - test ) when all groups were compared with group - a , b statistical level of significance ( using dunnett 's t - test ) when group - c , group - d , and group - e were compared with group - b in group - b animals , the activities of phase - ii biotransforming enzymes such as gst and udpgt were significantly ( p < 0.05 ) higher [ figure 5b and c compared to group - a rats . in group - d animals , when groups f - h rats were compared with group - a rats , there was no significant change of gst activity in group - f and group - g rats . udpgt activity was decreased to near normal values in groups c - e rats when compared to group - b rats which had about 2 times higher udpgt activity than that in group - a rats . sod activity was significantly ( p < 0.05 ) higher in groups c - e rats when compared to group - b rats , upon asos treatment . similar trend was found in case of catalase activity in carcinogen treated rats which received asos [ figure 5e ] , except in group - d rats . upon asos administration ( except aso-2 which had even less activity of catalase than the carcinogen control animals ) in groups - c and - e rats , the activity was found to enhance toward the normal value . no predominant statistically significant difference in the activity of catalase was observed in groups f - h rats compared to group - a rats . all the carcinogen control animals showed 100% incidences of haf and tumors along with hns scattered predominantly on liver surface . aso-1/aso-3 treatment in carcinogen treated rats showed no haf and tumor in 40% of the rats and remaining 60% of the rats of those groups showed haf without any tumor incidence and reddish hns much smaller in size and less in number compared to grey - white , larger hns of group - b rats . this might be by modulating some genetic pathways in which igf - ii gene has a predominant role in cell signaling for hcc development . application of aso-2 in carcinogen - treated rats was found to be ineffective to control hcc development as the group of rats had 100% incidence of haf and tumors in liver . overexpression of igf - ii has been found in hcc and in hepatic lesions . in group - b and group - d rats , overexpression of igf - ii gene was observed in focal lesions and in some scattered hepatocytes . rats of group - c and group - e ( the rats of those two groups in which haf were seen ) had very few scattered igf - ii gene - expressed hepatocytes , suggesting silencing of igf - ii gene by aso-1/aso-3 in those rats to a great extent . further confocal microscopic images of the liver sections showed that asos reached the hepatocytes within 2 h and the quantity of oligomers increased with time ( at least up to 12 h ) . fesem data depict that there was a loss of structural integrity in the livers of carcinogen control rats . aso-1/aso-3 treatment in carcinogen treated rats show hepatocytes with more compact cytoplasm with finer granular structure . nuclei were positioned more centrally in the hepatocytes as in carcinogen control animals , but space between the hepatocytes was more similar to those in normal control rats . cyt.p-450 is an isoenzyme of a collection of cyt.p-450 monooxygenases , which catalyze different types of the oxidation reaction . our data suggest that oxidizing activities through cyt.p-450 enzymes might be suppressed due to the presence of carcinogen and unlike aso-1/-3 treatment , aso-2 administration had no marked effect to improve cyt.p-450 enzyme activity in carcinogen treated rats . , aso-1 control rats ) might be due to the use of cyt.p-450 enzymes much more in livers of those rats during the degradation of the phosphorothioate oligomer or its products compared with the rats of groups g and h which showed no predominant change in cyt.p-450 contents . unlike carcinogen control rats , the rats treated with aso-1/aso-3 decreased gst activity significantly toward the normal value . however , carcinogen treated animals received aso-2 ( group - d ) had gst activity similar to that of carcinogen control animals , indicating gst activity was not altered by the administration of aso-2 . a significantly ( p < 0.05 ) higher udpgt activity in carcinogen control rats than normal control rats was reduced to near normal value upon application of aso-1/-3 , suggesting that aso-1/-3 influence the enzyme to inactivate xenobiotics in carcinogen treated rats . catalase activity was not significantly different between normal control rats and igf - ii aso control rats . in carcinogen control animals , carcinogen treated animals received aso-1/aso-3 enhanced catalase activities toward the normal control value compared to carcinogen control animals . however , the application of aso-2 in carcinogen treated animals did not show any enhancement , rather reduced the catalase activity in carcinogen treated rats . sod activity did not vary significantly in aso treated rats ( aso control ) compared to normal animals , indicating that in normal rats aso-1/-2/-3 had no effect on sod . the findings suggest that administration of aso-1/-2/-3 had induced sod activity directly or indirectly by triggering some other enzyme systems in carcinogen treated rats . out of the three asos used here to block igf - ii gene overexpression , aso-1 was found to control most significantly the development and/or progress of hepatic cancer . function of antisense oligomers in vivo depends on many facts such as mrna - antisense oligomer duplex formation , rate of binding of oligomers with mrna , stability of the formed duplex in vivo etc .

in british columbia ( bc ) , canada , the ethnic breakdown of people with tuberculosis ( tb ) has changed over the last several decades . since 2010 , there has been an average of 269 active tb cases per year in bc , more than 70 percent of which affected immigrants . in 2013 , the majority of tb cases were in chinese- , punjabi- , tagalog- , korean- , and vietnamese - speaking immigrants , with the highest proportion of tb cases being immigrants from mainland china . immigrants also accounted for the majority of persons starting treatment for latent tb infection ( ltbi)64.1 percent in 2011 . most active tb cases in immigrants developed from ltbi that had been acquired in their countries of origin based on genotyping studies [ 35 ] . the who has recommended treating ltbi in immigrants as a strategy that can further reduce active tb cases in low incidence settings such as bc . initiation of and adherence to ltbi treatment are generally low in immigrants to north america [ 79 ] . in canada , drug therapy for ltbi is usually four to nine months depending upon the regimen used . incomplete courses of treatment are less effective in preventing tb disease ; thus , adherence to and completion of the entire course of ltbi therapy are essential [ 10 , 11 ] . low treatment adherence among immigrants partially depends on their knowledge and perceptions of ltbi [ 1216 ] , which can be influenced by the use of culturally specific educational materials and cultural brokers [ 17 , 18 ] . providing accessible , culturally sensitive messaging through a variety of formats is hypothesized to increase rates of treatment initiation and consequently increase acceptance and completion of ltbi treatment [ 19 , 20 ] . because chinese immigrants account for the highest proportion of tb cases in bc , the qualitative part of the study focused on chinese immigrants . previous studies have identified some issues related to knowledge and perceptions of ltbi and its treatment among chinese immigrants in north america [ 14 , 16 ] . these studies focused on active tb and reported that chinese immigrants ' knowledge of the transmission of tb bacteria and prevention of tb disease was low . chinese immigrants generally believed in western health care providers ' abilities and knowledge and were willing to seek care from them but preferred traditional chinese medicine . moreover , access to health insurance and the stigma associated with tb led to people not seeking medical care even if they suspected that they had tb [ 14 , 16 ] . the objectives of this research were to ( a ) document knowledge levels of patients in a tb clinic ; ( b ) identify chinese immigrants ' knowledge and perceptions of ltbi ; ( c ) specify messages that would be most appropriate for ltbi education in this population ; and ( d ) identify the most appropriate format of health promotional materials to address the specific needs of this population . as part of a larger tb education project that included developing culturally relevant and informative materials for a diverse group of immigrants , we conducted a mixed methods study . it included a patient survey at the provincial tb clinics in greater vancouver that evaluated knowledge of ltbi and treatment of it and a qualitative approach using fgd for chinese immigrants [ 22 , 23 ] . a survey in english of patients who visited the bc centre for disease control ( bccdc ) tb clinics was conducted from june 2013 to june 2014 . survey questions of knowledge , attitudes , and perceptions regarding ltbi and its treatment were developed and pilot - tested in five clerical staff from bccdc to establish clarity of questions . to estimate the level of knowledge only very basic facts about ltbi were included in the survey , and we considered a score of 80% or higher as representing adequate knowledge . after refining them , since no further edits were required , we then pilot - tested the questions for clarity with 20 patients . respondents to the survey included all individuals who presented to the tb clinics and were 18 years old or older . each respondent provided a unique identifier , which was used to exclude any duplicate surveys . using focus group discussions ( fgds ) , we then explored further details of knowledge and perceptions towards ltbi specifically in chinese immigrants . all participants self - selected either early ( well - settled ) immigrants or recent ( new ) immigrants according to their self - perceived levels of adjusting to the new culture , their social engagement with canadians , their perceptions of settlement , their feelings of belonging , and length of time residing in canada [ 14 , 26 , 27 ] . ethics approval was obtained from the behavioural research ethics board of the university of british columbia and the office of research ethics of simon fraser university . for survey respondents survey respondents who provided their emails and/or phone numbers were contacted for fgds . for fgds , introductory letters and consent forms written in simplified chinese were sent to each participant through email two weeks prior to the fgds . signed consent forms were collected at the focus groups . surveys were distributed by trained clerical staff at the bccdc tb clinics to all patients of 18 years old or older . data were input weekly into a microsoft office access database by a trained clinic clerk , reviewed for accuracy by a study team member , and then imported into a microsoft office excel dataset for data analysis . for fgds , a structured topic guide , informed by the literature , was developed in english and then translated into simplified chinese . four key questions guided the data collection : ( 1 ) what do you know about treatment options for latent tb infection ? ( 2 ) what have you heard in your family or community about latent tb infection and about the treatment of it ? ( 3 ) what might influence whether someone with latent tb infection takes treatment ? ( 4 ) how would you interest your community in learning about how to prevent active tb from latent tb infection ? the interviews were conducted in mandarin with four chinese volunteers recruited by one research assistant to ensure clarity of the topic guide questions . a mock fgd conducted in english under the supervision of an experienced coinvestigator who did not speak mandarin served to train two mandarin - speaking research assistants as facilitators . the purpose of this training was to ensure consistent processes used by the focus group facilitators during fgds . two fgds were then conducted in mandarin by the two facilitators one as the moderator and a second as note taker . prior to conducting the focus groups , a pamphlet was prepared describing basic facts about ltbi . it was distributed to participants after the first key question so that subsequent questions could be answered even if initial knowledge levels regarding ltbi had been low . anonymity was preserved by assigning and using a unique number to identify each study participant . all statistical analyses were executed with rstudio version 3.0.3 for windows ( rstudio inc . , demographic information of survey respondents was calculated as proportions of total respondents , and a mean knowledge score was calculated from six equally weighted questions on basic facts about ltbi . open coding of data from transcripts and field notes was performed in simplified chinese to preserve nuances during translation . codes , themes , and selected quotes that were particularly representative of the major themes were then translated into english . all transcripts were reviewed and coded by three mandarin - speaking research assistants who sought to identify patterns and trends in emergent themes relevant to ltbi and its treatment . they began by independently reviewing the fgds transcripts through line - by - line examination from which each research assistant identified relevant codes . codes were aggregated to decrease overlap , and disagreements were addressed by having the coders review the primary data together until consensus among them was reached . they continued to code the transcripts independently , consult with each other , and update the code book iteratively as necessary [ 29 , 30 ] . all codes were used to create a conceptual map from which four major themes emerged ( figure 1 ) . to establish themes , codes were grouped into subcategories and categories according to their meanings that were described in the code book , and lines were drawn to display the connections between codes and categories . the conceptual map was shared with our research team to ensure interpretations of the codes were meaningful , to discuss the themes , to relabel or regroup the data when necessary , and to determine if additional information needed to be solicited from the transcripts . quotes were then sorted into each category and subcategory , followed by comparing and contrasting quotes within and between groups to identify differences and similarities . one research assistant documented the process and the themes that emerged [ 22 , 23 ] . of 912 survey respondents , 67.1% were women , 91.7% had completed high school , and the largest age group among respondents was 2544 years old ( 49.3% ) . only 19.8% answered that ltbi had no symptoms ; 19.0% believed that ltbi did not spread from person to person ; and 44.0% knew that treating ltbi prevented tb . most patients ( 63.9% ) answered that ltbi was treated with prescribed tb medicine , but fewer ( 41.4% ) knew the duration of treatment . nearly half of respondents ( 43.8% ) answered that bcg vaccination conferred life - long immunity . the mean knowledge score was 40.0% ( 95% ci : 38.3% , 41.7% ) for all respondents ( table 1 ) . the first group included six early migrants who had immigrated to canada more than six years ago : two females and four males with a mean age of 40.3 years old . the second group included six recent migrants who had lived in canada for six or fewer years and all were female with a mean age of 34.5 years old . half had obtained a high school diploma ( table 2 ) . among the twenty - five potential participants contacted ( sixteen survey respondents and nine people recruited through advertising ) , twelve participated in this study to completion . the remaining thirteen ( all survey respondents ) either did not respond ( n = 8) or refused to participate ( n = 5 ) . four major themes emerged from the fgds : knowledge of ltbi ; concerns about the health system ; marginalization from society ; and ways of raising awareness ( figure 1 ) . participants in both focus groups expressed low levels of general knowledge concerning ltbi ( figure 1 ) . definition . all of the participants confused ltbi with active lung tb , and some recent migrants used the word furthermore , two recent migrants considered ltbi to be a lethal cancer . cause and transmission . in each focus group , participants frequently talked about ltbi as being caused by either bacteria or , even more commonly , by a germ . suggested risk factors for ltbi included when the immune system is weak , malnutrition , tiredness , and not sleeping well . participants in each group knew that tb could be transmitted through the air from the cough of a person with active tb , and most of them explicitly stated that tb bacteria could affect anyone , particularly in public places . one recent immigrant indicated that ltbi could be transmitted through food because food contains tb bacteria . diagnosis . participants in each group described the signs and symptoms of ltbi as equivalent to the symptoms of active tb : coughing , spitting up blood , only one early immigrant said that there was a skin test used to diagnose active tb since she had previously undergone the test . she expressed concerns about the validity of the skin test : i told him [ my family doctor ] that i was given the vaccine before . so , there are antibodies in my body . but , the skin test may neutralize the antigens . so , i asked him if it was really necessary for me to do this skin test . i told him [ my family doctor ] that i was given the vaccine before . so , i asked him if it was really necessary for me to do this skin test . all early immigrants and most recent immigrants believed that one could prevent ltbi from developing into active tb by doing exercises and having a balanced diet , for example , eating a lot of vitamin c in order to strengthen the immune system . in contrast , one recent migrant was less hopeful for patients with ltbi : if you have ltbi , i think you can do nothing . i mean , do not entice , do not trigger , or do not wake up the bacteria . after this information was provided to them , participants in both focus groups expressed concerns about taking medications : all the antibiotics have side effects , such as fungal infection , the cost of the medicines should not be too high , and the probability of developing active tb from ltbi is relatively low compared to side effects of the drugs . in addition , not having symptoms and not feeling sick were also mentioned in both groups as reasons to refuse any treatment . when they were asked which treatment(s ) they considered the most effective , some participants said traditional treatment methods were preferable while others disagreed and preferred to seek western interventions . the cost of the treatment was an important consideration for the majority of the participants and was mentioned in both groups . they worried that the costs of ltbi treatment were too high and represented a significant barrier to seeking care.for example , if i 'm poor , and the folk prescription is relatively cheaper , i 'll try it . but if i have the money , i 'll go to see a medical expert , definitely people who are rich wo n't abandon the treatment . for example , if i 'm poor , and the folk prescription is relatively cheaper , i 'll try it . but if i have the money , i 'll go to see a medical expert , definitely people who are rich wo n't abandon the treatment . participants in both groups discussed at length their concerns about the health system , which could hinder or encourage whether they sought health care for ltbi ( figure 1 ) . participants in both groups stated that in general they trusted medical professionals in canada to provide appropriate care . however , almost all participants questioned if their family doctors had enough knowledge of ltbi to diagnose this disease correctly . moreover , cultural and language barriers were highlighted by early immigrants as barriers that hindered effective communication with family doctors.the family doctor should communicate with us using the language or expressions that we can understand . for example , they can tell us eating garlic may be good for your health , which could be easy for us to understand we know that we chinese all eat garlic . so , i mean , they need to use the language that is similar to our daily language so we can accept it more easily . the family doctor should communicate with us using the language or expressions that we can understand . for example , they can tell us eating garlic may be good for your health , which could be easy for us to understand we know that we chinese all eat garlic . so , i mean , they need to use the language that is similar to our daily language so we can accept it more easily . waiting time . most early migrants commented on the long waiting times associated with the canadian health system and that decision - makers should make a concerted effort to address this . they indicated that they might go to other countries to treat the disease instead of waiting for a long time in canada . all of the recent migrants and most of the early migrants believed that people with ltbi would be marginalized from society ( figure 1 ) . many participants voiced that people with ltbi might be rejected by their neighbours , relatives , or friends : their first inclination will be to go away from me if i am diagnosed with ltbi . stigma . stigma associated with ltbi was mentioned as a portrayal of self - marginalization . some participants believed that their neighbours would panic and disclose a diagnosis of ltbi to others . they also worried that people might label them as sick persons who could transmit the disease.i'm afraid my neighbours will discriminate against me . they will tell everyone they know that i have this disease some of your friends will isolate you . even if i tell my friends i have ltbi , and it is not infectious , will they believe me ? . they will tell everyone they know that i have this disease some of your friends will isolate you . even if i tell my friends i have ltbi , and it is not infectious , will they believe me ? in order to protect themselves from stigma , most participants said that they would keep it a secret if they were diagnosed with ltbi . they would choose not to tell their neighbours or friends because it was not infectious . three early migrants disagreed , saying that if they had ltbi , it was their responsibility to tell their neighbours . so , i do not want them to guess what kind of disease i have . all participants would inform their families since they felt their family members could provide support to them , including helping figure out how to treat ltbi . all of the participants were interested in learning about ltbi and its treatment and had various suggestions to increase chinese immigrants ' awareness . ideas ranged from creating pamphlets to posting videos in mandarin and simplified chinese on websites that were popular for chinese communities including vansky , although past research has generalized inquiries regarding ltbi from research on active tb , our results support the need to study ltbi as a separate and specific phenomenon . chinese immigrants may be more likely to have negative attitudes towards ltbi treatment compared to tb treatment for several reasons . treating active tb patients can eliminate symptoms in a short time while treating people with ltbi can not improve their immediate well - being because ltbi causes no symptoms [ 9 , 31 ] . secondly , unlike active tb , ltbi is not contagious and thus the moral urgency of protecting others through one 's own treatment is mitigated . more importantly , participants in our study raised specific concerns about ltbi treatment in that the probability of developing active tb from ltbi was relatively low compared to side effects of the drugs . this concern regarding side effects has not been highlighted in studies of active tb probably because the perceived costs and benefits of taking medications to treat active tb are quite different from those of ltbi [ 14 , 16 ] . specific actions that can address each of our main findings are listed in table 3 . we identified the need for health promotional materials that are specifically tailored to addressing knowledge and perceptions of ltbi among the chinese immigrant community . in our study , tb knowledge was very low for all survey respondents and fgds participants . ltbi was often confused with tb disease , and this confusion may influence the demand for ltbi diagnosis and treatment , in that treatment may be considered unnecessary if ltbi is perceived as an incurable disease [ 32 , 33 ] . health educational materials such as videos or pamphlets with accurate , culturally relevant information on cause , transmission , diagnosis , treatment , and consequences of ltbi are required to address this knowledge gap [ 18 , 34 ] . health care providers should uncover how specific immigrant groups perceive ltbi treatment and provide them with materials to stress the distinction between ltbi and other diseases ( table 3 ) . such information may address misconceptions and barriers to treatment completion [ 13 , 20 ] . our study further uncovered several issues specifically related to chinese immigrants ' perceptions of the health care system that could also be addressed through health promotional materials . lack of familiarity with navigating the health system and negotiating its inefficiencies were perceived barriers to being tested for ltbi and/or taking ltbi treatment . for example , study participants did not know where to seek health care for ltbi . although medication for treating ltbi is free of charge in bc , many of the participants believed that cost was a barrier . they may be unable to or unwilling to seek health care because of these real and perceived barriers . to overcome these barriers , clear messaging on how to navigate the health system providing incentives and assistance for chinese immigrants could encourage treatment adherence . to facilitate effective physician - patient communication , identified as a priority by early migrants , family physicians who provide health care to chinese immigrants should be made aware of cultural differences in interpreting the meaning of ltbi and have access to culturally relevant health promotional tools , guidelines , or education and training programs ( table 3 ) . stigma related to ltbi arose in focus groups as a particularly important societal issue , and the reduction of stigma could be central to health promotion efforts in this area . a study on active tb conducted previously in alberta , canada , reported that many people in a local chinese community knew that they should help others reduce tb - related fears . however , in our study , none of the participants claimed to be willing to help others eliminate ltbi - related panic nor would they be open to receiving such help from others . since strong social support can enhance adherence to treatment , stigma is likely to reduce ltbi treatment adherence . patients may avoid seeking health care if they feel they must hide their ltbi diagnosis from their support networks . to address this problem , community - based educational materials such as an online video , perceived as one of the most appropriate mediums by participants , could be created to raise awareness of ltbi community - wide and consequently influence adherence to ltbi treatment ( table 3 ) [ 13 , 34 ] this study met the criteria for rigor of qualitative research , namely , trustworthiness including credibility , transferability , dependability , and confirmability . data triangulation drew from the mock fgd , field notes , survey results , and discussions with the research team members . member checking consisted of providing fgds participants with interpreted themes and codes through emails and validation of our interpretation of data through individual phone calls by the same research assistant who facilitated the fgds . to achieve transferability , our focus groups were assembled from both early and recent immigrants , as well as a diverse age range and mix of genders . this demographic diversity strengthened our ability to potentially capture knowledge and perceptions towards ltbi among immigrants who came from mainland china to the greater vancouver area . a dependability audit was conducted by the primary investigator and one coinvestigator through reviewing the activities of the researchers in the whole process . finally , a confirmability audit trail that included field notes , transcripts , and investigators ' memos was used to provide linkages between the raw data and the final conceptualizations . first , our survey at the time of the focus groups was conducted in english , so even though chinese immigrants frequently attended our clinic , few responded to the survey . secondly , due to limited resources , we were only able to conduct two fgds , so thematic saturation may not have been achieved , and participants ' perceptions may not represent chinese immigrants ' in rural parts of the province or in the rest of canada . thirdly , we may not have captured all perspectives because we necessarily omitted the perspectives of any potential participants who refused to participate due to their beliefs or lack of time . fourthly , the moderator of fgds was a recent chinese immigrant , and it is possible that early immigrants may not have identified with her . finally , during data analysis , coders may have been focused on particular areas relevant to ltbi and could potentially have ignored others . this study identified knowledge and perceptions of chinese immigrants towards ltbi and highlighted key areas of educational efforts to focus on . it points to the importance of raising awareness of ltbi and reducing ltbi related stigma in chinese immigrant communities . communication barriers with family doctors could be addressed during routine health services for this patient population to encourage and promote increased adherence to ltbi treatment with a goal of further reducing tb incidence in bc . ltbi and tb diseases are distinct although related issues and our findings support studying them independently . further research should include fgds in other immigrant groups to ascertain if their perceptions are similar and evaluation of health promotional materials specific to immigrants to ensure that these materials overcome the barriers identified in this study .

background . since most tuberculosis ( tb ) cases in immigrants to british columbia ( bc ) , canada , develop from latent tb infection ( ltbi ) , treating immigrants for ltbi can contribute to the eradication of tb . however , adherence to ltbi treatment is a challenge that is influenced by knowledge and perceptions . this research explores chinese immigrants ' knowledge and perceptions towards ltbi in greater vancouver . methods . this mixed methods study included a cross - sectional patient survey at bc 's provincial tb clinics and two focus group discussions ( fgds ) with chinese immigrants . data from fgds were coded and analyzed in simplified chinese . codes , themes , and selected quotes were then translated into english . results . the survey identified a mean basic knowledge score : 40.0% ( 95% ci : 38.3% , 41.7% ) . fgds confirmed that chinese immigrants ' knowledge of ltbi was low , and they confused it with tb disease to the extent of experiencing ltbi associated stigma . participants also expressed difficulties navigating the health system which impeded testing and treatment of ltbi . online videos were the preferred format for receiving health information . conclusion . we identified striking gaps in knowledge surrounding an ltbi diagnosis . concerns of stigma may influence acceptance and adherence of ltbi treatment in chinese immigrants . integrating these findings into routine health care is recommended .

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

in british columbia ( bc ) , canada , the ethnic breakdown of people with tuberculosis ( tb ) has changed over the last several decades . since 2010 , there has been an average of 269 active tb cases per year in bc , more than 70 percent of which affected immigrants . in 2013 , the majority of tb cases were in chinese- , punjabi- , tagalog- , korean- , and vietnamese - speaking immigrants , with the highest proportion of tb cases being immigrants from mainland china . immigrants also accounted for the majority of persons starting treatment for latent tb infection ( ltbi)64.1 percent in 2011 . most active tb cases in immigrants developed from ltbi that had been acquired in their countries of origin based on genotyping studies [ 35 ] . the who has recommended treating ltbi in immigrants as a strategy that can further reduce active tb cases in low incidence settings such as bc . initiation of and adherence to ltbi treatment are generally low in immigrants to north america [ 79 ] . in canada , drug therapy for ltbi is usually four to nine months depending upon the regimen used . incomplete courses of treatment are less effective in preventing tb disease ; thus , adherence to and completion of the entire course of ltbi therapy are essential [ 10 , 11 ] . low treatment adherence among immigrants partially depends on their knowledge and perceptions of ltbi [ 1216 ] , which can be influenced by the use of culturally specific educational materials and cultural brokers [ 17 , 18 ] . providing accessible , culturally sensitive messaging through a variety of formats is hypothesized to increase rates of treatment initiation and consequently increase acceptance and completion of ltbi treatment [ 19 , 20 ] . because chinese immigrants account for the highest proportion of tb cases in bc , the qualitative part of the study focused on chinese immigrants . previous studies have identified some issues related to knowledge and perceptions of ltbi and its treatment among chinese immigrants in north america [ 14 , 16 ] . these studies focused on active tb and reported that chinese immigrants ' knowledge of the transmission of tb bacteria and prevention of tb disease was low . chinese immigrants generally believed in western health care providers ' abilities and knowledge and were willing to seek care from them but preferred traditional chinese medicine . moreover , access to health insurance and the stigma associated with tb led to people not seeking medical care even if they suspected that they had tb [ 14 , 16 ] . the objectives of this research were to ( a ) document knowledge levels of patients in a tb clinic ; ( b ) identify chinese immigrants ' knowledge and perceptions of ltbi ; ( c ) specify messages that would be most appropriate for ltbi education in this population ; and ( d ) identify the most appropriate format of health promotional materials to address the specific needs of this population . as part of a larger tb education project that included developing culturally relevant and informative materials for a diverse group of immigrants , we conducted a mixed methods study . it included a patient survey at the provincial tb clinics in greater vancouver that evaluated knowledge of ltbi and treatment of it and a qualitative approach using fgd for chinese immigrants [ 22 , 23 ] . a survey in english of patients who visited the bc centre for disease control ( bccdc ) tb clinics was conducted from june 2013 to june 2014 . survey questions of knowledge , attitudes , and perceptions regarding ltbi and its treatment were developed and pilot - tested in five clerical staff from bccdc to establish clarity of questions . to estimate the level of knowledge only very basic facts about ltbi were included in the survey , and we considered a score of 80% or higher as representing adequate knowledge . after refining them , since no further edits were required , we then pilot - tested the questions for clarity with 20 patients . respondents to the survey included all individuals who presented to the tb clinics and were 18 years old or older . using focus group discussions ( fgds ) , we then explored further details of knowledge and perceptions towards ltbi specifically in chinese immigrants . all participants self - selected either early ( well - settled ) immigrants or recent ( new ) immigrants according to their self - perceived levels of adjusting to the new culture , their social engagement with canadians , their perceptions of settlement , their feelings of belonging , and length of time residing in canada [ 14 , 26 , 27 ] . ethics approval was obtained from the behavioural research ethics board of the university of british columbia and the office of research ethics of simon fraser university . for fgds , introductory letters and consent forms written in simplified chinese were sent to each participant through email two weeks prior to the fgds . signed consent forms were collected at the focus groups . surveys were distributed by trained clerical staff at the bccdc tb clinics to all patients of 18 years old or older . data were input weekly into a microsoft office access database by a trained clinic clerk , reviewed for accuracy by a study team member , and then imported into a microsoft office excel dataset for data analysis . for fgds , a structured topic guide , informed by the literature , was developed in english and then translated into simplified chinese . four key questions guided the data collection : ( 1 ) what do you know about treatment options for latent tb infection ? ( 2 ) what have you heard in your family or community about latent tb infection and about the treatment of it ? ( 3 ) what might influence whether someone with latent tb infection takes treatment ? ( 4 ) how would you interest your community in learning about how to prevent active tb from latent tb infection ? a mock fgd conducted in english under the supervision of an experienced coinvestigator who did not speak mandarin served to train two mandarin - speaking research assistants as facilitators . the purpose of this training was to ensure consistent processes used by the focus group facilitators during fgds . two fgds were then conducted in mandarin by the two facilitators one as the moderator and a second as note taker . prior to conducting the focus groups , a pamphlet was prepared describing basic facts about ltbi . it was distributed to participants after the first key question so that subsequent questions could be answered even if initial knowledge levels regarding ltbi had been low . , demographic information of survey respondents was calculated as proportions of total respondents , and a mean knowledge score was calculated from six equally weighted questions on basic facts about ltbi . open coding of data from transcripts and field notes was performed in simplified chinese to preserve nuances during translation . codes , themes , and selected quotes that were particularly representative of the major themes were then translated into english . all transcripts were reviewed and coded by three mandarin - speaking research assistants who sought to identify patterns and trends in emergent themes relevant to ltbi and its treatment . they began by independently reviewing the fgds transcripts through line - by - line examination from which each research assistant identified relevant codes . codes were aggregated to decrease overlap , and disagreements were addressed by having the coders review the primary data together until consensus among them was reached . they continued to code the transcripts independently , consult with each other , and update the code book iteratively as necessary [ 29 , 30 ] . to establish themes , codes were grouped into subcategories and categories according to their meanings that were described in the code book , and lines were drawn to display the connections between codes and categories . the conceptual map was shared with our research team to ensure interpretations of the codes were meaningful , to discuss the themes , to relabel or regroup the data when necessary , and to determine if additional information needed to be solicited from the transcripts . quotes were then sorted into each category and subcategory , followed by comparing and contrasting quotes within and between groups to identify differences and similarities . of 912 survey respondents , 67.1% were women , 91.7% had completed high school , and the largest age group among respondents was 2544 years old ( 49.3% ) . only 19.8% answered that ltbi had no symptoms ; 19.0% believed that ltbi did not spread from person to person ; and 44.0% knew that treating ltbi prevented tb . most patients ( 63.9% ) answered that ltbi was treated with prescribed tb medicine , but fewer ( 41.4% ) knew the duration of treatment . the mean knowledge score was 40.0% ( 95% ci : 38.3% , 41.7% ) for all respondents ( table 1 ) . the first group included six early migrants who had immigrated to canada more than six years ago : two females and four males with a mean age of 40.3 years old . the second group included six recent migrants who had lived in canada for six or fewer years and all were female with a mean age of 34.5 years old . among the twenty - five potential participants contacted ( sixteen survey respondents and nine people recruited through advertising ) , twelve participated in this study to completion . four major themes emerged from the fgds : knowledge of ltbi ; concerns about the health system ; marginalization from society ; and ways of raising awareness ( figure 1 ) . definition . all of the participants confused ltbi with active lung tb , and some recent migrants used the word furthermore , two recent migrants considered ltbi to be a lethal cancer . in each focus group , participants frequently talked about ltbi as being caused by either bacteria or , even more commonly , by a germ . suggested risk factors for ltbi included when the immune system is weak , malnutrition , tiredness , and not sleeping well . participants in each group knew that tb could be transmitted through the air from the cough of a person with active tb , and most of them explicitly stated that tb bacteria could affect anyone , particularly in public places . diagnosis . participants in each group described the signs and symptoms of ltbi as equivalent to the symptoms of active tb : coughing , spitting up blood , only one early immigrant said that there was a skin test used to diagnose active tb since she had previously undergone the test . all early immigrants and most recent immigrants believed that one could prevent ltbi from developing into active tb by doing exercises and having a balanced diet , for example , eating a lot of vitamin c in order to strengthen the immune system . i mean , do not entice , do not trigger , or do not wake up the bacteria . after this information was provided to them , participants in both focus groups expressed concerns about taking medications : all the antibiotics have side effects , such as fungal infection , the cost of the medicines should not be too high , and the probability of developing active tb from ltbi is relatively low compared to side effects of the drugs . in addition , not having symptoms and not feeling sick were also mentioned in both groups as reasons to refuse any treatment . they worried that the costs of ltbi treatment were too high and represented a significant barrier to seeking care.for example , if i 'm poor , and the folk prescription is relatively cheaper , i 'll try it . but if i have the money , i 'll go to see a medical expert , definitely people who are rich wo n't abandon the treatment . for example , if i 'm poor , and the folk prescription is relatively cheaper , i 'll try it . participants in both groups discussed at length their concerns about the health system , which could hinder or encourage whether they sought health care for ltbi ( figure 1 ) . however , almost all participants questioned if their family doctors had enough knowledge of ltbi to diagnose this disease correctly . for example , they can tell us eating garlic may be good for your health , which could be easy for us to understand we know that we chinese all eat garlic . so , i mean , they need to use the language that is similar to our daily language so we can accept it more easily . the family doctor should communicate with us using the language or expressions that we can understand . so , i mean , they need to use the language that is similar to our daily language so we can accept it more easily . most early migrants commented on the long waiting times associated with the canadian health system and that decision - makers should make a concerted effort to address this . all of the recent migrants and most of the early migrants believed that people with ltbi would be marginalized from society ( figure 1 ) . many participants voiced that people with ltbi might be rejected by their neighbours , relatives , or friends : their first inclination will be to go away from me if i am diagnosed with ltbi . stigma associated with ltbi was mentioned as a portrayal of self - marginalization . some participants believed that their neighbours would panic and disclose a diagnosis of ltbi to others . even if i tell my friends i have ltbi , and it is not infectious , will they believe me ? they will tell everyone they know that i have this disease some of your friends will isolate you . even if i tell my friends i have ltbi , and it is not infectious , will they believe me ? so , i do not want them to guess what kind of disease i have . all of the participants were interested in learning about ltbi and its treatment and had various suggestions to increase chinese immigrants ' awareness . ideas ranged from creating pamphlets to posting videos in mandarin and simplified chinese on websites that were popular for chinese communities including vansky , although past research has generalized inquiries regarding ltbi from research on active tb , our results support the need to study ltbi as a separate and specific phenomenon . chinese immigrants may be more likely to have negative attitudes towards ltbi treatment compared to tb treatment for several reasons . treating active tb patients can eliminate symptoms in a short time while treating people with ltbi can not improve their immediate well - being because ltbi causes no symptoms [ 9 , 31 ] . secondly , unlike active tb , ltbi is not contagious and thus the moral urgency of protecting others through one 's own treatment is mitigated . more importantly , participants in our study raised specific concerns about ltbi treatment in that the probability of developing active tb from ltbi was relatively low compared to side effects of the drugs . this concern regarding side effects has not been highlighted in studies of active tb probably because the perceived costs and benefits of taking medications to treat active tb are quite different from those of ltbi [ 14 , 16 ] . specific actions that can address each of our main findings are listed in table 3 . we identified the need for health promotional materials that are specifically tailored to addressing knowledge and perceptions of ltbi among the chinese immigrant community . ltbi was often confused with tb disease , and this confusion may influence the demand for ltbi diagnosis and treatment , in that treatment may be considered unnecessary if ltbi is perceived as an incurable disease [ 32 , 33 ] . health educational materials such as videos or pamphlets with accurate , culturally relevant information on cause , transmission , diagnosis , treatment , and consequences of ltbi are required to address this knowledge gap [ 18 , 34 ] . health care providers should uncover how specific immigrant groups perceive ltbi treatment and provide them with materials to stress the distinction between ltbi and other diseases ( table 3 ) . our study further uncovered several issues specifically related to chinese immigrants ' perceptions of the health care system that could also be addressed through health promotional materials . lack of familiarity with navigating the health system and negotiating its inefficiencies were perceived barriers to being tested for ltbi and/or taking ltbi treatment . for example , study participants did not know where to seek health care for ltbi . although medication for treating ltbi is free of charge in bc , many of the participants believed that cost was a barrier . they may be unable to or unwilling to seek health care because of these real and perceived barriers . to overcome these barriers , clear messaging on how to navigate the health system providing incentives and assistance for chinese immigrants could encourage treatment adherence . to facilitate effective physician - patient communication , identified as a priority by early migrants , family physicians who provide health care to chinese immigrants should be made aware of cultural differences in interpreting the meaning of ltbi and have access to culturally relevant health promotional tools , guidelines , or education and training programs ( table 3 ) . stigma related to ltbi arose in focus groups as a particularly important societal issue , and the reduction of stigma could be central to health promotion efforts in this area . a study on active tb conducted previously in alberta , canada , reported that many people in a local chinese community knew that they should help others reduce tb - related fears . however , in our study , none of the participants claimed to be willing to help others eliminate ltbi - related panic nor would they be open to receiving such help from others . since strong social support can enhance adherence to treatment , stigma is likely to reduce ltbi treatment adherence . patients may avoid seeking health care if they feel they must hide their ltbi diagnosis from their support networks . to address this problem , community - based educational materials such as an online video , perceived as one of the most appropriate mediums by participants , could be created to raise awareness of ltbi community - wide and consequently influence adherence to ltbi treatment ( table 3 ) [ 13 , 34 ] this study met the criteria for rigor of qualitative research , namely , trustworthiness including credibility , transferability , dependability , and confirmability . data triangulation drew from the mock fgd , field notes , survey results , and discussions with the research team members . this demographic diversity strengthened our ability to potentially capture knowledge and perceptions towards ltbi among immigrants who came from mainland china to the greater vancouver area . a dependability audit was conducted by the primary investigator and one coinvestigator through reviewing the activities of the researchers in the whole process . finally , a confirmability audit trail that included field notes , transcripts , and investigators ' memos was used to provide linkages between the raw data and the final conceptualizations . first , our survey at the time of the focus groups was conducted in english , so even though chinese immigrants frequently attended our clinic , few responded to the survey . secondly , due to limited resources , we were only able to conduct two fgds , so thematic saturation may not have been achieved , and participants ' perceptions may not represent chinese immigrants ' in rural parts of the province or in the rest of canada . fourthly , the moderator of fgds was a recent chinese immigrant , and it is possible that early immigrants may not have identified with her . finally , during data analysis , coders may have been focused on particular areas relevant to ltbi and could potentially have ignored others . this study identified knowledge and perceptions of chinese immigrants towards ltbi and highlighted key areas of educational efforts to focus on . it points to the importance of raising awareness of ltbi and reducing ltbi related stigma in chinese immigrant communities . communication barriers with family doctors could be addressed during routine health services for this patient population to encourage and promote increased adherence to ltbi treatment with a goal of further reducing tb incidence in bc . ltbi and tb diseases are distinct although related issues and our findings support studying them independently . further research should include fgds in other immigrant groups to ascertain if their perceptions are similar and evaluation of health promotional materials specific to immigrants to ensure that these materials overcome the barriers identified in this study .

one of the most popular and effective methods for motion tracking in cardiac images is optical flow ( of ) . this technique is utilized for the purpose of motion estimation of 2d cardiac images in many studies.[16 ] of calculation was first introduced by horn and schunck . this technique is one of the most important and well - known methods in the context of machine vision . of there are four groups of methods for of calculation : differential methods , block - matching methods , energy - based methods , and phase - based methods . most of these methods are based on the assumption that intensity of each region of a moving image remains constant in consecutive frames . in fact , in of formulation , the main idea is that the intensity of a set of pixels is preserved between the times t and t+t . the intensity of an image may be inconstant for some reasons like motions of the object ( translation or rotation ) , motions of the light source , or variations of brightness in the entire image . in echocardiography images , the intensity constancy assumption is violated in many regions because of complicated motions of beating heart , including translation , rotation , shear , contraction , and expansion . these complex motions , leading to displacement of the heart myocardial fibers in the direction of transducer , may result in entering or removing some pixels in some areas in the next frame . in fact , cyclic variations of the echocardiography images , as a function of the cardiac muscle behavior , depend on the angle of myocardial fibers respect to ultrasonic wave propagation . in addition , the intensity of image may alter for the effect of large displacements like the displacements of mitral valve . also , speckle noise and its variations in each frame can influence the intensity . intensity inconstancy , which leads to violation of the basic assumption of of calculations , is an important source of error in motion estimation . considering the intensity variations of moving patterns in of computations could bring about more precise and less erroneous motion estimations . in some of the previous studies , the effect of intensity variation in of computations is surveyed.[101319 ] these studies resulted in more accurate estimations than common of techniques . nevertheless , none of them have presented a method for considering the intensity inconstancy in of in echocardiography images . this matter motivated us to present a new method for considering intensity variations in echocardiography sequences . a physical model is presented for infrared images , which models the intensity variationsas an exponential function . in another approach , namely generalized dynamic imaging model ( gdim ) , the hypothesis is that the intensity varies as product of a multiplier in previous amount of intensity , and an offset is added to it . in other words , the intensity variation is modeled with a multiplier and an offsetwhich is more accurate than the other works considering the intensity variations just as a multiplier or just an offset . in gdim , initially , these quantities are unknown and then , will be computed associated with two more unknowns belonging to of ; the unknowns that are related to of are velocity field components ( u , v ) . also , there are some other researches accomplished in this context , which consider brightness inconstancy using different constraints and models.[2224 ] finding a precise mathematical function as a model of intensity variation in echocardiography images is difficult due to the fact that these images have unique specifications and myocardial fibers have complicated motions in every cardiac cycle . these fibers have a non - uniform motion , in different directions and also different sizes in each direction . as mentioned above , cyclic variations in echocardiography images are a function of the cardiac muscle motion and depend on the orientation of myocardial fibers toward ultrasonic wave . none of the mentioned approaches has considered the specifications and variations of echocardiograms and they have not addressed the problem of intensity inconstancy in this category of images . therefore , in this research , we motivated to propose a method which considers the variations in echocardiography images . to state the matter differently , in this approach , the intensity variation that occurs between two consecutive frames is calculated pixel - by - pixel in the image and afterwards , the effect of the variation is combined with of calculations . in this study , we investigate whether our proposed method improves the motion estimation of echocardiography images by of techniques . in subsequent parts of this paper , in section 2 , firstlythe of method and its equations which are required for cardiac motion estimation are described ; then our proposed method will be clarified . in section 3 , validation results are expressed ; these results first arise from a comparison between our method and two otherof - based methods and then from a comparison between the proposed method and expert point of view as a golden standard . finally , section 4 is assigned to summarizing and conclusionof the paper and future works . the main idea of of is based on the constancy of image intensity in consecutive frames . if i(x , y , t ) is the intensity of pixelsat location expressed by ( x , y ) and time t , the basic assumption of of can be declared by ( 1 ) : i(x , y , t ) = i(x + x , y + y , t + t ) ( 1 ) in this equation , x and y are determined as below : ( u , v ) are the of or velocity components in the horizontal and vertical directions at the point ( x , y ) , and x , y are displacements at mentioned directions , respectively . first - order taylor expansion of the right hand of ( 1 ) will result in ( 2 ) : in this equation , ix , iy are spatial derivatives of intensity in x , y directions , respectively , and it is the temporal derivative of intensity . in ( 2 ) , there are two unknowns ( u , v ) in one equation . consequently , the solution of this equation is not possible with usual methods . some methods are proposed to solve the ( 2 ) in an efficient way . in this paper , we use the method proposed by lucas and kanade which estimates the motion locally assuming it to be invariant within a spatial window . they suggested that this equation could be solved by minimization methods like weighted least squares . the advantage of this method is that the of calculations are not implemented in the entire image . in other words , the weighted least squares equationis represented in ( 3 ) : in this equation , x = ( x , y ) and w(x ) is a 2d window with dimension of ll , including n pixels of xi that is the domain containing the neighborhood of x. minimizing this equation with respect to v = ( u , v ) , ( 4 ) is obtained : a = [ i(x1), ,i(xn ) ] ( 5 ) w = diag[w(x1), ,w(xn ) ] ( 6 ) b = (it(x1), ,it(xn ) ) ( 7 ) the solution of ( 4 ) is expressed in ( 9 ) : in all of these equations , the intensity variation influence is not considered . in this study , a coarse - to - fine approach is implemented . number of the coarse - to - fine pyramid levels in our implementation is selected by comparison of efficiency between different choices . it means that a coarse - to - fine pyramid with four levels has the maximum accuracy and needs the minimum calculation time . each frame of the image is sub - sampled by the factor 1/2 at each level and a pyramid of images with four different resolutions is created . at the next step , the motion estimation algorithm , mentioned in previous equations , is accomplished starting at coarsest level ( the level having less resolution ) continuing to finest level ( the level having the most resolution ) . the estimation of each level transfers to the next level as an initial estimation ; the initial value in first level of the pyramid is set to zero . the aim of this research is combining the intensity variation influence with mentioned equations to improve the motion estimation in echocardiography images . in this study subsequently , the obtained values are replaced with the right hand of ( 2 ) that is zero . this means that in our calculations ( 2 ) is altered to ( 10 ) , as below : in this equation , i is the intensity variation in the pixel located at ( x , y ) and at the time t. in fact , i is calculated locally at each pixel of the image . the considerable point in ( 10 ) actually , ( x,y ) is the displacement vector or the motion field of each pixel which is in close relation with the principal unknown of the of equation , namely the velocity vector ( u , v ) . consequently , we do not have ( x,y ) for computation of i at the first step . as a result , an estimation of these unknowns firstly , for each frame of the sequence , the motion estimation algorithm based on the weighted least squares approach is applied to every level of the coarse - to - fine pyramid , from the coarsest to finest level , respectively . at each level , the frame is up - sampled with the factor 2 and the algorithm is applied again . this process is repeated while reaching to the pyramid apex , namely the finest level . the initial value for ( x,y ) at the first level is considered zero , and in the other levels , the initial value is obtained by doubling the estimated value in previous level . in figure 1 , motion estimation steps in a four level coarse to fine pyramid is demonstrated . in this figure , g00 and g10 are the initial sizes of two consecutive frames that in each level are sub sampled with the factor . g01 , g02 , g03 and g11 , g12 , g13 are the coarser levels of frame 1 and 2 , respectively . in addition , dx and dy are the same ( x,y ) which are updated in each level of the pyramid . for validation of the proposed method , the algorithm was implemented on real echocardiography images . data is acquired from this site : www.yale.edu/imaging/echo_atlas/contents/index.html and the resolution of imaging is 3.5 mhz . our method was compared with two more of - based methods , gdim and lucas - kanade . all of the methods have comparable parameter settings . in the calculations related to our proposed method and lucus - kanade method , the optimum values obtained for the window w(x ) dimensions were 1717 pixels . it was obtained by calculation of the optimum value of mean square error ( mse ) when a given displacement is applied to the image . the criterion of comparison , mse , is shown in ( 11 ) : in this equation , t(k ) is the set of true displacement values , c(k ) is the set of calculated values , and e(k ) is the error of each point . we selected a number of landmarks on a frame of echocardiography sequence to draw a curve calculated by our method . these landmarks were selected on endocardial border of heart and were interpolated by b - spline curves . we compared it with expert defined curve by calculation of three distance metrics , mean , rms and hausdorff , between two curves . motion components were estimated by our proposed method and two other methods , namely lucas - kanade and gdim . the lucas - kanade method is one of the common of techniques and the intensity variation is not considered in it . the of calculations of the lucas - kanade method are implemented by ( 2 ) . nevertheless , in the gdim method , the intensity variation is considered as a multiplier in first frame 's intensity and an offset . in other words , the of equationrelated to this method is written as ( 12 ) , while in our proposed method , the intensity variation is considered locally and pixel - by - pixel : in this equation , m,c respectively , are the multiplier and offset values . moreover , u , v are the unknowns in this equation . actually , these four parameters are calculated simultaneously using a least squares approach by ( 13 ) : the complete procedure of this calculation is expressed in the related reference . practically , determination of a ground truth for heart motions is a challenging issue in validation of tracking methods . to evaluate the proposed method for echocardiography images , owing to the fact that the real value of displacements in different regions of an echocardiography is not available , three given displacements , 5,10 , and 15 pixels , were applied on this image for validation purpose . our proposed method and two other methods ( lucas - kanade and gdim ) were implemented to estimate the mentioned given displacements . we observed that the mse of our method was clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied displacements in addition of displacement error , we computed angular error for the proposed method and compared it with two other mentioned algorithms . we applied three different optional angles , 0 , 45 , 75 , on an echocardiography image and estimated them with three methods . table 2 shows the mse of estimated angles with three algorithms ( proposed , lucas - kanade and gdim ) . it is observed that the angular error related to our method is clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied angles finally , we implemented our proposed method on consecutive frames of a sequence of echocardiography images with a frame rate of 24 frames per second . figure 2 shows the of calculated by our method between frames 12 , 13 of this sequence which are related to expansion phase ( diastole ) . in figure 2 , expansion of left ventricle can be observed by velocity arrows computed by our proposed of estimation method . for more appropriate visualization of of arrows frame 12 of an expanding heart motion estimation by the proposed method in an echocardiography image ; optical flow is calculated between frames 12 and 13 to evaluate the proposed method for real heart motions in echocardiography images , we drew the endocardial border of the left ventricle calculated with the proposed method and compared it with the manual curve defined by an expert who was blind to automatic estimation result . for this purpose , at first , we defined a set of landmarks on the endocardial border and interpolated them by b - spline curves ; secondly , we compared it with the curve defined by the expert . we calculated three distance metrics , namely mean , root mean square ( rms ) and hausdorff distance , between automatic and manually defined curves . mean distance is defined as ( 14 ) : in this equation , a and b are radial distances from a common central point and second metric distance , rms , is defined as ( 15 ) : the last and the most precise metric , haussdorff distance , is defined as ( 16 ) : where d is the euclidean distance between automatic and manual curves . in figure 3 , panels ( a ) to ( c ) show the b - spline interpolated curve related to frame 13 of the echocardiographic sequence calculated by lucas - kanade , gdim and the proposed method , respectively , and panel ( d ) shows the expert defined curve . table 3 shows the results of the comparison of three automatically defined curves with the manually defined one . it is observed that the haussdorff distance between the estimated curve defined by the proposed method and the manually defined curve , as a golden standard , is 4.81 pixels less than this distance for lucas - kanade and 2.28 pixels less than the gdim method . accordingly , we can say the proposed method is appropriately working when compared with the other methods . b - spline interpolated curve related to frame 13 of the echocardiography sequence calculated by ( a ) lucas - kanade ; ( b ) gdim ; ( c ) the proposed method ; and ( d ) expert defined curve distance metrics between three automatic and one expert defined curves practically , determination of a ground truth for heart motions is a challenging issue in validation of tracking methods . to evaluate the proposed method for echocardiography images , owing to the fact that the real value of displacements in different regions of an echocardiography is not available , three given displacements , 5,10 , and 15 pixels , were applied on this image for validation purpose . our proposed method and two other methods ( lucas - kanade and gdim ) were implemented to estimate the mentioned given displacements . we observed that the mse of our method was clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied displacements in addition of displacement error , we computed angular error for the proposed method and compared it with two other mentioned algorithms . we applied three different optional angles , 0 , 45 , 75 , on an echocardiography image and estimated them with three methods . table 2 shows the mse of estimated angles with three algorithms ( proposed , lucas - kanade and gdim ) . it is observed that the angular error related to our method is clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied angles finally , we implemented our proposed method on consecutive frames of a sequence of echocardiography images with a frame rate of 24 frames per second . figure 2 shows the of calculated by our method between frames 12 , 13 of this sequence which are related to expansion phase ( diastole ) . in figure 2 , expansion of left ventricle can be observed by velocity arrows computed by our proposed of estimation method . for more appropriate visualization of of arrows frame 12 of an expanding heart motion estimation by the proposed method in an echocardiography image ; optical flow is calculated between frames 12 and 13 to evaluate the proposed method for real heart motions in echocardiography images , we drew the endocardial border of the left ventricle calculated with the proposed method and compared it with the manual curve defined by an expert who was blind to automatic estimation result . for this purpose , at first , we defined a set of landmarks on the endocardial border and interpolated them by b - spline curves ; secondly , we compared it with the curve defined by the expert . we calculated three distance metrics , namely mean , root mean square ( rms ) and hausdorff distance , between automatic and manually defined curves . mean distance is defined as ( 14 ) : in this equation , a and b are radial distances from a common central point and second metric distance , rms , is defined as ( 15 ) : the last and the most precise metric , haussdorff distance , is defined as ( 16 ) : where d is the euclidean distance between automatic and manual curves . in figure 3 , panels ( a ) to ( c ) show the b - spline interpolated curve related to frame 13 of the echocardiographic sequence calculated by lucas - kanade , gdim and the proposed method , respectively , and panel ( d ) shows the expert defined curve . table 3 shows the results of the comparison of three automatically defined curves with the manually defined one . it is observed that the haussdorff distance between the estimated curve defined by the proposed method and the manually defined curve , as a golden standard , is 4.81 pixels less than this distance for lucas - kanade and 2.28 pixels less than the gdim method . accordingly , we can say the proposed method is appropriately working when compared with the other methods . b - spline interpolated curve related to frame 13 of the echocardiography sequence calculated by ( a ) lucas - kanade ; ( b ) gdim ; ( c ) the proposed method ; and ( d ) expert defined curve distance metrics between three automatic and one expert defined curves cardiac motions in echocardiography images may lead to variation of intensity in different areas of the image . intensity variations result in violation of the main assumption of the of calculations . in this paper , we considered intensity variation in of calculations and tried to compensate the effect of these variations to improve the estimations in echocardiography images . in our proposed method , the intensity variation was considered locally and pixel - by - pixel since heart has non uniform and complicated motions and definition of a mathematical function to model the intensity pattern is a problematic issue . we evaluated our method by comparison with two other of - based tracking methods , lucas - kanade and gdim . in addition , we compared the proposed method with expert point of view and observed that the curve which was defined by our method was close to the curve which was defined by expert , when compared to two other methods . this research can be a prelude to approach a specific model of intensity variation for echocardiography images . investigating the way of intensity variation in different regions of echocardiography images may lead to obtaining such a model . paria torkashvand received the b.s degree in electrical engineering from khaje nasireddin toosi university , tehran , iran , in 2007 and the m.s . degree in medical engineering from iran university of science and technology , tehran , iran in 2010 . e - mail : paria.torkashvand@gmail.com hamid behnam received the b.s . degree in electrical engineering from iran university of science and technology , tehran , iran , in 1988 , the m.s . degree in medical engineering from sharif university of technology , tehran , iran , in 1992 , the ph.d . degree in applied electronics from tokyo institute of technology , tokyo , japan in 1998 . since 1998 till 2004 , he was a researcher at iran research organization for science and technology and from 2004 he has been a faculty member at iran university of science and technology , in tehran , iran . his research interests are ultrasound in medicine , medical image processing and medical signal processing . e - mail : behnam@iust.ac.ir zahra alizadeh sani earned a medical degree at mashhad university of medical sciences , mashhad , iran in 1998 . she was resident of cardio vascular diseases in mashhad university of medical sciences , mashhad , iran in oct . completed fellowships in echocardiography ( sep.2007-mar.2009 ) , at iran university of medical sciences , and in basic cardiac mr ( june . her research interests are cardiovascular mri , physics , pulse sequence , clinical application , cardiovascular ct , angiography , physics , technique , clinical application echocardiography , physics , clinical application . since 2009 , she works as an assistant professor of cardiovascular imaging in cardiovascular imaging department , shaheed rajaei cardiovascular and medical center , tehran university of medical sciences .

the quantitative analysis of cardiac motions in echocardiography images is a noteworthy issue in processing of these images . cardiac motions can be estimated by optical flow ( of ) computation in different regions of image which is based on the assumption that the intensity of a moving pattern remains constant in consecutive frames . however , in echocardiographic sequences , this assumption may be violated because of unique specifications of ultrasound . there are some methodsapplying the brightness variation effect in of . almost all of them have presented a mathematical brightness variation model globally in the images . nevertheless , there is not a brightness variation model for echocardiographic images in these methods . therefore , we are looking for a method to apply brightness variations locally in different regions of the image . in this study , we proposed a method to modify ausual of technique by considering intensity variation . to evaluate this method , we implement two other of - based methods , one usual of method and a modified of method applying brightness variation as a multiplier and an offset ( generalized dynamic imaging model [ gdim ] ) and compare them with ours . these algorithms and ours were implemented on real 2d echocardiograms . our method resulted in more accurate estimations than two others . at last , we compared our method with expertspoint of view and observed that three distance metrics between them was appropriately smaller than other methods . the haussdorff distance between the estimated curve defined by the proposed method and the expert defined curve is 4.81 pixels less than this distance for lucas - kanade and 2.28 pixels less than gdim .

INTRODUCTION MATERIALS AND METHODS RESULTS Validation Results for Applied Motions in Echocardiography Images CONCLUSION BIOGRAPHIES

one of the most popular and effective methods for motion tracking in cardiac images is optical flow ( of ) . this technique is utilized for the purpose of motion estimation of 2d cardiac images in many studies. this technique is one of the most important and well - known methods in the context of machine vision . of there are four groups of methods for of calculation : differential methods , block - matching methods , energy - based methods , and phase - based methods . most of these methods are based on the assumption that intensity of each region of a moving image remains constant in consecutive frames . in fact , in of formulation , the main idea is that the intensity of a set of pixels is preserved between the times t and t+t . the intensity of an image may be inconstant for some reasons like motions of the object ( translation or rotation ) , motions of the light source , or variations of brightness in the entire image . in echocardiography images , the intensity constancy assumption is violated in many regions because of complicated motions of beating heart , including translation , rotation , shear , contraction , and expansion . these complex motions , leading to displacement of the heart myocardial fibers in the direction of transducer , may result in entering or removing some pixels in some areas in the next frame . in fact , cyclic variations of the echocardiography images , as a function of the cardiac muscle behavior , depend on the angle of myocardial fibers respect to ultrasonic wave propagation . in addition , the intensity of image may alter for the effect of large displacements like the displacements of mitral valve . also , speckle noise and its variations in each frame can influence the intensity . intensity inconstancy , which leads to violation of the basic assumption of of calculations , is an important source of error in motion estimation . considering the intensity variations of moving patterns in of computations could bring about more precise and less erroneous motion estimations . in some of the previous studies , the effect of intensity variation in of computations is surveyed. [101319 ] these studies resulted in more accurate estimations than common of techniques . nevertheless , none of them have presented a method for considering the intensity inconstancy in of in echocardiography images . this matter motivated us to present a new method for considering intensity variations in echocardiography sequences . a physical model is presented for infrared images , which models the intensity variationsas an exponential function . in another approach , namely generalized dynamic imaging model ( gdim ) , the hypothesis is that the intensity varies as product of a multiplier in previous amount of intensity , and an offset is added to it . in other words , the intensity variation is modeled with a multiplier and an offsetwhich is more accurate than the other works considering the intensity variations just as a multiplier or just an offset . also , there are some other researches accomplished in this context , which consider brightness inconstancy using different constraints and models. [2224 ] finding a precise mathematical function as a model of intensity variation in echocardiography images is difficult due to the fact that these images have unique specifications and myocardial fibers have complicated motions in every cardiac cycle . these fibers have a non - uniform motion , in different directions and also different sizes in each direction . as mentioned above , cyclic variations in echocardiography images are a function of the cardiac muscle motion and depend on the orientation of myocardial fibers toward ultrasonic wave . none of the mentioned approaches has considered the specifications and variations of echocardiograms and they have not addressed the problem of intensity inconstancy in this category of images . therefore , in this research , we motivated to propose a method which considers the variations in echocardiography images . to state the matter differently , in this approach , the intensity variation that occurs between two consecutive frames is calculated pixel - by - pixel in the image and afterwards , the effect of the variation is combined with of calculations . in this study , we investigate whether our proposed method improves the motion estimation of echocardiography images by of techniques . in subsequent parts of this paper , in section 2 , firstlythe of method and its equations which are required for cardiac motion estimation are described ; then our proposed method will be clarified . in section 3 , validation results are expressed ; these results first arise from a comparison between our method and two otherof - based methods and then from a comparison between the proposed method and expert point of view as a golden standard . the main idea of of is based on the constancy of image intensity in consecutive frames . if i(x , y , t ) is the intensity of pixelsat location expressed by ( x , y ) and time t , the basic assumption of of can be declared by ( 1 ) : i(x , y , t ) = i(x + x , y + y , t + t ) ( 1 ) in this equation , x and y are determined as below : ( u , v ) are the of or velocity components in the horizontal and vertical directions at the point ( x , y ) , and x , y are displacements at mentioned directions , respectively . first - order taylor expansion of the right hand of ( 1 ) will result in ( 2 ) : in this equation , ix , iy are spatial derivatives of intensity in x , y directions , respectively , and it is the temporal derivative of intensity . in ( 2 ) , there are two unknowns ( u , v ) in one equation . consequently , the solution of this equation is not possible with usual methods . in this paper , we use the method proposed by lucas and kanade which estimates the motion locally assuming it to be invariant within a spatial window . the advantage of this method is that the of calculations are not implemented in the entire image . in other words , the weighted least squares equationis represented in ( 3 ) : in this equation , x = ( x , y ) and w(x ) is a 2d window with dimension of ll , including n pixels of xi that is the domain containing the neighborhood of x. minimizing this equation with respect to v = ( u , v ) , ( 4 ) is obtained : a = [ i(x1), ,i(xn ) ] ( 5 ) w = diag[w(x1), ,w(xn ) ] ( 6 ) b = (it(x1), ,it(xn ) ) ( 7 ) the solution of ( 4 ) is expressed in ( 9 ) : in all of these equations , the intensity variation influence is not considered . in this study , a coarse - to - fine approach is implemented . number of the coarse - to - fine pyramid levels in our implementation is selected by comparison of efficiency between different choices . each frame of the image is sub - sampled by the factor 1/2 at each level and a pyramid of images with four different resolutions is created . the aim of this research is combining the intensity variation influence with mentioned equations to improve the motion estimation in echocardiography images . in this study subsequently , the obtained values are replaced with the right hand of ( 2 ) that is zero . this means that in our calculations ( 2 ) is altered to ( 10 ) , as below : in this equation , i is the intensity variation in the pixel located at ( x , y ) and at the time t. in fact , i is calculated locally at each pixel of the image . the considerable point in ( 10 ) actually , ( x,y ) is the displacement vector or the motion field of each pixel which is in close relation with the principal unknown of the of equation , namely the velocity vector ( u , v ) . consequently , we do not have ( x,y ) for computation of i at the first step . as a result , an estimation of these unknowns firstly , for each frame of the sequence , the motion estimation algorithm based on the weighted least squares approach is applied to every level of the coarse - to - fine pyramid , from the coarsest to finest level , respectively . at each level , the frame is up - sampled with the factor 2 and the algorithm is applied again . the initial value for ( x,y ) at the first level is considered zero , and in the other levels , the initial value is obtained by doubling the estimated value in previous level . in this figure , g00 and g10 are the initial sizes of two consecutive frames that in each level are sub sampled with the factor . g01 , g02 , g03 and g11 , g12 , g13 are the coarser levels of frame 1 and 2 , respectively . for validation of the proposed method , the algorithm was implemented on real echocardiography images . data is acquired from this site : www.yale.edu/imaging/echo_atlas/contents/index.html and the resolution of imaging is 3.5 mhz . our method was compared with two more of - based methods , gdim and lucas - kanade . all of the methods have comparable parameter settings . in the calculations related to our proposed method and lucus - kanade method , the optimum values obtained for the window w(x ) dimensions were 1717 pixels . it was obtained by calculation of the optimum value of mean square error ( mse ) when a given displacement is applied to the image . we selected a number of landmarks on a frame of echocardiography sequence to draw a curve calculated by our method . we compared it with expert defined curve by calculation of three distance metrics , mean , rms and hausdorff , between two curves . motion components were estimated by our proposed method and two other methods , namely lucas - kanade and gdim . the lucas - kanade method is one of the common of techniques and the intensity variation is not considered in it . the of calculations of the lucas - kanade method are implemented by ( 2 ) . nevertheless , in the gdim method , the intensity variation is considered as a multiplier in first frame 's intensity and an offset . in other words , the of equationrelated to this method is written as ( 12 ) , while in our proposed method , the intensity variation is considered locally and pixel - by - pixel : in this equation , m,c respectively , are the multiplier and offset values . moreover , u , v are the unknowns in this equation . actually , these four parameters are calculated simultaneously using a least squares approach by ( 13 ) : the complete procedure of this calculation is expressed in the related reference . practically , determination of a ground truth for heart motions is a challenging issue in validation of tracking methods . to evaluate the proposed method for echocardiography images , owing to the fact that the real value of displacements in different regions of an echocardiography is not available , three given displacements , 5,10 , and 15 pixels , were applied on this image for validation purpose . our proposed method and two other methods ( lucas - kanade and gdim ) were implemented to estimate the mentioned given displacements . we observed that the mse of our method was clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied displacements in addition of displacement error , we computed angular error for the proposed method and compared it with two other mentioned algorithms . we applied three different optional angles , 0 , 45 , 75 , on an echocardiography image and estimated them with three methods . table 2 shows the mse of estimated angles with three algorithms ( proposed , lucas - kanade and gdim ) . it is observed that the angular error related to our method is clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied angles finally , we implemented our proposed method on consecutive frames of a sequence of echocardiography images with a frame rate of 24 frames per second . in figure 2 , expansion of left ventricle can be observed by velocity arrows computed by our proposed of estimation method . for more appropriate visualization of of arrows frame 12 of an expanding heart motion estimation by the proposed method in an echocardiography image ; optical flow is calculated between frames 12 and 13 to evaluate the proposed method for real heart motions in echocardiography images , we drew the endocardial border of the left ventricle calculated with the proposed method and compared it with the manual curve defined by an expert who was blind to automatic estimation result . for this purpose , at first , we defined a set of landmarks on the endocardial border and interpolated them by b - spline curves ; secondly , we compared it with the curve defined by the expert . we calculated three distance metrics , namely mean , root mean square ( rms ) and hausdorff distance , between automatic and manually defined curves . mean distance is defined as ( 14 ) : in this equation , a and b are radial distances from a common central point and second metric distance , rms , is defined as ( 15 ) : the last and the most precise metric , haussdorff distance , is defined as ( 16 ) : where d is the euclidean distance between automatic and manual curves . in figure 3 , panels ( a ) to ( c ) show the b - spline interpolated curve related to frame 13 of the echocardiographic sequence calculated by lucas - kanade , gdim and the proposed method , respectively , and panel ( d ) shows the expert defined curve . table 3 shows the results of the comparison of three automatically defined curves with the manually defined one . it is observed that the haussdorff distance between the estimated curve defined by the proposed method and the manually defined curve , as a golden standard , is 4.81 pixels less than this distance for lucas - kanade and 2.28 pixels less than the gdim method . accordingly , we can say the proposed method is appropriately working when compared with the other methods . b - spline interpolated curve related to frame 13 of the echocardiography sequence calculated by ( a ) lucas - kanade ; ( b ) gdim ; ( c ) the proposed method ; and ( d ) expert defined curve distance metrics between three automatic and one expert defined curves practically , determination of a ground truth for heart motions is a challenging issue in validation of tracking methods . to evaluate the proposed method for echocardiography images , owing to the fact that the real value of displacements in different regions of an echocardiography is not available , three given displacements , 5,10 , and 15 pixels , were applied on this image for validation purpose . our proposed method and two other methods ( lucas - kanade and gdim ) were implemented to estimate the mentioned given displacements . we observed that the mse of our method was clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied displacements in addition of displacement error , we computed angular error for the proposed method and compared it with two other mentioned algorithms . we applied three different optional angles , 0 , 45 , 75 , on an echocardiography image and estimated them with three methods . table 2 shows the mse of estimated angles with three algorithms ( proposed , lucas - kanade and gdim ) . it is observed that the angular error related to our method is clearly less than two other methods . mean square error for three different methods of optical flow calculation in an echocardiography image for three applied angles finally , we implemented our proposed method on consecutive frames of a sequence of echocardiography images with a frame rate of 24 frames per second . figure 2 shows the of calculated by our method between frames 12 , 13 of this sequence which are related to expansion phase ( diastole ) . for more appropriate visualization of of arrows frame 12 of an expanding heart motion estimation by the proposed method in an echocardiography image ; optical flow is calculated between frames 12 and 13 to evaluate the proposed method for real heart motions in echocardiography images , we drew the endocardial border of the left ventricle calculated with the proposed method and compared it with the manual curve defined by an expert who was blind to automatic estimation result . for this purpose , at first , we defined a set of landmarks on the endocardial border and interpolated them by b - spline curves ; secondly , we compared it with the curve defined by the expert . we calculated three distance metrics , namely mean , root mean square ( rms ) and hausdorff distance , between automatic and manually defined curves . mean distance is defined as ( 14 ) : in this equation , a and b are radial distances from a common central point and second metric distance , rms , is defined as ( 15 ) : the last and the most precise metric , haussdorff distance , is defined as ( 16 ) : where d is the euclidean distance between automatic and manual curves . in figure 3 , panels ( a ) to ( c ) show the b - spline interpolated curve related to frame 13 of the echocardiographic sequence calculated by lucas - kanade , gdim and the proposed method , respectively , and panel ( d ) shows the expert defined curve . table 3 shows the results of the comparison of three automatically defined curves with the manually defined one . it is observed that the haussdorff distance between the estimated curve defined by the proposed method and the manually defined curve , as a golden standard , is 4.81 pixels less than this distance for lucas - kanade and 2.28 pixels less than the gdim method . accordingly , we can say the proposed method is appropriately working when compared with the other methods . b - spline interpolated curve related to frame 13 of the echocardiography sequence calculated by ( a ) lucas - kanade ; ( b ) gdim ; ( c ) the proposed method ; and ( d ) expert defined curve distance metrics between three automatic and one expert defined curves cardiac motions in echocardiography images may lead to variation of intensity in different areas of the image . intensity variations result in violation of the main assumption of the of calculations . in this paper , we considered intensity variation in of calculations and tried to compensate the effect of these variations to improve the estimations in echocardiography images . in our proposed method , the intensity variation was considered locally and pixel - by - pixel since heart has non uniform and complicated motions and definition of a mathematical function to model the intensity pattern is a problematic issue . we evaluated our method by comparison with two other of - based tracking methods , lucas - kanade and gdim . in addition , we compared the proposed method with expert point of view and observed that the curve which was defined by our method was close to the curve which was defined by expert , when compared to two other methods . this research can be a prelude to approach a specific model of intensity variation for echocardiography images . investigating the way of intensity variation in different regions of echocardiography images may lead to obtaining such a model . paria torkashvand received the b.s degree in electrical engineering from khaje nasireddin toosi university , tehran , iran , in 2007 and the m.s . degree in electrical engineering from iran university of science and technology , tehran , iran , in 1988 , the m.s . degree in medical engineering from sharif university of technology , tehran , iran , in 1992 , the ph.d . completed fellowships in echocardiography ( sep.2007-mar.2009 ) , at iran university of medical sciences , and in basic cardiac mr ( june .

the persistent rise in the proportion of chronic dialysis patients resulting from diabetic kidney disease in asian countries , including japan , over the past 20 years has been associated with higher mortality and is widely recognized as a major public health concern . to combat this problem , intensive efforts are underway to clarify the evolving management contributing to the amelioration of the development and progression of diabetic kidney disease . angiotensin system ( ras ) with either angiotensinconverting enzyme inhibitors ( acei ) or angiotensin receptor blockers ( arb ) , peroxisome proliferatoractivated receptor ( ppar ) alpha agonist and , recently , paricalcitol have been reported to prevent the development and progression of diabetic kidney disease . we focus on the reason why a global pandemic of endstage renal disease ( esrd ) attributed to diabetes has continued , as well as how we can challenge diabetic kidney disease in clinical practice . the world health organization ( who ) estimated that more than 300 million people will have diabetes by 2025 , resulting in a pandemic that threatens to collapse socioeconomic recourses . more recently , the baker idi heart and diabetes institute estimated that the world prevalence of diabetes among adults ( aged 2079 years ) will increase to 7.7% , and 439 million adults by 2030 . similarly , the number of people who have diabetes or who are suspected of having diabetes has increased by 1.6fold over the past decade , and this trend is suspected to continue in japan . as a result , the number of people who have diabetic kidney disease has increased and diabetic esrd , in particular , has been the main cause of newly introduced diseases to chronic dialysis since 1998 , and the trend is still continuing in japan . in contrast , although the number of new cases of esrd with diabetes has increased , the rate of new cases of esrd requiring dialysis among americans diagnosed with diabetes fell 35% between 1996 and 2007 , according to a study by the us centers for disease control and prevention . here , we raise several reasons to explain the discrepancy between asian and western countries ( table 1 ) . increased numbers of patients with diabetes ; patients higher ignorance when receiving treatment ; poorly controlled blood glucose , blood pressure and lipids ; lower rate of preventable screening for indication of developing diabetic kidney disease ; and the aging process could contribute to an increased rate of new cases of esrd requiring dialysis in japan . as compared with western countries , the proportion of patients with diabetes receiving a recommended medical evaluation , such as an annual urinary albumin measurement , has increased from 21.7% in 2000 to 27.2% in 2006 in the shiga prefecture , japan . furthermore , even in hospitals and clinics where diabetes specialists were taking care of patients , almost half of the diabetic patients were not receiving the measurement of urinary albumin from january 2004 to july 2005 . in other words , the measurement of urinary albumin is still neglected in clinical practice , although many clinicians know the importance of albuminuria as an indicator of diabetic kidney disease as well as a sensitive , accessible predictor of cardiovascular risk . in addition , esrd in japan has been regarded as a geriatric disorder and the mean age of newly diagnosed esrd patients with diabetes was 2.5 years older than that in the usa ( mean age 65.7 years in japan vs 63.2 year in the usa ) , suggesting that the aging process could also contribute to an increased incidence of diabetic esrd in japan . the earliest clinical sign of diabetic kidney disease is an elevated urinary albumin excretion , referred to as microalbuminuria . microalbuminuria is defined as an albumin excretion rate ( aer ) of 20199 g / min in a timed or a 24h urine collection ( equivalent to urinary albumin creatinine ratio [ acr ] of 30299 mg / g creatinine in a random spot sample ) . microalbuminuria progresses to overt proteinuria , leading to a decline in renal function defined as glomerular filtration rate ( gfr ) . generally , overt proteinuria inexorably progresses to esrd 68 years after the detection of overt proteinuria . thus , microalbuminuria in diabetic patients has been recognized as a predictor of progression to esrd . based on the data from over 5000 patients who were followed from the first diagnosis of type 2 diabetes in the united kingdom prospective diabetes study ( ukpds ) , annual transition rates from one stage to another stage of diabetic kidney disease were approximately 2% at each stage . furthermore , microalbuminuria has been shown to be closely associated with a higher risk for cardiovascular morbidity and mortality . indeed , the cardiovascular mortality in type 2 diabetic patients with microalbuminuria has been reported to be twofold higher than that in patients with normoalbuminuria . therefore , microalbuminuria is an important therapeutic target to improve the prognosis of renal and cardiovascular risk in diabetic patients . based on landmark clinical trials , intensive regimens of glucose control have been shown to reduce the development and progression of diabetic kidney disease . furthermore , the persistence of microvascular benefits in patients , who were previously intensively treated , was reported in the followup study of the diabetes control and complications trial ( dcct ) in the epidemiology of diabetes interventions and complications ( edic ) and of the ukpds , although their glycemic control has been equivalent to that of previous control arm subjects during follow up . recent trials in patients with more longstanding type 2 diabetes have also confirmed the benefit of intensifying glucose control on development and/or progression of microvascular complications , including diabetic kidney disease . the veterans affairs diabetes trial ( vadt ) showed significant reductions in albuminuria with intensive glycemic control ( achieved median hba1c 6.9% ) compared with standard glycemic control , although intensifying glucose control failed to affect other primary and secondary endpoints beneficially . the action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation ( advance ) trial has also shown that intensifying glucose control to achieve a hba1c level of < 6.5% did provide the benefit of reducing the risk of both the development and progression of diabetic kidney disease . as compared with standard control , intensive control was associated with a significant reduction in newonset microalbuminuria by 9% . furthermore , intensifying glucose control resulted in a significant reduction in renal events by 21% , including new or worsening diabetic kidney disease defined as the development of overt proteinuria , renal replacement therapy or death from renal causes , although the incidence of the doubling of serum creatinine level did not differ . unfortunately , this study also failed to reduce the incidence of major macrovascular events defined as myocardial infarction , stroke or cardiovascular death with intensive control as compared with standard control . nevertheless , the reduction in the incidence of diabetic kidney disease might have long term benefits on cardiovascular disease , because diabetic patients with kidney disease have a higher risk of macrovascular disease . however , the results of action to control cardiovascular risk in diabetes ( accord ) showed that the risk of death was increased by nearnormal glycemic control with intensifying treatment as compared with standard control without the reduction of cardiovascular events , although recent analyses from the accord trial have shown that the risk of development of overt proteinuria was 31% lower with intensive therapy at transition and 28% lower at study end than with standard therapy . as reported in the posthoc epidemiological analysis of the accord study , we need to pay attention to what the benefit of intensifying glucose control for diabetic patients is . it must be weighed against the risks of intensive glycemic control , including allcause and cardiovascular diseaserelated mortality , weight gain , and incidence of severe hypoglycemic episodes . furthermore , a recent subanalysis of the advance trial clearly showed that severe hypoglycemia was strongly associated with an increased risk of a range of adverse clinical outcomes , including macrovascular and/or microvascular events as well as death . intensive glycemic treatment targeting a hba1c goal level of 6.0% or less could be beneficial for individuals who are younger and have newly diagnosed diabetes . however , a conservative hba1c targeting the 7% range might be appropriate in older individuals who have established diabetes , cardiovascular disease and major risk factors for cardiovascular disease . therefore , the goals for managing elderly patients with diabetes , especially type 2 diabetes , should be individualized according to the patient s age , disease stage and other comorbid conditions . indeed , the american diabetes association 2011 recommends a hba1c level below or around 7% to reduce microvascular and macrovascular complications of diabetes in patients soon after the diagnosis of diabetes . strict blood pressure control of < 130/80 mmhg is universally recommended in patients with diabetes to lower incidences of stroke , heart failure , diabetesrelated death , retinal photocoagulation and to reduce the risk of micro or overt proteinuria . in the recent advance study , the reduction of blood pressure from 140/73 mmhg ( control group ) to 136/73 mmhg ( indapamide perindopril group ) was shown to reduce the risks of a major macro or microvascular ( mostly new microalbuminuria ) event , death from cardiovascular disease and death from any cause after 4.3 years of follow up , extending the early findings of the ukpds to an even lower blood pressure . therefore , targeting blood pressure < 130/80 mmhg appears to be appropriate in type 2 diabetics to fight against the development and progression of diabetic kidney disease . in diabetic patients with microalbuminuria or overt proteinuria , ras inhibitors play a pivotal role in the prevention and treatment of diabetic kidney disease . landmark studies with type 1 and type 2 diabetic patients at various stages of diabetic kidney diseases have well provided the clinical evidence that treatment with ras inhibitors did slow the progressive decrease in gfr , reduce proteinuria and microalbuminuria , prevent progression from one stage of diabetic kidney disease to others , and reduce cardiovascular mortality and morbidity as shown in figure 1 . as described in the next section , recent studies have shown the effectiveness of arb , not only to reduce the progression of diabetic kidney disease to esrd , but also to revert the progressive course . landmark studies showing the effectiveness of renin angiotensin system inhibitors on diabetic kidney disease , and cardiovascular mortality and morbidity . dual ras blockade with acei and arb might be more effective in reducing proteinuria compared with monotherapy in patients with diabetic kidney diseases . based on the ongoing telmisartan alone and in combination with the ramipril global endpoint trial ( ontarget ) , although combination therapy with ramipril and telmisartan reduces proteinuria than monotherapy , it worsens major renal outcomes including dialysis , doubling of serum creatinine and death . thus , combination ras blockade should not be used in diabetic patients , especially elderly type 2 diabetic patients with normo and/or microalbuminuria . first , acei or arb should be used and their dosage should be increased to obtain an optimal antialbuminuric and/or proteinuric response . combination treatment with both acei and arb should be prescribed by a nephrologist , and given to those patients with overt proteinuria and/or massive proteinuria despite the use of maximum dosages of acei or arb . in those diabetic patients , monitoring of renal function is needed , and the treatment should be stopped in the event of acute kidney injury , low blood pressure and/or high potassium level . however , the effect of combination treatment with aliskiren and arb in type 2 diabetic patients with overt diabetic kidney disease was recently reported . in the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study , 599 patients with diabetic kidney disease with overt proteinuria were treated with losartan 100 mg , followed by the addition of a placebo or aliskiren ( 300 mg ) . as a result , treatment with 300 mg of aliskiren daily reduced the mean urinary acr by 20% as compared with the placebo , with a reduction of 50% or more in 24.7% of the patients who received aliskiren as compared with 12.5% of those who received the placebo . at present , the aliskiren trial in type 2 diabetes using cardiorenal endpoints ( altitude ) to confirm the effectiveness of combination treatment with either an acei or an arb plus aliskiren on both renal and cardiovascular events is ongoing , in which diabetic patients with proteinuria and a history of cardiovascular disease were enrolled . although ras inhibitors have become the mainstays of treating established diabetic kidney disease , the beneficial effects of these agents on the early phases of diabetic kidney disease is unclear . bilous et al . examined the effect of candesartan on microalbuminuria and albumin excretion rates , either before renal disease began or in its earliest stages based on data from the diabetic retinopathy candesartan trials ( direct ) randomized trials . the incidence of microalbuminuria in type 1 diabetes was 5% in both the candesartan and placebo groups , and that of microalbuminuria in type 2 diabetics was 12% in the candesartan group compared with 13% in the placebo group . mann et al . also examined the longterm renal effects of another arb , telmisartan , in adults who were intolerant to acei , but had a high risk of vascular disease without albuminuria at baseline . although treatment with telmisartan significantly reduced the risk for new microalbuminuria , overt proteinuria , or both , the reduction in albuminuria was not associated with less progression of renal disease , including dialysis or doubling of serum creatinine . therefore , to decide whether we need to use ras inhibitors in hypertensive and diabetic patients , the degree of the patient s vascular and renal risk must be assessed in addition to taking into account the efficacy on the functions of the cardiovascular system and diabetic kidney disease . based on landmark clinical trials , intensive regimens of glucose control have been shown to reduce the development and progression of diabetic kidney disease . furthermore , the persistence of microvascular benefits in patients , who were previously intensively treated , was reported in the followup study of the diabetes control and complications trial ( dcct ) in the epidemiology of diabetes interventions and complications ( edic ) and of the ukpds , although their glycemic control has been equivalent to that of previous control arm subjects during follow up . recent trials in patients with more longstanding type 2 diabetes have also confirmed the benefit of intensifying glucose control on development and/or progression of microvascular complications , including diabetic kidney disease . the veterans affairs diabetes trial ( vadt ) showed significant reductions in albuminuria with intensive glycemic control ( achieved median hba1c 6.9% ) compared with standard glycemic control , although intensifying glucose control failed to affect other primary and secondary endpoints beneficially . the action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation ( advance ) trial has also shown that intensifying glucose control to achieve a hba1c level of < 6.5% did provide the benefit of reducing the risk of both the development and progression of diabetic kidney disease . as compared with standard control , intensive control was associated with a significant reduction in newonset microalbuminuria by 9% . furthermore , intensifying glucose control resulted in a significant reduction in renal events by 21% , including new or worsening diabetic kidney disease defined as the development of overt proteinuria , renal replacement therapy or death from renal causes , although the incidence of the doubling of serum creatinine level did not differ . unfortunately , this study also failed to reduce the incidence of major macrovascular events defined as myocardial infarction , stroke or cardiovascular death with intensive control as compared with standard control . nevertheless , the reduction in the incidence of diabetic kidney disease might have long term benefits on cardiovascular disease , because diabetic patients with kidney disease have a higher risk of macrovascular disease . however , the results of action to control cardiovascular risk in diabetes ( accord ) showed that the risk of death was increased by nearnormal glycemic control with intensifying treatment as compared with standard control without the reduction of cardiovascular events , although recent analyses from the accord trial have shown that the risk of development of overt proteinuria was 31% lower with intensive therapy at transition and 28% lower at study end than with standard therapy . as reported in the posthoc epidemiological analysis of the accord study , we need to pay attention to what the benefit of intensifying glucose control for diabetic patients is . it must be weighed against the risks of intensive glycemic control , including allcause and cardiovascular diseaserelated mortality , weight gain , and incidence of severe hypoglycemic episodes . furthermore , a recent subanalysis of the advance trial clearly showed that severe hypoglycemia was strongly associated with an increased risk of a range of adverse clinical outcomes , including macrovascular and/or microvascular events as well as death . intensive glycemic treatment targeting a hba1c goal level of 6.0% or less could be beneficial for individuals who are younger and have newly diagnosed diabetes . however , a conservative hba1c targeting the 7% range might be appropriate in older individuals who have established diabetes , cardiovascular disease and major risk factors for cardiovascular disease . therefore , the goals for managing elderly patients with diabetes , especially type 2 diabetes , should be individualized according to the patient s age , disease stage and other comorbid conditions . indeed , the american diabetes association 2011 recommends a hba1c level below or around 7% to reduce microvascular and macrovascular complications of diabetes in patients soon after the diagnosis of diabetes . strict blood pressure control of < 130/80 mmhg is universally recommended in patients with diabetes to lower incidences of stroke , heart failure , diabetesrelated death , retinal photocoagulation and to reduce the risk of micro or overt proteinuria . in the recent advance study , the reduction of blood pressure from 140/73 mmhg ( control group ) to 136/73 mmhg ( indapamide perindopril group ) was shown to reduce the risks of a major macro or microvascular ( mostly new microalbuminuria ) event , death from cardiovascular disease and death from any cause after 4.3 years of follow up , extending the early findings of the ukpds to an even lower blood pressure . therefore , targeting blood pressure < 130/80 mmhg appears to be appropriate in type 2 diabetics to fight against the development and progression of diabetic kidney disease . in diabetic patients with microalbuminuria or overt proteinuria , ras inhibitors play a pivotal role in the prevention and treatment of diabetic kidney disease . landmark studies with type 1 and type 2 diabetic patients at various stages of diabetic kidney diseases have well provided the clinical evidence that treatment with ras inhibitors did slow the progressive decrease in gfr , reduce proteinuria and microalbuminuria , prevent progression from one stage of diabetic kidney disease to others , and reduce cardiovascular mortality and morbidity as shown in figure 1 . as described in the next section , recent studies have shown the effectiveness of arb , not only to reduce the progression of diabetic kidney disease to esrd , but also to revert the progressive course . landmark studies showing the effectiveness of renin angiotensin system inhibitors on diabetic kidney disease , and cardiovascular mortality and morbidity . dual ras blockade with acei and arb might be more effective in reducing proteinuria compared with monotherapy in patients with diabetic kidney diseases . based on the ongoing telmisartan alone and in combination with the ramipril global endpoint trial ( ontarget ) , although combination therapy with ramipril and telmisartan reduces proteinuria than monotherapy , it worsens major renal outcomes including dialysis , doubling of serum creatinine and death . thus , combination ras blockade should not be used in diabetic patients , especially elderly type 2 diabetic patients with normo and/or microalbuminuria . first , acei or arb should be used and their dosage should be increased to obtain an optimal antialbuminuric and/or proteinuric response . combination treatment with both acei and arb should be prescribed by a nephrologist , and given to those patients with overt proteinuria and/or massive proteinuria despite the use of maximum dosages of acei or arb . in those diabetic patients , monitoring of renal function is needed , and the treatment should be stopped in the event of acute kidney injury , low blood pressure and/or high potassium level . however , the effect of combination treatment with aliskiren and arb in type 2 diabetic patients with overt diabetic kidney disease was recently reported . in the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study , 599 patients with diabetic kidney disease with overt proteinuria were treated with losartan 100 mg , followed by the addition of a placebo or aliskiren ( 300 mg ) . as a result , treatment with 300 mg of aliskiren daily reduced the mean urinary acr by 20% as compared with the placebo , with a reduction of 50% or more in 24.7% of the patients who received aliskiren as compared with 12.5% of those who received the placebo . at present , the aliskiren trial in type 2 diabetes using cardiorenal endpoints ( altitude ) to confirm the effectiveness of combination treatment with either an acei or an arb plus aliskiren on both renal and cardiovascular events is ongoing , in which diabetic patients with proteinuria and a history of cardiovascular disease were enrolled . although ras inhibitors have become the mainstays of treating established diabetic kidney disease , the beneficial effects of these agents on the early phases of diabetic kidney disease is unclear . bilous et al . examined the effect of candesartan on microalbuminuria and albumin excretion rates , either before renal disease began or in its earliest stages based on data from the diabetic retinopathy candesartan trials ( direct ) randomized trials . the incidence of microalbuminuria in type 1 diabetes was 5% in both the candesartan and placebo groups , and that of microalbuminuria in type 2 diabetics was 12% in the candesartan group compared with 13% in the placebo group . mann et al . also examined the longterm renal effects of another arb , telmisartan , in adults who were intolerant to acei , but had a high risk of vascular disease without albuminuria at baseline . although treatment with telmisartan significantly reduced the risk for new microalbuminuria , overt proteinuria , or both , the reduction in albuminuria was not associated with less progression of renal disease , including dialysis or doubling of serum creatinine . therefore , to decide whether we need to use ras inhibitors in hypertensive and diabetic patients , the degree of the patient s vascular and renal risk must be assessed in addition to taking into account the efficacy on the functions of the cardiovascular system and diabetic kidney disease . we carried out a prospective observational followup study including a total of 216 japanese type 2 diabetic patients with microalbuminuria . in our study , we used the definition of remission / regression of microalbuminuria similar to that of the perkins et al . the remission was defined as a shift of the aer from microalbuminuria to normoalbuminuria , and the regression was defined as a 50% reduction in the aer from baseline . the 6year cumulative incidence of progression from microalbuminuria to overt proteinuria was 28% ( 95% ci 1937 ) , whereas those for remission and regression were 51% ( 95% ci 4260 ) and 54% ( 95% ci 4563 ) , respectively ( figure 2 ) . in the pooled logistic regression analysis , each modifiable factor was trisected according to the number of patients and was applied as three categories in the analysis . the results showed that microalbuminuria of short duration , the use of ras blockades , a hba1c level of < 7.35% and lower systolic blood pressure < 130 mmhg were identified to be independent factors associated with remission / regression of microalbuminuria . angiotensinii receptor blockers have also been shown to induce remission and regression of microalbuminuria in japanese type 2 diabetic patients . in the shiga microalbuminuria reduction trial , 150 patients with microalbuminuria were randomly assigned to either the valsartan group or the amlodipine group and followed for 24 weeks . during the study , however , the frequency of patients who achieved remission or regression of microalbuminuria was significantly higher in the valsartan group than in the amlodipine group ( remission 23 vs 11% , p = 0.011 ; regression 34 vs 16% , p = 0.008 ) . in another japanese incipient to overt : angiotensin ii blocker , telmisartan , investigation on type 2 diabetic nephropathy ( innovation ) study , microalbuminuria remission at final observation occurred in 21.2% of patients with 80 mg of telmisartan , 12.8% of patients with 40 mg of telmisartan and 1.2% of patients with a placebo ( both telmisartan doses vs placebo , p < 0.001 ) . in addition , patients receiving 80 or 40 mg of telmisartan achieved superior renoprotection , shown by lower transition rates to overt nephropathy , compared with the placebo . taken together , these results strongly indicate that ras blockade by using arb not only prevent the progression of microalbuminuria to overt proteinuria , but also induce remission and regression of microalbuminuria in japanese type 2 diabetic patients . a prospective observational followup study including a total of 216 japanese type 2 diabetic patients with microalbuminuria was carried out to follow the change of stage of microalbuminuria for 6 years . the remission was defined as a shift of the albumin excretion rate ( aer ) from microalbuminuria to normoalbuminuria , and the regression was defined as 50% reduction in the aer from baseline . similar to ours , the steno2 study also reported that a high proportion of patients with microalbuminuria returned to normoalbuminuria with a multifactorial intervention in 151 type 2 diabetic patients with microalbuminuria . after a mean of 7.8 years of follow up , 46 ( 31% ) patients returned to normoalbuminuria , 58 ( 38% ) patients still had microalbuminuria and 47 ( 31% ) patients progressed to overt proteinuria . lower hba1c , starting antihypertensive therapy and starting ras inhibitor drugs during the follow up were independently associated with the remission of microalbuminuria . recent analysis , especially regarding the effect of lowering blood pressure , clearly showed that more than half of all type 2 diabetic patients with microalbuminuria and macroalbuminuria returned to normoalbuminuria with any blood pressure lowering drugs in the advance study . however , more patients assigned to perindopril indapamide treatment than those assigned to placebo treatment achieved remission to normoalbuminuria . to explore the clinical impact of a reduction of microalbuminuria , we expanded the follow up by a 2 years beyond our previous study and examined whether remission and regression of microalbuminuria could translate into risk reduction of renal and cardiovascular events . the primary evaluation consisted of combined incidence defined as cardiovascular death , and first hospitalization for renal and cardiovascular events . a secondary evaluation was the kidney function as determined by the annual decline rates of estimated gfr ( egfr ) . during the 8year followup period , the number of first occurrences of outcomes in subgroups , who achieved remission of microalbuminuria , was 11 events and 36 events in the nonremission group . the pooled logistic analysis adjusted by sex , age , the initial aer levels , a history of cardiovascular disease , current smoking , hba1c , total cholesterol , blood pressure , the use of ras inhibitors , the use of lipid lowering drugs and body mass index ( bmi ) showed that the risk for outcomes in patients , who achieved remission , was 0.25 ( 95% ci 0.070.87 ) as compared with those whose microalbuminuric stage did not change during the follow up , whereas that in patients who progressed to overt proteinuria was 2.55 ( 95% ci 1.046.30 ) ( figure 3 ) . even though failing to achieve the remission , the number of the first occurrences of outcomes in subgroups stratified by a 50% reduction of urinary albumin excretion was 12 events in the regression group and 35 events in the nonregression group . meier estimation showed that the cumulative incidence of evaluated events was significantly lower in the regression group than in the nonregression group . the 8year cumulative incidence of these outcomes in the regression group showed a 59% decrease compared with the nonregression group . the adjusted risk for outcomes in patients who achieved the regression was 0.41 ( 95% ci 0.150.96 ) as compared with those whose microalbuminuric stage did not achieve the regression during the follow up . a prospective observational followup study including a total of 216 japanese type 2 diabetic patients with microalbuminuria was carried out to explore the clinical impact of remission and progression of microalbuminuria . the primary evaluation consisted of combined incidence defined as cardiovascular death and first hospitalization for renal and cardiovascular events . the pooled logistic analysis adjusted by sex , age , the initial albumin excretion rate levels , a history of cardiovascular disease , current smoking , hba1c , total cholesterol , triglyceride , highdensity lipoprotein cholesterol , systolic blood pressure , diastolic blood pressure , the use of renin angiotensin system inhibitors , the use of lipid lowering drugs and body mass index showed that the risk for outcomes in patients , who achieved remission , was 0.25 ( 95% ci 0.070.87 ) as compared with those whose microalbuminuric stage did not change during the follow up , whereas that in patients , who progressed to overt proteinuria , was 2.55 ( 95% ci 1.046.30 ) . as suspected , the annual decline rate of egfr in the progression group ( median 4.2 ml / min / year ) was significantly faster than in the nonchange group ( 2.4 ml / min / year ) , whereas the annual decline rate of egfr in the remission group was significantly slower , 1.1 ml / min / year , which is almost identical with the decline rate by normal aging reported in healthy people . the effect of reducing microalbuminuria on kidney function was also reported in the steno2 study aforementioned . the patients who reverted to normoalbuminuria had an egfr decline of 2.3 ml / min / year ; however , those who still had microalbuminuria lost 3.7 ml / min / year of egfr , and those who progressed to overt proteinuria showed the highest decline in egfr of 5.4 ml / min / year . these results show that the remission of microalbuminuria is closely related to the improvement of renal function in the long term . physicians have to take care of diabetic patients with an aggressive multifactorial management plan as early as possible after the development of microalbuminuria ( table 2 ) . the clinical target , which is important and effective for diabetic kidney disease , is to achieve the remission and/or regression of microalbuminuria . furthermore , reducing microalbuminuria results in a risk reduction of not only renal , but also cardiovascular events .

abstractduring the past 10 years , a global pandemic of endstage renal disease ( esrd ) attributed to diabetes mellitus has changed the therapeutic strategies based on landmark trials that have shown that diabetic micro and macrovascular complications might be preventable . however , the remaining risk of the progression of diabetic kidney disease to esrd is still high , despite newly introduced antidiabetic , antihypertensive and dyslipidemic drugs in the 21st century . here , we show the importance of targeting remission and regression of microalbuminuria in type 2 diabetic patients . to achieve the remission and regression of microalbuminuria , physicians have revised the management strategy of diabetic patients and have to act immediately . early detection of microalbuminuria with continuous screening , the use of renin angiotensin system blockades , and targets for hba1c of < 7.35% and systolic blood pressure of < 130 mmhg are closely associated with the remission and regression of microalbuminuria , resulting in protection against the progression of diabetic kidney disease , as well as cardiovascular events . our concept of the natural history of diabetic kidney disease has to be modified by our results and others . reducing microalbuminuria is therefore considered to be an important therapeutic target and could be a pivotal biomarker of therapeutic success in diabetic patients . ( j diabetes invest , doi:10.1111/j.20401124.2011.00112.x , 2011 )

Introduction Epidemiology Screening methods for diabetic nephropathy Therapeutic strategy of diabetic kidney disease Targeting euglycemia Blood pressure control with RAS inhibitors Remission and regression of early stage of diabetic kidney disease and cardiorenal protection Conclusion

the persistent rise in the proportion of chronic dialysis patients resulting from diabetic kidney disease in asian countries , including japan , over the past 20 years has been associated with higher mortality and is widely recognized as a major public health concern . to combat this problem , intensive efforts are underway to clarify the evolving management contributing to the amelioration of the development and progression of diabetic kidney disease . angiotensin system ( ras ) with either angiotensinconverting enzyme inhibitors ( acei ) or angiotensin receptor blockers ( arb ) , peroxisome proliferatoractivated receptor ( ppar ) alpha agonist and , recently , paricalcitol have been reported to prevent the development and progression of diabetic kidney disease . we focus on the reason why a global pandemic of endstage renal disease ( esrd ) attributed to diabetes has continued , as well as how we can challenge diabetic kidney disease in clinical practice . similarly , the number of people who have diabetes or who are suspected of having diabetes has increased by 1.6fold over the past decade , and this trend is suspected to continue in japan . as a result , the number of people who have diabetic kidney disease has increased and diabetic esrd , in particular , has been the main cause of newly introduced diseases to chronic dialysis since 1998 , and the trend is still continuing in japan . in other words , the measurement of urinary albumin is still neglected in clinical practice , although many clinicians know the importance of albuminuria as an indicator of diabetic kidney disease as well as a sensitive , accessible predictor of cardiovascular risk . the earliest clinical sign of diabetic kidney disease is an elevated urinary albumin excretion , referred to as microalbuminuria . thus , microalbuminuria in diabetic patients has been recognized as a predictor of progression to esrd . based on the data from over 5000 patients who were followed from the first diagnosis of type 2 diabetes in the united kingdom prospective diabetes study ( ukpds ) , annual transition rates from one stage to another stage of diabetic kidney disease were approximately 2% at each stage . indeed , the cardiovascular mortality in type 2 diabetic patients with microalbuminuria has been reported to be twofold higher than that in patients with normoalbuminuria . therefore , microalbuminuria is an important therapeutic target to improve the prognosis of renal and cardiovascular risk in diabetic patients . based on landmark clinical trials , intensive regimens of glucose control have been shown to reduce the development and progression of diabetic kidney disease . furthermore , the persistence of microvascular benefits in patients , who were previously intensively treated , was reported in the followup study of the diabetes control and complications trial ( dcct ) in the epidemiology of diabetes interventions and complications ( edic ) and of the ukpds , although their glycemic control has been equivalent to that of previous control arm subjects during follow up . recent trials in patients with more longstanding type 2 diabetes have also confirmed the benefit of intensifying glucose control on development and/or progression of microvascular complications , including diabetic kidney disease . the action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation ( advance ) trial has also shown that intensifying glucose control to achieve a hba1c level of < 6.5% did provide the benefit of reducing the risk of both the development and progression of diabetic kidney disease . nevertheless , the reduction in the incidence of diabetic kidney disease might have long term benefits on cardiovascular disease , because diabetic patients with kidney disease have a higher risk of macrovascular disease . however , the results of action to control cardiovascular risk in diabetes ( accord ) showed that the risk of death was increased by nearnormal glycemic control with intensifying treatment as compared with standard control without the reduction of cardiovascular events , although recent analyses from the accord trial have shown that the risk of development of overt proteinuria was 31% lower with intensive therapy at transition and 28% lower at study end than with standard therapy . as reported in the posthoc epidemiological analysis of the accord study , we need to pay attention to what the benefit of intensifying glucose control for diabetic patients is . furthermore , a recent subanalysis of the advance trial clearly showed that severe hypoglycemia was strongly associated with an increased risk of a range of adverse clinical outcomes , including macrovascular and/or microvascular events as well as death . indeed , the american diabetes association 2011 recommends a hba1c level below or around 7% to reduce microvascular and macrovascular complications of diabetes in patients soon after the diagnosis of diabetes . in the recent advance study , the reduction of blood pressure from 140/73 mmhg ( control group ) to 136/73 mmhg ( indapamide perindopril group ) was shown to reduce the risks of a major macro or microvascular ( mostly new microalbuminuria ) event , death from cardiovascular disease and death from any cause after 4.3 years of follow up , extending the early findings of the ukpds to an even lower blood pressure . therefore , targeting blood pressure < 130/80 mmhg appears to be appropriate in type 2 diabetics to fight against the development and progression of diabetic kidney disease . in diabetic patients with microalbuminuria or overt proteinuria , ras inhibitors play a pivotal role in the prevention and treatment of diabetic kidney disease . landmark studies with type 1 and type 2 diabetic patients at various stages of diabetic kidney diseases have well provided the clinical evidence that treatment with ras inhibitors did slow the progressive decrease in gfr , reduce proteinuria and microalbuminuria , prevent progression from one stage of diabetic kidney disease to others , and reduce cardiovascular mortality and morbidity as shown in figure 1 . as described in the next section , recent studies have shown the effectiveness of arb , not only to reduce the progression of diabetic kidney disease to esrd , but also to revert the progressive course . landmark studies showing the effectiveness of renin angiotensin system inhibitors on diabetic kidney disease , and cardiovascular mortality and morbidity . thus , combination ras blockade should not be used in diabetic patients , especially elderly type 2 diabetic patients with normo and/or microalbuminuria . combination treatment with both acei and arb should be prescribed by a nephrologist , and given to those patients with overt proteinuria and/or massive proteinuria despite the use of maximum dosages of acei or arb . in those diabetic patients , monitoring of renal function is needed , and the treatment should be stopped in the event of acute kidney injury , low blood pressure and/or high potassium level . however , the effect of combination treatment with aliskiren and arb in type 2 diabetic patients with overt diabetic kidney disease was recently reported . at present , the aliskiren trial in type 2 diabetes using cardiorenal endpoints ( altitude ) to confirm the effectiveness of combination treatment with either an acei or an arb plus aliskiren on both renal and cardiovascular events is ongoing , in which diabetic patients with proteinuria and a history of cardiovascular disease were enrolled . although ras inhibitors have become the mainstays of treating established diabetic kidney disease , the beneficial effects of these agents on the early phases of diabetic kidney disease is unclear . the incidence of microalbuminuria in type 1 diabetes was 5% in both the candesartan and placebo groups , and that of microalbuminuria in type 2 diabetics was 12% in the candesartan group compared with 13% in the placebo group . although treatment with telmisartan significantly reduced the risk for new microalbuminuria , overt proteinuria , or both , the reduction in albuminuria was not associated with less progression of renal disease , including dialysis or doubling of serum creatinine . therefore , to decide whether we need to use ras inhibitors in hypertensive and diabetic patients , the degree of the patient s vascular and renal risk must be assessed in addition to taking into account the efficacy on the functions of the cardiovascular system and diabetic kidney disease . based on landmark clinical trials , intensive regimens of glucose control have been shown to reduce the development and progression of diabetic kidney disease . recent trials in patients with more longstanding type 2 diabetes have also confirmed the benefit of intensifying glucose control on development and/or progression of microvascular complications , including diabetic kidney disease . the action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation ( advance ) trial has also shown that intensifying glucose control to achieve a hba1c level of < 6.5% did provide the benefit of reducing the risk of both the development and progression of diabetic kidney disease . furthermore , intensifying glucose control resulted in a significant reduction in renal events by 21% , including new or worsening diabetic kidney disease defined as the development of overt proteinuria , renal replacement therapy or death from renal causes , although the incidence of the doubling of serum creatinine level did not differ . nevertheless , the reduction in the incidence of diabetic kidney disease might have long term benefits on cardiovascular disease , because diabetic patients with kidney disease have a higher risk of macrovascular disease . however , the results of action to control cardiovascular risk in diabetes ( accord ) showed that the risk of death was increased by nearnormal glycemic control with intensifying treatment as compared with standard control without the reduction of cardiovascular events , although recent analyses from the accord trial have shown that the risk of development of overt proteinuria was 31% lower with intensive therapy at transition and 28% lower at study end than with standard therapy . as reported in the posthoc epidemiological analysis of the accord study , we need to pay attention to what the benefit of intensifying glucose control for diabetic patients is . furthermore , a recent subanalysis of the advance trial clearly showed that severe hypoglycemia was strongly associated with an increased risk of a range of adverse clinical outcomes , including macrovascular and/or microvascular events as well as death . however , a conservative hba1c targeting the 7% range might be appropriate in older individuals who have established diabetes , cardiovascular disease and major risk factors for cardiovascular disease . indeed , the american diabetes association 2011 recommends a hba1c level below or around 7% to reduce microvascular and macrovascular complications of diabetes in patients soon after the diagnosis of diabetes . in the recent advance study , the reduction of blood pressure from 140/73 mmhg ( control group ) to 136/73 mmhg ( indapamide perindopril group ) was shown to reduce the risks of a major macro or microvascular ( mostly new microalbuminuria ) event , death from cardiovascular disease and death from any cause after 4.3 years of follow up , extending the early findings of the ukpds to an even lower blood pressure . therefore , targeting blood pressure < 130/80 mmhg appears to be appropriate in type 2 diabetics to fight against the development and progression of diabetic kidney disease . in diabetic patients with microalbuminuria or overt proteinuria , ras inhibitors play a pivotal role in the prevention and treatment of diabetic kidney disease . landmark studies with type 1 and type 2 diabetic patients at various stages of diabetic kidney diseases have well provided the clinical evidence that treatment with ras inhibitors did slow the progressive decrease in gfr , reduce proteinuria and microalbuminuria , prevent progression from one stage of diabetic kidney disease to others , and reduce cardiovascular mortality and morbidity as shown in figure 1 . as described in the next section , recent studies have shown the effectiveness of arb , not only to reduce the progression of diabetic kidney disease to esrd , but also to revert the progressive course . landmark studies showing the effectiveness of renin angiotensin system inhibitors on diabetic kidney disease , and cardiovascular mortality and morbidity . thus , combination ras blockade should not be used in diabetic patients , especially elderly type 2 diabetic patients with normo and/or microalbuminuria . combination treatment with both acei and arb should be prescribed by a nephrologist , and given to those patients with overt proteinuria and/or massive proteinuria despite the use of maximum dosages of acei or arb . in those diabetic patients , monitoring of renal function is needed , and the treatment should be stopped in the event of acute kidney injury , low blood pressure and/or high potassium level . however , the effect of combination treatment with aliskiren and arb in type 2 diabetic patients with overt diabetic kidney disease was recently reported . in the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study , 599 patients with diabetic kidney disease with overt proteinuria were treated with losartan 100 mg , followed by the addition of a placebo or aliskiren ( 300 mg ) . at present , the aliskiren trial in type 2 diabetes using cardiorenal endpoints ( altitude ) to confirm the effectiveness of combination treatment with either an acei or an arb plus aliskiren on both renal and cardiovascular events is ongoing , in which diabetic patients with proteinuria and a history of cardiovascular disease were enrolled . although ras inhibitors have become the mainstays of treating established diabetic kidney disease , the beneficial effects of these agents on the early phases of diabetic kidney disease is unclear . the incidence of microalbuminuria in type 1 diabetes was 5% in both the candesartan and placebo groups , and that of microalbuminuria in type 2 diabetics was 12% in the candesartan group compared with 13% in the placebo group . although treatment with telmisartan significantly reduced the risk for new microalbuminuria , overt proteinuria , or both , the reduction in albuminuria was not associated with less progression of renal disease , including dialysis or doubling of serum creatinine . therefore , to decide whether we need to use ras inhibitors in hypertensive and diabetic patients , the degree of the patient s vascular and renal risk must be assessed in addition to taking into account the efficacy on the functions of the cardiovascular system and diabetic kidney disease . we carried out a prospective observational followup study including a total of 216 japanese type 2 diabetic patients with microalbuminuria . in our study , we used the definition of remission / regression of microalbuminuria similar to that of the perkins et al . the remission was defined as a shift of the aer from microalbuminuria to normoalbuminuria , and the regression was defined as a 50% reduction in the aer from baseline . the results showed that microalbuminuria of short duration , the use of ras blockades , a hba1c level of < 7.35% and lower systolic blood pressure < 130 mmhg were identified to be independent factors associated with remission / regression of microalbuminuria . angiotensinii receptor blockers have also been shown to induce remission and regression of microalbuminuria in japanese type 2 diabetic patients . during the study , however , the frequency of patients who achieved remission or regression of microalbuminuria was significantly higher in the valsartan group than in the amlodipine group ( remission 23 vs 11% , p = 0.011 ; regression 34 vs 16% , p = 0.008 ) . taken together , these results strongly indicate that ras blockade by using arb not only prevent the progression of microalbuminuria to overt proteinuria , but also induce remission and regression of microalbuminuria in japanese type 2 diabetic patients . a prospective observational followup study including a total of 216 japanese type 2 diabetic patients with microalbuminuria was carried out to follow the change of stage of microalbuminuria for 6 years . the remission was defined as a shift of the albumin excretion rate ( aer ) from microalbuminuria to normoalbuminuria , and the regression was defined as 50% reduction in the aer from baseline . similar to ours , the steno2 study also reported that a high proportion of patients with microalbuminuria returned to normoalbuminuria with a multifactorial intervention in 151 type 2 diabetic patients with microalbuminuria . lower hba1c , starting antihypertensive therapy and starting ras inhibitor drugs during the follow up were independently associated with the remission of microalbuminuria . recent analysis , especially regarding the effect of lowering blood pressure , clearly showed that more than half of all type 2 diabetic patients with microalbuminuria and macroalbuminuria returned to normoalbuminuria with any blood pressure lowering drugs in the advance study . to explore the clinical impact of a reduction of microalbuminuria , we expanded the follow up by a 2 years beyond our previous study and examined whether remission and regression of microalbuminuria could translate into risk reduction of renal and cardiovascular events . during the 8year followup period , the number of first occurrences of outcomes in subgroups , who achieved remission of microalbuminuria , was 11 events and 36 events in the nonremission group . the pooled logistic analysis adjusted by sex , age , the initial aer levels , a history of cardiovascular disease , current smoking , hba1c , total cholesterol , blood pressure , the use of ras inhibitors , the use of lipid lowering drugs and body mass index ( bmi ) showed that the risk for outcomes in patients , who achieved remission , was 0.25 ( 95% ci 0.070.87 ) as compared with those whose microalbuminuric stage did not change during the follow up , whereas that in patients who progressed to overt proteinuria was 2.55 ( 95% ci 1.046.30 ) ( figure 3 ) . even though failing to achieve the remission , the number of the first occurrences of outcomes in subgroups stratified by a 50% reduction of urinary albumin excretion was 12 events in the regression group and 35 events in the nonregression group . a prospective observational followup study including a total of 216 japanese type 2 diabetic patients with microalbuminuria was carried out to explore the clinical impact of remission and progression of microalbuminuria . the pooled logistic analysis adjusted by sex , age , the initial albumin excretion rate levels , a history of cardiovascular disease , current smoking , hba1c , total cholesterol , triglyceride , highdensity lipoprotein cholesterol , systolic blood pressure , diastolic blood pressure , the use of renin angiotensin system inhibitors , the use of lipid lowering drugs and body mass index showed that the risk for outcomes in patients , who achieved remission , was 0.25 ( 95% ci 0.070.87 ) as compared with those whose microalbuminuric stage did not change during the follow up , whereas that in patients , who progressed to overt proteinuria , was 2.55 ( 95% ci 1.046.30 ) . as suspected , the annual decline rate of egfr in the progression group ( median 4.2 ml / min / year ) was significantly faster than in the nonchange group ( 2.4 ml / min / year ) , whereas the annual decline rate of egfr in the remission group was significantly slower , 1.1 ml / min / year , which is almost identical with the decline rate by normal aging reported in healthy people . these results show that the remission of microalbuminuria is closely related to the improvement of renal function in the long term . physicians have to take care of diabetic patients with an aggressive multifactorial management plan as early as possible after the development of microalbuminuria ( table 2 ) . the clinical target , which is important and effective for diabetic kidney disease , is to achieve the remission and/or regression of microalbuminuria .

most cancer patients suffer anaemia during the course of the disease or the treatment , in particular those patients receiving myelosuppressive chemotherapy . severe anaemia ( < 8 g / dl ) can lead to the onset of cardiovascular and respiratory symptoms , such as tachycardia , hypotension and dyspnoea , although less severe anaemia can also produce a range of symptoms such as fatigue , exercise intolerance , decreased appetite and reduced general welfare . in addition , anaemia has been shown to influence the evolution of disease , increasing the risk of progression and decreasing overall survival [ 3 , 4 ] . significant progress has been made in the prevention and management of chemotherapy - induced anaemia . current therapeutic options include iron supplementation , administration of erythropoiesis - stimulating agents ( esas ) and blood transfusion . several guidelines have been published with specific recommendations [ 57 ] , like the 2007 european organization for research and treatment of cancer ( eortc ) guidelines . the european cancer anaemia survey ( ecas ) , the first major epidemiological study on anaemia conducted in 2001 , revealed a high prevalence and incidence , and highlighted that many patients ( some of them with haemoglobin [ hb ] < 10 g / dl and/or symptomatic anaemia ) received no specific treatment . since then , some studies in particular countries like france , germany and belgium , have provided updated and country - specific information on anaemia management and fulfilment of guidelines . in the spanish context , there is no recent data available in this field . this study aims to provide accurate and relevant information on the prevalence and patterns of treatment of anaemia in spanish cancer patients treated with systemic therapy with or without radiation therapy in the clinical practice . this was an observational , multicenter , cross - sectional study in 21 spanish hospitals . we consecutively included all patients who attended the clinic during a particular day of the week from may 19 to may 23 , 2008 ( to be chosen by each site according to their availability ) , and fulfilled selection criteria : age 18 years ; patients diagnosed with non - myeloid tumour ( except for myelodysplastic syndromes ) , undergoing treatment with systemic therapy ( ct , hormone therapy or immunotherapy ) , with or without radiation therapy ; patients who had received the last cycle of treatment within the previous 4 weeks ; patients who had a blood test ( including hb value ) performed on the date of the visit or within the past 72 h ; non - hospitalized patients . the protocol was approved by an independent ethics committee , and all patients gave their written informed consent before enrolment . data on clinical characteristics , type of cancer , treatment for anaemia and hb levels were collected for the baseline visit and for the past 4 weeks . we defined the degree of anaemia according to world health organization ( who ) as follows : mild anaemia , hb between 11.9 and 10 g / dl ; moderate anaemia , hb between 9.9 and 8 g / dl ; severe anaemia , hb < 8 g / dl . the prevalence of anaemia in the entire study population and in specific subgroups ( by type and stage of tumour , age category , sex , presence or absence of symptoms and type and characteristics of systemic therapy ) was described . treatments for anaemia received in the 4 weeks prior to inclusion and provided on that day were also described ( no treatment , transfusion , iron supplements or vitamins , esas ) . we performed an exploratory analysis to analyze the relationship between patient characteristics or treatment and the presence of anaemia . for qualitative variables , the relationship was assessed using chi square tests ( or fisher s exact test if it was applicable ) , and for quantitative variables , using student s t tests . statistical analyses were performed with the sas package - version 9.0 ( sas institute , cary , nc , usa ) . the study included 214 patients with a median age of 63 years ( range 2091 ) , 58 % women and 73 % with solid tumours . the most common tumour types were breast ( 37.6 % ) , gastrointestinal ( 23.6 % ) and lung cancer ( 19.7 % ) . most patients ( 91 % ) were receiving ct ( of which 26 % received platinum compounds ) , 8.5 % hormone therapy , and 8.6 % immunotherapy ( table 1).table 1characteristics of the patients and treatment administered non - myeloid malignancies n = 214 non - myeloid malignancies n = 214age ( years ) , median ( range)63 ( 20 ; 91)chemotherapy , n ( % ) 193 ( 91.0)women , n ( % ) 124 ( 57.9)adjuvant37 ( 20.7)reason for visit , n ( % ) advanced stage142 ( 79.3 ) chemotherapy160 ( 74.8 ) line of advanced stage , n ( % ) monitoring visit55 ( 25.7 ) first76 ( 53.9 ) radiotherapy10 ( 4.7 ) second37 ( 26.2 ) therapy ( antiemetics )5 ( 2.3 ) third16 ( 11.3 ) treatment of complications1 ( 0.5 ) more than third12 ( 8.5 ) rbc transfusion1 ( 0.5)intention , n ( % ) other reasons3 ( 1.4 ) curative87 ( 46.8)time since diagnosis ( years ) , median ( range)0.7 ( 0.120 ) palliative99 ( 53.2)primary tumour , n ( % ) cycle number , median ( range)4 ( 1 ; 19 ) solid tumour157 ( 73.4)type of therapy , n ( % ) breast59 ( 37.6 ) monotherapy69 ( 36.7 ) gastrointestinal37 ( 23.6 ) polychemotherapy119 ( 63.3 ) lung31 ( 19.7)use of platinums , n ( % ) other30 ( 19.1 ) with platinum40 ( 25.8 ) haematological tumour57 ( 26.6 ) without platinum115 ( 74.2 ) lymphoma14 ( 24.6)radiotherapy , n ( % ) 21 ( 10.0 ) myeloma31 ( 54.4)hormonal therapy , n ( % ) 18 ( 8.5 ) other12 ( 21.0)immunotherapy , n ( % ) 18 ( 8.6)metastasis ( only solid tumours ) , n ( % ) 98 ( 63.2)targeted therapy , n ( % ) 20 ( 9.5)stage iii iv ( only haematological tumours ) , n ( % ) 27 ( 65.8)other systemic treatments , n ( % ) 17 ( 8.1)ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviationvalid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 characteristics of the patients and treatment administered ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviation valid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 the main reason for the visit was ct administration ( 74.8 % ) , followed by routine follow - up visit ( 25.7 % ) , radiotherapy administration ( 4.7 % ) and other reasons ( 5.2 % ) . the median time since diagnosis was 8.4 months ( range 1.2240 ) . overall , 48.1 % of patients ( 95 % ci 45.258.6 ) had anaemia ( of whom 13 % had levels < 10 g / dl and 31 % were symptomatic ) ( fig . 1prevalence of anaemia in cancer patients treated with systemic therapy prevalence of anaemia in cancer patients treated with systemic therapy median hb level in the overall sample was 12.0 g / dl ( range 7.416.1 ) ( table 2 ) . the proportion of patients who reported symptoms of anaemia in the current visit was the same as in the previous 4 weeks . only three patients with symptoms at the current visit did not have anaemia according to the laboratory test.table 2haematological parameters and symptoms of anaemiavariable haemoglobin ( g / dl)valid n214mean ( sd)12.06 ( 1.60)median ( range)12.0 ( 7.4 ; 16.1)leukocytes ( 10/l)valid n214mean ( sd)5.91 ( 2.97)median ( range)5.0 ( 1.6 ; 17.7)platelets ( 10/l)valid n214mean ( sd)230.44 ( 115.49)median ( range)213.0 ( 22.5 ; 927.0)during the last 4 weeks , has the patient had symptoms related to anaemia?valid n206 ( 100.0 % ) yes34 ( 16.5 % ) no172 ( 83.5 % ) in the current visit , has the patient symptoms related to anaemia?valid n208 ( 100.0 % ) yes35 ( 16.8 % ) no173 ( 83.2 % ) sd standard deviation haematological parameters and symptoms of anaemia sd standard deviation there was a trend towards a higher prevalence of anaemia in women ( 52 vs. 42 % in men , p = 0.140 ) and in patients with solid tumours ( 52 vs. 37 % in patients with haematological malignancies , p = 0.094 ) ( fig . 2 ) . no association between anaemia and age , stage or tumour type was observed ( data not shown ) . a higher prevalence was found in patients with symptoms of anaemia in the last 4 weeks ( 82 vs. 40 % in patients without symptoms , p < 0.0001 ) , and in patients treated with ct ( 51 vs. 21 % in patients treated with hormonal therapy or immunotherapy , p = 0.013 ) , platinum - based ct ( 70 vs. 47 % in patients with non - platinum based ct , p = 0.013 ) or palliative ct ( 61 vs. 39 % in patients with curative ct , p = 0.003 ) ( fig . 2prevalence of anaemia in different subgroups according to patient characteristics , type of tumour , symptoms related to anaemia and systemic therapy prevalence of anaemia in different subgroups according to patient characteristics , type of tumour , symptoms related to anaemia and systemic therapy there were no differences in the prevalence of anaemia between patients with ct receiving monotherapy or combination therapy ( 52 vs. 49 % , p = 0.457 ) . the administration of concomitant radiotherapy was not related to anaemia either ( 52 vs. 48 % , p = 0.944 ) . the proportion of patients with anaemia was significantly higher in patients with more than three lines of ct ( 83 vs. 46 , 54 and 44 % in patients undergoing first- , second- and third - line of ct , respectively , p = 0.006 ) and in patients in the fourth or subsequent ct cycles , compared with patients in the first three cycles ( 58 vs. 41 % , p = 0.028 ) . no significant differences were found in mean leukocyte or platelet count between patients with and without anaemia ( data not shown ) . in patients with anaemia ( n = 103 ) , the management in the previous 4 weeks was as follows : 62.1 % received no specific treatment ( of whom 91.9 % had mild anaemia ) , 24.0 % received esa , 13.6 % received iron supplementation and 8.7 % received transfusion ( fig . the use of transfusion in the previous 4 weeks was higher in patients with moderate or severe anaemia , compared to patients with mild anaemia ( 23.1 vs. 5.6 % , p < 0.0001 ) . thereby , 37.5 % of patients who received transfusion during the previous 4 weeks ( 3 out of 8) still had moderate or severe anaemia.fig . 3therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) in the current visit , the percentage of patients without any treatment for anaemia increased to 74.8 % ( of whom 94.9 % had mild anaemia ) . sixty - nine percent of the patients with moderate or severe anaemia received treatment , compared with only 18 % of patients with mild anaemia ( p < 0.0001 ) . only two patients were prescribed a transfusion at the study visit ( one received only transfusion and the other one received transfusion , iron and esa ) , both having moderate or severe anaemia . among the 25 patients who received any anaemia treatment at the study visit , 18 patients received esa s ( 17.5 % of anaemic subjects ) . darbepoetin alfa was the most commonly used agent ( 13 patients , four patients received epoetin beta , and 1 patient received epoetin alfa ) . the use of esa s was more frequent in patients with moderate or severe anaemia ( 46 vs. 13 % in patients with mild anaemia , p = 0.012 ) . among patients prescribed esa s , the mean hb in patients with mild or moderate - severe anaemia was 10.5 and 8.2 g / dl , respectively . iron supplementation was used in 13 patients ( 12.6 % of anaemic subjects ) . the study included 214 patients with a median age of 63 years ( range 2091 ) , 58 % women and 73 % with solid tumours . the most common tumour types were breast ( 37.6 % ) , gastrointestinal ( 23.6 % ) and lung cancer ( 19.7 % ) . most patients ( 91 % ) were receiving ct ( of which 26 % received platinum compounds ) , 8.5 % hormone therapy , and 8.6 % immunotherapy ( table 1).table 1characteristics of the patients and treatment administered non - myeloid malignancies n = 214 non - myeloid malignancies n = 214age ( years ) , median ( range)63 ( 20 ; 91)chemotherapy , n ( % ) 193 ( 91.0)women , n ( % ) 124 ( 57.9)adjuvant37 ( 20.7)reason for visit , n ( % ) advanced stage142 ( 79.3 ) chemotherapy160 ( 74.8 ) line of advanced stage , n ( % ) monitoring visit55 ( 25.7 ) first76 ( 53.9 ) radiotherapy10 ( 4.7 ) second37 ( 26.2 ) therapy ( antiemetics )5 ( 2.3 ) third16 ( 11.3 ) treatment of complications1 ( 0.5 ) more than third12 ( 8.5 ) rbc transfusion1 ( 0.5)intention , n ( % ) other reasons3 ( 1.4 ) curative87 ( 46.8)time since diagnosis ( years ) , median ( range)0.7 ( 0.120 ) palliative99 ( 53.2)primary tumour , n ( % ) cycle number , median ( range)4 ( 1 ; 19 ) solid tumour157 ( 73.4)type of therapy , n ( % ) breast59 ( 37.6 ) monotherapy69 ( 36.7 ) gastrointestinal37 ( 23.6 ) polychemotherapy119 ( 63.3 ) lung31 ( 19.7)use of platinums , n ( % ) other30 ( 19.1 ) with platinum40 ( 25.8 ) haematological tumour57 ( 26.6 ) without platinum115 ( 74.2 ) lymphoma14 ( 24.6)radiotherapy , n ( % ) 21 ( 10.0 ) myeloma31 ( 54.4)hormonal therapy , n ( % ) 18 ( 8.5 ) other12 ( 21.0)immunotherapy , n ( % ) 18 ( 8.6)metastasis ( only solid tumours ) , n ( % ) 98 ( 63.2)targeted therapy , n ( % ) 20 ( 9.5)stage iii iv ( only haematological tumours ) , n ( % ) 27 ( 65.8)other systemic treatments , n ( % ) 17 ( 8.1)ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviationvalid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 characteristics of the patients and treatment administered ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviation valid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 the main reason for the visit was ct administration ( 74.8 % ) , followed by routine follow - up visit ( 25.7 % ) , radiotherapy administration ( 4.7 % ) and other reasons ( 5.2 % ) . the median time since diagnosis was 8.4 months ( range 1.2240 ) . overall , 48.1 % of patients ( 95 % ci 45.258.6 ) had anaemia ( of whom 13 % had levels < 10 g / dl and 31 % were symptomatic ) ( fig 1prevalence of anaemia in cancer patients treated with systemic therapy prevalence of anaemia in cancer patients treated with systemic therapy median hb level in the overall sample was 12.0 g / dl ( range 7.416.1 ) ( table 2 ) . the proportion of patients who reported symptoms of anaemia in the current visit was the same as in the previous 4 weeks . only three patients with symptoms at the current visit did not have anaemia according to the laboratory test.table 2haematological parameters and symptoms of anaemiavariable haemoglobin ( g / dl)valid n214mean ( sd)12.06 ( 1.60)median ( range)12.0 ( 7.4 ; 16.1)leukocytes ( 10/l)valid n214mean ( sd)5.91 ( 2.97)median ( range)5.0 ( 1.6 ; 17.7)platelets ( 10/l)valid n214mean ( sd)230.44 ( 115.49)median ( range)213.0 ( 22.5 ; 927.0)during the last 4 weeks , has the patient had symptoms related to anaemia?valid n206 ( 100.0 % ) yes34 ( 16.5 % ) no172 ( 83.5 % ) in the current visit , has the patient symptoms related to anaemia?valid n208 ( 100.0 % ) yes35 ( 16.8 % ) no173 ( 83.2 % ) sd standard deviation haematological parameters and symptoms of anaemia sd standard deviation there was a trend towards a higher prevalence of anaemia in women ( 52 vs. 42 % in men , p = 0.140 ) and in patients with solid tumours ( 52 vs. 37 % in patients with haematological malignancies , p = 0.094 ) ( fig . 2 ) . no association between anaemia and age , stage or tumour type was observed ( data not shown ) . a higher prevalence was found in patients with symptoms of anaemia in the last 4 weeks ( 82 vs. 40 % in patients without symptoms , p < 0.0001 ) , and in patients treated with ct ( 51 vs. 21 % in patients treated with hormonal therapy or immunotherapy , p = 0.013 ) , platinum - based ct ( 70 vs. 47 % in patients with non - platinum based ct , p = 0.013 ) or palliative ct ( 61 vs. 39 % in patients with curative ct , p = 0.003 ) ( fig . 2prevalence of anaemia in different subgroups according to patient characteristics , type of tumour , symptoms related to anaemia and systemic therapy prevalence of anaemia in different subgroups according to patient characteristics , type of tumour , symptoms related to anaemia and systemic therapy there were no differences in the prevalence of anaemia between patients with ct receiving monotherapy or combination therapy ( 52 vs. 49 % , p = 0.457 ) . the administration of concomitant radiotherapy was not related to anaemia either ( 52 vs. 48 % , p = 0.944 ) . the proportion of patients with anaemia was significantly higher in patients with more than three lines of ct ( 83 vs. 46 , 54 and 44 % in patients undergoing first- , second- and third - line of ct , respectively , p = 0.006 ) and in patients in the fourth or subsequent ct cycles , compared with patients in the first three cycles ( 58 vs. 41 % , p = 0.028 ) . no significant differences were found in mean leukocyte or platelet count between patients with and without anaemia ( data not shown ) . in patients with anaemia ( n = 103 ) , the management in the previous 4 weeks was as follows : 62.1 % received no specific treatment ( of whom 91.9 % had mild anaemia ) , 24.0 % received esa , 13.6 % received iron supplementation and 8.7 % received transfusion ( fig . the use of transfusion in the previous 4 weeks was higher in patients with moderate or severe anaemia , compared to patients with mild anaemia ( 23.1 vs. 5.6 % , p < 0.0001 ) . thereby , 37.5 % of patients who received transfusion during the previous 4 weeks ( 3 out of 8) still had moderate or severe anaemia.fig . 3therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) in the current visit , the percentage of patients without any treatment for anaemia increased to 74.8 % ( of whom 94.9 % had mild anaemia ) . sixty - nine percent of the patients with moderate or severe anaemia received treatment , compared with only 18 % of patients with mild anaemia ( p < 0.0001 ) . only two patients were prescribed a transfusion at the study visit ( one received only transfusion and the other one received transfusion , iron and esa ) , both having moderate or severe anaemia . among the 25 patients who received any anaemia treatment at the study visit , 18 patients received esa s ( 17.5 % of anaemic subjects ) . darbepoetin alfa was the most commonly used agent ( 13 patients , four patients received epoetin beta , and 1 patient received epoetin alfa ) . the use of esa s was more frequent in patients with moderate or severe anaemia ( 46 vs. 13 % in patients with mild anaemia , p = 0.012 ) . among patients prescribed esa s , the mean hb in patients with mild or moderate - severe anaemia was 10.5 and 8.2 g / dl , respectively . iron supplementation was used in 13 patients ( 12.6 % of anaemic subjects ) . we present information on the prevalence and management of anaemia in an unselected cohort of cancer patients undergoing systemic therapy in the spanish setting . we found a prevalence of anaemia ( defined as hb < 12 g / dl ) of approximately 50 % , which is similar to that found in other contemporary studies from european countries [ 911 ] . in the ecas , conducted in 2001 , the prevalence of anaemia at enrolment was lower ( 39 % ) , but the proportion of anaemic subjects not receiving treatment was comparable to ours ( more than 60 % ) . recent data from france suggest a better management of anaemia in this country , with esa administration in approximately two - third of anaemic patients , and only 17 % non - treated . in the belgian study with the same design as the present one , the prevalence of anaemia was comparable ( 56 % ) , but a higher relative proportion of moderate versus mild anaemia was found ( threefold to that of the spanish cohort ) . they also observed more anaemia in patients with haematological malignancies ( 73 % ) compared to solid tumours , which disagrees with our results . the fact that they did not exclude patients with myelodysplastic syndromes ( at high risk of anaemia ) can partially explain this finding , but probably there are other differences involved [ for example , more use of platinum - based regimens ( 37 vs. 26 % in our cohort ) , or more proportion of advanced disease ] . regarding anaemia management , there were also important disparities with our results : a higher proportion of treated patients ( almost 50 % ) and more transfusions . we observed a strong association between ct administration and anaemia , as previously reported in the ecas . among ct - related factors , more than three lines of treatment or more than three cycles were associated to a higher risk of anaemia . similarly , during ecas , the prevalence of anaemia increased progressively from cycle 1 to cycle 6 . the observed association with palliative ct probably reflects the degree of severity of the disease and its impact on erythropoiesis . interestingly , the prevalence of anaemia was not different in patients receiving mono - ct as opposed to poly - ct , but it was higher in patients receiving platinum - based regimens ( 70 vs. 47 % ) . we did not find any significant influence of radiotherapy administration . a model developed by barret et al . to predict anaemia development in cancer patients initiating ct included the following variables as the most important factors : low initial hb ( 12.9 g / dl in females , and 13.4 g / dl in males ) ; cancer type other than gastrointestinal / colorectal cancer ; treatment with platinum ct , and female gender . we did not analyze the prevalence by tumour subtype , due to sample size limitation , but we confirm a strong association with platinum - based ct . the eortc guidelines recommend to treat all patients with hb levels below 9 g / dl ( who were only a small percentage of our sample ) , and all symptomatic patients with levels between 9 and 11 g / dl . the anaemia cancer treatment ( a.c.t . ) study was a retrospective , extensive review of anaemic patients with cancer treated with esas investigating patterns of use according to eortc guidelines . it concluded that , in 2007 , most european patients were treated per guidelines , except for low iron supplementation rates . a prospective german study in cancer patients with anaemia observed that physicians preferred esa as first - line treatment in patients with solid tumours , but in patients with lymphoproliferative malignancies , transfusions or correction of underlying disorders was preferred . around one - third of anaemic subjects were symptomatic , which means that at least the same amount of individuals would benefit from receiving esas . since only 17 % of patients were being treated with erythropoietic agents , we can conclude that the eortc guidelines were still not fully implemented during 2008 in the clinical practice of spanish oncology or oncohematology services . current guidelines of the spanish society of medical oncology ( seom ) about the use of esas in cancer patients were published in 2009 , after the study completion . they established that , since esa treatment increases the hb level and decreases the red blood cell transfusion requirements , esas should be used within the approved indications in patients undergoing chemotherapy treatment , beginning at an hb level below 11 g / dl and maintaining it around 12 g / dl , with iron supplements if necessary . thus , our data also shows that management of anaemia in spain needs to be updated according to local guidelines . despite the concerns about use of esas , all meta - analyses considering only clinical trials performed under the approved indication ( patients receiving chemotherapy ) support their safety and clinical benefit in the treatment of chemotherapy - induced anaemia [ 1823 ] , including a reduction in transfusion needs both in solid tumours and in haematological malignancies [ 24 , 25 ] . in the larger meta - analysis conducted until now , the use of esas was neutral with regard to survival and disease progression . in this meta - analysis , it was also demonstrated that the use of esas is associated with an increased risk of venous thromboembolism ( odds ratio = 1.4 ) . regarding the increase in the risk for thromboembolic events [ 23 , 27 ] , it must be taken into account specially in patients with cardiovascular disease , in whom the esa dose must be adjusted according to patient characteristics to avoid an excessive change in plasma volume and maintaining a consistent hb level . the main limitations of our study are the limited sample size , that does not allow to explore differences in the prevalence and management of anaemia between tumour subtypes , for example , and the cross - sectional design , which does not allow for causal inferences . in spanish hospitals , about half of patients with non - myeloid malignancies undergoing systemic therapy suffer from anaemia . chemotherapy administration , especially if it contains platinum compounds or represents an advanced - line therapy , is associated with a high risk of anaemia . our results show that approximately two - third of patients with anaemia do not receive specific treatment and that esas are underused according to current eortc and local seom guidelines . since the treatment of anaemia with esas ( and adequate iron supplementation ) can significantly improve patients quality of life and may also improve the clinical outcome , it is advisable to review and optimize anaemia management in the clinical practice of spanish oncology and hematology services . the following investigators are members of the anaemia day study group and also participated in this study : co - authors . this study has been conducted by the above - signed authors and the anaemia day study group : dra . jls is consultant for novartis , bms , pfizer and amgen , and has received funding from novartis and bms . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

backgroundthe present study aimed to provide updated data on anaemia prevalence and management in cancer patients undergoing systemic therapy in spain.methodsthis was a multicenter , observational , cross - sectional study performed in 2008 . eligible patients were 18 years , with non - myeloid malignancies treated with systemic therapy [ chemotherapy ( ct ) , hormonal therapy or immunotherapy ] . anaemia was defined according to who as haemoglobin ( hb ) < 12 g / dl.resultsthe study included 214 patients with a median age of 63 years ( range 2091 ) , 58 % women , 73 % with solid tumours , and 79 % with advanced disease . ct was used in 91 % of patients ( 26 % with platinum compounds ) , hormonal therapy in 8.5 % , and immunotherapy in 8.5 % . in our study , 48.1 % of patients [ 95 % confidence interval ( ci ) 45.258.6 ] showed anaemia ( 31 % symptomatic ) : 42.0 % mild ( 10 hb 11.9 g / dl ) , 5.6 % moderate ( 8 hb 9.9 g / dl ) , and 0.5 % severe ( hb < 8 g / dl ) . a higher prevalence was observed in patients treated with ct ( 51 vs. 20 % , p = 0.01 ) , platinum - based ct ( 70 vs. 47 % , p = 0.01 ) or palliative ct ( 61 vs. 39 % , p = 0.003 ) . anaemia was also more frequent in patients with more than three lines of ct ( 83 % ) and in the fourth or subsequent ct cycle ( 58 % ) . management in the previous 4 weeks in patients with anaemia was : 62 % did not receive treatment ( 92 % mild ) , 24 % received erythropoiesis - stimulating agents ( esas ) , 14 % received iron and 8.7 % received transfusion.conclusionsin spanish hospitals , about half of patients with non - myeloid malignancies undergoing systemic therapy fulfilled anaemia criteria ( 87 % mild ) . approximately two - third of patients with anaemia do not receive specific treatment and esa use is below current guidelines .

Introduction Patients and methods Results Study population Prevalence of anaemia Factors related to anaemia Anaemia management Discussion Conclusion Appendix Conflict of interest Open Access

this study aims to provide accurate and relevant information on the prevalence and patterns of treatment of anaemia in spanish cancer patients treated with systemic therapy with or without radiation therapy in the clinical practice . we consecutively included all patients who attended the clinic during a particular day of the week from may 19 to may 23 , 2008 ( to be chosen by each site according to their availability ) , and fulfilled selection criteria : age 18 years ; patients diagnosed with non - myeloid tumour ( except for myelodysplastic syndromes ) , undergoing treatment with systemic therapy ( ct , hormone therapy or immunotherapy ) , with or without radiation therapy ; patients who had received the last cycle of treatment within the previous 4 weeks ; patients who had a blood test ( including hb value ) performed on the date of the visit or within the past 72 h ; non - hospitalized patients . we defined the degree of anaemia according to world health organization ( who ) as follows : mild anaemia , hb between 11.9 and 10 g / dl ; moderate anaemia , hb between 9.9 and 8 g / dl ; severe anaemia , hb < 8 g / dl . the study included 214 patients with a median age of 63 years ( range 2091 ) , 58 % women and 73 % with solid tumours . most patients ( 91 % ) were receiving ct ( of which 26 % received platinum compounds ) , 8.5 % hormone therapy , and 8.6 % immunotherapy ( table 1).table 1characteristics of the patients and treatment administered non - myeloid malignancies n = 214 non - myeloid malignancies n = 214age ( years ) , median ( range)63 ( 20 ; 91)chemotherapy , n ( % ) 193 ( 91.0)women , n ( % ) 124 ( 57.9)adjuvant37 ( 20.7)reason for visit , n ( % ) advanced stage142 ( 79.3 ) chemotherapy160 ( 74.8 ) line of advanced stage , n ( % ) monitoring visit55 ( 25.7 ) first76 ( 53.9 ) radiotherapy10 ( 4.7 ) second37 ( 26.2 ) therapy ( antiemetics )5 ( 2.3 ) third16 ( 11.3 ) treatment of complications1 ( 0.5 ) more than third12 ( 8.5 ) rbc transfusion1 ( 0.5)intention , n ( % ) other reasons3 ( 1.4 ) curative87 ( 46.8)time since diagnosis ( years ) , median ( range)0.7 ( 0.120 ) palliative99 ( 53.2)primary tumour , n ( % ) cycle number , median ( range)4 ( 1 ; 19 ) solid tumour157 ( 73.4)type of therapy , n ( % ) breast59 ( 37.6 ) monotherapy69 ( 36.7 ) gastrointestinal37 ( 23.6 ) polychemotherapy119 ( 63.3 ) lung31 ( 19.7)use of platinums , n ( % ) other30 ( 19.1 ) with platinum40 ( 25.8 ) haematological tumour57 ( 26.6 ) without platinum115 ( 74.2 ) lymphoma14 ( 24.6)radiotherapy , n ( % ) 21 ( 10.0 ) myeloma31 ( 54.4)hormonal therapy , n ( % ) 18 ( 8.5 ) other12 ( 21.0)immunotherapy , n ( % ) 18 ( 8.6)metastasis ( only solid tumours ) , n ( % ) 98 ( 63.2)targeted therapy , n ( % ) 20 ( 9.5)stage iii iv ( only haematological tumours ) , n ( % ) 27 ( 65.8)other systemic treatments , n ( % ) 17 ( 8.1)ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviationvalid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 characteristics of the patients and treatment administered ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviation valid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 the main reason for the visit was ct administration ( 74.8 % ) , followed by routine follow - up visit ( 25.7 % ) , radiotherapy administration ( 4.7 % ) and other reasons ( 5.2 % ) . overall , 48.1 % of patients ( 95 % ci 45.258.6 ) had anaemia ( of whom 13 % had levels < 10 g / dl and 31 % were symptomatic ) ( fig . 1prevalence of anaemia in cancer patients treated with systemic therapy prevalence of anaemia in cancer patients treated with systemic therapy median hb level in the overall sample was 12.0 g / dl ( range 7.416.1 ) ( table 2 ) . only three patients with symptoms at the current visit did not have anaemia according to the laboratory test.table 2haematological parameters and symptoms of anaemiavariable haemoglobin ( g / dl)valid n214mean ( sd)12.06 ( 1.60)median ( range)12.0 ( 7.4 ; 16.1)leukocytes ( 10/l)valid n214mean ( sd)5.91 ( 2.97)median ( range)5.0 ( 1.6 ; 17.7)platelets ( 10/l)valid n214mean ( sd)230.44 ( 115.49)median ( range)213.0 ( 22.5 ; 927.0)during the last 4 weeks , has the patient had symptoms related to anaemia?valid n206 ( 100.0 % ) yes34 ( 16.5 % ) no172 ( 83.5 % ) in the current visit , has the patient symptoms related to anaemia?valid n208 ( 100.0 % ) yes35 ( 16.8 % ) no173 ( 83.2 % ) sd standard deviation haematological parameters and symptoms of anaemia sd standard deviation there was a trend towards a higher prevalence of anaemia in women ( 52 vs. 42 % in men , p = 0.140 ) and in patients with solid tumours ( 52 vs. 37 % in patients with haematological malignancies , p = 0.094 ) ( fig . a higher prevalence was found in patients with symptoms of anaemia in the last 4 weeks ( 82 vs. 40 % in patients without symptoms , p < 0.0001 ) , and in patients treated with ct ( 51 vs. 21 % in patients treated with hormonal therapy or immunotherapy , p = 0.013 ) , platinum - based ct ( 70 vs. 47 % in patients with non - platinum based ct , p = 0.013 ) or palliative ct ( 61 vs. 39 % in patients with curative ct , p = 0.003 ) ( fig . 2prevalence of anaemia in different subgroups according to patient characteristics , type of tumour , symptoms related to anaemia and systemic therapy prevalence of anaemia in different subgroups according to patient characteristics , type of tumour , symptoms related to anaemia and systemic therapy there were no differences in the prevalence of anaemia between patients with ct receiving monotherapy or combination therapy ( 52 vs. 49 % , p = 0.457 ) . the proportion of patients with anaemia was significantly higher in patients with more than three lines of ct ( 83 vs. 46 , 54 and 44 % in patients undergoing first- , second- and third - line of ct , respectively , p = 0.006 ) and in patients in the fourth or subsequent ct cycles , compared with patients in the first three cycles ( 58 vs. 41 % , p = 0.028 ) . in patients with anaemia ( n = 103 ) , the management in the previous 4 weeks was as follows : 62.1 % received no specific treatment ( of whom 91.9 % had mild anaemia ) , 24.0 % received esa , 13.6 % received iron supplementation and 8.7 % received transfusion ( fig . the use of transfusion in the previous 4 weeks was higher in patients with moderate or severe anaemia , compared to patients with mild anaemia ( 23.1 vs. 5.6 % , p < 0.0001 ) . 3therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) in the current visit , the percentage of patients without any treatment for anaemia increased to 74.8 % ( of whom 94.9 % had mild anaemia ) . the study included 214 patients with a median age of 63 years ( range 2091 ) , 58 % women and 73 % with solid tumours . most patients ( 91 % ) were receiving ct ( of which 26 % received platinum compounds ) , 8.5 % hormone therapy , and 8.6 % immunotherapy ( table 1).table 1characteristics of the patients and treatment administered non - myeloid malignancies n = 214 non - myeloid malignancies n = 214age ( years ) , median ( range)63 ( 20 ; 91)chemotherapy , n ( % ) 193 ( 91.0)women , n ( % ) 124 ( 57.9)adjuvant37 ( 20.7)reason for visit , n ( % ) advanced stage142 ( 79.3 ) chemotherapy160 ( 74.8 ) line of advanced stage , n ( % ) monitoring visit55 ( 25.7 ) first76 ( 53.9 ) radiotherapy10 ( 4.7 ) second37 ( 26.2 ) therapy ( antiemetics )5 ( 2.3 ) third16 ( 11.3 ) treatment of complications1 ( 0.5 ) more than third12 ( 8.5 ) rbc transfusion1 ( 0.5)intention , n ( % ) other reasons3 ( 1.4 ) curative87 ( 46.8)time since diagnosis ( years ) , median ( range)0.7 ( 0.120 ) palliative99 ( 53.2)primary tumour , n ( % ) cycle number , median ( range)4 ( 1 ; 19 ) solid tumour157 ( 73.4)type of therapy , n ( % ) breast59 ( 37.6 ) monotherapy69 ( 36.7 ) gastrointestinal37 ( 23.6 ) polychemotherapy119 ( 63.3 ) lung31 ( 19.7)use of platinums , n ( % ) other30 ( 19.1 ) with platinum40 ( 25.8 ) haematological tumour57 ( 26.6 ) without platinum115 ( 74.2 ) lymphoma14 ( 24.6)radiotherapy , n ( % ) 21 ( 10.0 ) myeloma31 ( 54.4)hormonal therapy , n ( % ) 18 ( 8.5 ) other12 ( 21.0)immunotherapy , n ( % ) 18 ( 8.6)metastasis ( only solid tumours ) , n ( % ) 98 ( 63.2)targeted therapy , n ( % ) 20 ( 9.5)stage iii iv ( only haematological tumours ) , n ( % ) 27 ( 65.8)other systemic treatments , n ( % ) 17 ( 8.1)ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviationvalid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 characteristics of the patients and treatment administered ecog eastern cooperative oncology group , rbc red blood cells , sd standard deviation valid n = 155 , valid n = 41 , valid n = 179 , valid n = 141 , valid n = 186 , valid n = 188 , valid n = 155 , valid n = 211 , valid n = 213 , valid n = 210 the main reason for the visit was ct administration ( 74.8 % ) , followed by routine follow - up visit ( 25.7 % ) , radiotherapy administration ( 4.7 % ) and other reasons ( 5.2 % ) . overall , 48.1 % of patients ( 95 % ci 45.258.6 ) had anaemia ( of whom 13 % had levels < 10 g / dl and 31 % were symptomatic ) ( fig 1prevalence of anaemia in cancer patients treated with systemic therapy prevalence of anaemia in cancer patients treated with systemic therapy median hb level in the overall sample was 12.0 g / dl ( range 7.416.1 ) ( table 2 ) . only three patients with symptoms at the current visit did not have anaemia according to the laboratory test.table 2haematological parameters and symptoms of anaemiavariable haemoglobin ( g / dl)valid n214mean ( sd)12.06 ( 1.60)median ( range)12.0 ( 7.4 ; 16.1)leukocytes ( 10/l)valid n214mean ( sd)5.91 ( 2.97)median ( range)5.0 ( 1.6 ; 17.7)platelets ( 10/l)valid n214mean ( sd)230.44 ( 115.49)median ( range)213.0 ( 22.5 ; 927.0)during the last 4 weeks , has the patient had symptoms related to anaemia?valid n206 ( 100.0 % ) yes34 ( 16.5 % ) no172 ( 83.5 % ) in the current visit , has the patient symptoms related to anaemia?valid n208 ( 100.0 % ) yes35 ( 16.8 % ) no173 ( 83.2 % ) sd standard deviation haematological parameters and symptoms of anaemia sd standard deviation there was a trend towards a higher prevalence of anaemia in women ( 52 vs. 42 % in men , p = 0.140 ) and in patients with solid tumours ( 52 vs. 37 % in patients with haematological malignancies , p = 0.094 ) ( fig . a higher prevalence was found in patients with symptoms of anaemia in the last 4 weeks ( 82 vs. 40 % in patients without symptoms , p < 0.0001 ) , and in patients treated with ct ( 51 vs. 21 % in patients treated with hormonal therapy or immunotherapy , p = 0.013 ) , platinum - based ct ( 70 vs. 47 % in patients with non - platinum based ct , p = 0.013 ) or palliative ct ( 61 vs. 39 % in patients with curative ct , p = 0.003 ) ( fig . the proportion of patients with anaemia was significantly higher in patients with more than three lines of ct ( 83 vs. 46 , 54 and 44 % in patients undergoing first- , second- and third - line of ct , respectively , p = 0.006 ) and in patients in the fourth or subsequent ct cycles , compared with patients in the first three cycles ( 58 vs. 41 % , p = 0.028 ) . in patients with anaemia ( n = 103 ) , the management in the previous 4 weeks was as follows : 62.1 % received no specific treatment ( of whom 91.9 % had mild anaemia ) , 24.0 % received esa , 13.6 % received iron supplementation and 8.7 % received transfusion ( fig . the use of transfusion in the previous 4 weeks was higher in patients with moderate or severe anaemia , compared to patients with mild anaemia ( 23.1 vs. 5.6 % , p < 0.0001 ) . 3therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) therapeutic management of anaemia ( previous 4 weeks and current visit ) in patients with hb < 12 g / dl at study inclusion ( n = 103 ) in the current visit , the percentage of patients without any treatment for anaemia increased to 74.8 % ( of whom 94.9 % had mild anaemia ) . the use of esa s was more frequent in patients with moderate or severe anaemia ( 46 vs. 13 % in patients with mild anaemia , p = 0.012 ) . the fact that they did not exclude patients with myelodysplastic syndromes ( at high risk of anaemia ) can partially explain this finding , but probably there are other differences involved [ for example , more use of platinum - based regimens ( 37 vs. 26 % in our cohort ) , or more proportion of advanced disease ] . interestingly , the prevalence of anaemia was not different in patients receiving mono - ct as opposed to poly - ct , but it was higher in patients receiving platinum - based regimens ( 70 vs. 47 % ) . in spanish hospitals , about half of patients with non - myeloid malignancies undergoing systemic therapy suffer from anaemia . our results show that approximately two - third of patients with anaemia do not receive specific treatment and that esas are underused according to current eortc and local seom guidelines .

search for diabetes in youth ( search ) is a multicenter observational study of youth diagnosed with diabetes before age 20 years . participants were recruited from four geographically defined populations in ohio , washington , south carolina , and colorado , health plan enrollees in hawaii and california , and indian health service beneficiaries from four american indian populations . all registered patients were invited by mail , telephone , or at a clinic visit to complete a brief initial survey , and all survey completers whose diabetes was not secondary to other conditions were invited by telephone or at a clinic visit to complete a baseline research visit . participants whose diabetes was incident in 20022005 who completed the baseline study visit were invited for follow - up visits at 12 , 24 , and 60 months after their baseline study visit . during these visits , participants provided informed consent or , for those younger than 18 years old , assent and completed questionnaires . for metabolically stable participants ( i.e. , no episode of diabetic ketoacidosis during the previous month ) , fasting blood samples were drawn after a minimum 8-h overnight fast . these analyses include data from baseline visits of the 2001 ( prevalent ) cohort and from the baseline and 12-month follow - up visits of the 20022005 ( incident ) cohorts . baseline visits were completed by 45.7% of the eligible cohort ( 5,294 visits/11,592 subjects ) . after excluding 290 participants ( 5.5% ) without at least one pedsql module completed by youth or parent , the final baseline sample included 5,004 participants ( 2,462 prevalent and 2,542 incident subjects ) . these data were used to calculate mcids . of the 20022005 incident cohorts who completed the baseline and 12-month follow - up visits ( n = 1,449 , 57% of incident sample with baseline visits ) , 47 ( 3.2% ) without at least one pedsql module were also excluded , resulting in a final sample of 1,402 participants to identify predictors of qol change over time . youth and their parents reported on youths generic and health - related qol using the pedsql generic core and diabetes module ( 3 ) . all youth aged 5 years and older completed an age - appropriate self - report version of the two pedsql modules . items were read to the child or given to the child to read , depending on the child s reading skills . in addition , parents completed a parent - proxy version of the measures for all youth aged younger than 18 years at the time of their visit . thus , participants aged 18 years and older had one score for each module ( self - report ) , participants aged between 5 and 17 years received two scores for each module ( youth and parent report ) , and participants aged younger than 5 years had one score for each module ( parent report only ) . for all versions , items assess the degree of difficulty youth experience with different aspects of everyday living , including physical symptoms and emotional , social , and academic functioning ( generic core ) , as well as treatment adherence and barriers , diabetes - related worries , and communication with others about diabetes ( diabetes module ) . item scores were transformed , with subscale and total scale scores ranging from 0 to 100 . higher scores reflect better qol . the pedsql generic core and diabetes module are psychometrically sound ( 3 ) . demographic and medical information was collected by self - report or parent report and medical record review . those who self - reported being of hispanic ethnicity were categorized as hispanic , regardless of race , and the remainder were categorized by self - reported race . if no or multiple races were endorsed , participants were categorized as other / unknown race / ethnicity . health insurance was categorized as private , state / federally funded , other ( including indian health service , student health clinics , military , or other / unknown sources ) , or none based on self - report . clinical data , including physical measurement and fasting blood samples , were obtained at the baseline search study visit . glycemic control was assessed via glycated hemoglobin ( a1c ) using high - performance liquid chromatography ( tosoh bioscience , montgomeryville , pa ) . bmi was calculated from height and weight measured at the initial study visit , and z - scores ( zbmi ) were calculated using centers for disease control and prevention algorithms ( 14,15 ) . diabetes type was determined by physician diagnosis , and comorbid medical diagnoses were based on self - report or parent report . the mean ( sd ) and scale reliability ( cronbach ) values were calculated for each subscale and the total scores for the generic core and the diabetes module . mcids were calculated using a commonly used distribution - based method : one standard error of measurement ( sem ) ( 12,16 ) . the following equation is used to calculate the sem : sem = sd [ 1 ] , where is the total score or subscale reliability estimate . the sem estimates the variation in scores due to the measurement precision in the scale and assumes that a change in scores smaller than the value of the sem likely results from measurement error rather than a meaningful increase or decrease in the value of the construct being measured ( in this case , qol ) . a score change greater than or equal to the value of the sem represents meaningful variation in the measured construct that is likely not due to measurement error . mcids for various qol ( 17,18 ) measures , including the mcid for the pedsql across a range of pediatric chronic conditions ( 13 ) , have been calculated using this approach . this statistical methodology produces mcids that are expressed in the same units of measurement as the qol score ( 19 ) . the sem and resulting mcids were calculated for the sample of participants and parents for the total and subscale scores of the generic core and diabetes module and were also established separately for each diabetes type . the result is a single value that represents the magnitude of change in scores on qol measures that is detectable to the individual with the chronic condition . thus , the mcid represents a clinically meaningful or important difference in qol that can be referenced in clinical practice and research . to examine predictors of change in qol , two sets of regression analyses were conducted using data from the subset of participants diagnosed from 20022005 with a 12-month follow - up visit and complete data at both time points ( total n = 1,402 [ n range 1,0721,094 ] across the two modules and two reporters ) . general linear regression models were constructed to determine the demographic and clinical characteristics that predicted the amount and direction of improvement or worsening in pedsql change scores . pedsql change scores were calculated as total score at the follow - up visit minus total score at the baseline visit . predictors were demographic and clinical variables that have previously demonstrated associations with qol , including age , sex , insurance coverage , race / ethnicity , diabetes type , diabetes duration , z - bmi , and a1c , all of which were assessed at the baseline visit , and models controlled for pedsql scores at baseline . next , to determine the characteristics that predicted clinically meaningful improvement in qol , participants pedsql change scores for 1 year were categorized as positive ( score increase 1 mcid ) , negative ( score decrease 1 mcid ) , or no change . binary logistic regression models were constructed to predict positive qol change of at least 1 mcid compared with negative / no qol change , controlling for all measured demographic and medical variables noted above , time from baseline to follow - up , and baseline pedsql scores . separate analyses were conducted for parent and child report of the generic core and diabetes module total scores . the mean ( sd ) and scale reliability ( cronbach ) values were calculated for each subscale and the total scores for the generic core and the diabetes module . mcids were calculated using a commonly used distribution - based method : one standard error of measurement ( sem ) ( 12,16 ) . the following equation is used to calculate the sem : sem = sd [ 1 ] , where is the total score or subscale reliability estimate . the sem estimates the variation in scores due to the measurement precision in the scale and assumes that a change in scores smaller than the value of the sem likely results from measurement error rather than a meaningful increase or decrease in the value of the construct being measured ( in this case , qol ) . a score change greater than or equal to the value of the sem represents meaningful variation in the measured construct that is likely not due to measurement error . mcids for various qol ( 17,18 ) measures , including the mcid for the pedsql across a range of pediatric chronic conditions ( 13 ) , have been calculated using this approach . this statistical methodology produces mcids that are expressed in the same units of measurement as the qol score ( 19 ) . the sem and resulting mcids were calculated for the sample of participants and parents for the total and subscale scores of the generic core and diabetes module and were also established separately for each diabetes type . the result is a single value that represents the magnitude of change in scores on qol measures that is detectable to the individual with the chronic condition . thus , the mcid represents a clinically meaningful or important difference in qol that can be referenced in clinical practice and research . to examine predictors of change in qol , two sets of regression analyses were conducted using data from the subset of participants diagnosed from 20022005 with a 12-month follow - up visit and complete data at both time points ( total n = 1,402 [ n range 1,0721,094 ] across the two modules and two reporters ) . general linear regression models were constructed to determine the demographic and clinical characteristics that predicted the amount and direction of improvement or worsening in pedsql change scores . pedsql change scores were calculated as total score at the follow - up visit minus total score at the baseline visit . predictors were demographic and clinical variables that have previously demonstrated associations with qol , including age , sex , insurance coverage , race / ethnicity , diabetes type , diabetes duration , z - bmi , and a1c , all of which were assessed at the baseline visit , and models controlled for pedsql scores at baseline . next , to determine the characteristics that predicted clinically meaningful improvement in qol , participants pedsql change scores for 1 year were categorized as positive ( score increase 1 mcid ) , negative ( score decrease 1 mcid ) , or no change . binary logistic regression models were constructed to predict positive qol change of at least 1 mcid compared with negative / no qol change , controlling for all measured demographic and medical variables noted above , time from baseline to follow - up , and baseline pedsql scores . separate analyses were conducted for parent and child report of the generic core and diabetes module total scores . the study sample was composed of 5,004 children , adolescents , and young adults diagnosed with type 1 or type 2 diabetes in 2002 through 2005 ( n = 4,393 ) or whose diabetes was prevalent in 2001 ( youth were a mean age of 12.5 years ( sd , 4.7 ; range , 222 years ) , 51.3% female , 67.9% non - hispanic white , and 19.3% were publically insured ( medicaid / medicare ) . the demographic and clinical characteristics of participants in the study sample stratified by diabetes type are reported in table 1 . baseline demographic and clinical characteristics of 5,004 study participants , by diabetes type : search for diabetes in youth study , 20012005 the internal consistency of the pedsql total scores for the sample was excellent for the generic core ( youth report : = 0.87 , parent report : = 0.88 ) and diabetes module ( youth report : = 0.85 , parent report : = 0.88 ) . in most cases , the youth- and parent - reported total and subscale scores were consistently higher for type 1 diabetes than for type 2 diabetes , although the sds were quite large for both groups . mcids for the pedsql generic core and diabetes module at the baseline study visit , by reporter ( youth / parent ) and diabetes type : search for diabetes in youth study , 20012005 the pedsql was completed by 4,041 participant parent pairs for participants aged between 5 and 17 years , 551 participants aged 18 years or older , and 412 parents of participants aged younger than 5 years . the mcid for the generic core total score was 4.83 for youth - report and 5.16 for parent - report ( table 2 ) . for youth with type 1 diabetes , the generic core mcids were 4.72 for youth - report and 4.88 for parent - report . the generic core mcids were higher for those with type 2 diabetes compared the mcids for youth with type 1 diabetes ; mcids among youth with type 2 diabetes were 5.41 for youth - report and 6.27 for parent - report . the mcid for the diabetes module total score was 5.34 for youth - report and 4.64 for parent - report . the diabetes module mcids for participants with type 1 diabetes were 5.27 for youth - report and 4.54 for parent - report . as with the generic core mcids , the diabetes module mcids were higher among youth with type 2 diabetes : 5.96 for youth - report and 6.06 for parent - report . longitudinal analyses were conducted with data from 1,402 youth with at least one parent- or youth - reported score on the generic core or diabetes module at the 12-month follow - up visit ( n range 1,0721,984 across the two modules and two reporters ; table 3 ) . this subset comprises 55% of the 20022005 incident cohorts that were included in the baseline visit sample for this study . the 12-month follow - up visits occurred on average 14.6 months ( sd 3.1 [ range 6.028.1 ] ) after the initial visit . the youth in the longitudinal analysis were younger and had shorter disease duration ( due to the exclusion of the participants whose diabetes was prevalent in 2001 ) , and lower zbmi ( all p < 0.05 ) than youth in the larger cross - sectional sample used to calculate mcids at baseline . linear regression analysis of predictors of change in pedsql total scores * between the baseline and 12-month follow - up visit : search for diabetes in youth study , 20022005 incident cases in the linear regression models , improvements in youth - reported generic core and diabetes module pedsql change scores during 1 year were predicted by lower baseline qol ( = 0.50 and = 0.52 , respectively ; p < 0.0001 ) , male sex ( = 1.66 and = 1.93 , respectively ; p < 0.05 ) , and being privately insured ( = 2.25 and = 2.28 , respectively ; p < 0.01 ) . for parent - reported scores , lower baseline qol ( = 0.47 , p < 0.0001 ) , private insurance ( = 2.46 , p < 0.01 ) , diagnosis of type 1 diabetes ( = 4.36 , p < 0.01 ) , and lower baseline a1c ( = 0.98 , p < 0.001 ) predicted greater improvements in generic core change scores . lower baseline qol ( = 0.40 , p < 0.0001 ) , lower baseline a1c ( = 0.84 , p < 0.001 ) , and longer duration of diabetes ( = 0.12 , p < 0.01 ) predicted greater improvement in diabetes module change scores . across scales , reporters , and diabetes type , approximately one - third of the sample had pedsql change scores that decreased by 1 mcid , one - third had no significant qol change , and one - third had change scores that increased by 1 mcid . in the logistic regression models predicting a clinically meaningful improvement ( 1 mcid ) in pedsql change scores ( table 4 ) , baseline qol scores significantly predicted an improvement for both youth- and parent - reports on the generic core and diabetes module ( or range 0.920.94 , all p < 0.0001 ) . for youth - report , there were no additional predictors of clinically meaningful improvements on the generic core beyond baseline qol scores , whereas clinically meaningful improvements in diabetes module scores were also predicted by being privately insured ( or 0.68 [ 95% ci 0.480.98 ] , p < 0.05 ) . for parent - report , beyond baseline qol scores , clinically meaningful improvements on the generic core were also predicted by private insurance ( or 0.65 [ 0.450.94 ] , p < 0.05 ) , lower bmi ( 0.86 [ 0.741.00 ] , p < 0.05 ) , and lower a1c ( 0.85 [ 0.770.95 ] , p < 0.01 ) , whereas clinically meaningful improvements on the diabetes module were also predicted by lower a1c ( 0.87 [ 0.790.97 ] , p < 0.05 ) . logistic regression analysis to identify predictors of 1 mcid improvement on pedsql total score between the baseline and 12-month follow - up study visits : search for diabetes in youth study , 20022005 incident cases the study sample was composed of 5,004 children , adolescents , and young adults diagnosed with type 1 or type 2 diabetes in 2002 through 2005 ( n = 4,393 ) or whose diabetes was prevalent in 2001 ( youth were a mean age of 12.5 years ( sd , 4.7 ; range , 222 years ) , 51.3% female , 67.9% non - hispanic white , and 19.3% were publically insured ( medicaid / medicare ) . the demographic and clinical characteristics of participants in the study sample stratified by diabetes type are reported in table 1 . baseline demographic and clinical characteristics of 5,004 study participants , by diabetes type : search for diabetes in youth study , 20012005 the internal consistency of the pedsql total scores for the sample was excellent for the generic core ( youth report : = 0.87 , parent report : = 0.88 ) and diabetes module ( youth report : = 0.85 , parent report : = 0.88 ) . in most cases , the youth- and parent - reported total and subscale scores were consistently higher for type 1 diabetes than for type 2 diabetes , although the sds were quite large for both groups . mcids for the pedsql generic core and diabetes module at the baseline study visit , by reporter ( youth / parent ) and diabetes type : search for diabetes in youth study , 20012005 parent pairs for participants aged between 5 and 17 years , 551 participants aged 18 years or older , and 412 parents of participants aged younger than 5 years . the mcid for the generic core total score was 4.83 for youth - report and 5.16 for parent - report ( table 2 ) . for youth with type 1 diabetes , the generic core mcids were 4.72 for youth - report and 4.88 for parent - report . the generic core mcids were higher for those with type 2 diabetes compared the mcids for youth with type 1 diabetes ; mcids among youth with type 2 diabetes were 5.41 for youth - report and 6.27 for parent - report . the mcid for the diabetes module total score was 5.34 for youth - report and 4.64 for parent - report . the diabetes module mcids for participants with type 1 diabetes were 5.27 for youth - report and 4.54 for parent - report . as with the generic core mcids , the diabetes module mcids were higher among youth with type 2 diabetes : 5.96 for youth - report and 6.06 for parent - report . longitudinal analyses were conducted with data from 1,402 youth with at least one parent- or youth - reported score on the generic core or diabetes module at the 12-month follow - up visit ( n range 1,0721,984 across the two modules and two reporters ; table 3 ) . this subset comprises 55% of the 20022005 incident cohorts that were included in the baseline visit sample for this study . the 12-month follow - up visits occurred on average 14.6 months ( sd 3.1 [ range 6.028.1 ] ) after the initial visit . the youth in the longitudinal analysis were younger and had shorter disease duration ( due to the exclusion of the participants whose diabetes was prevalent in 2001 ) , and lower zbmi ( all p < 0.05 ) than youth in the larger cross - sectional sample used to calculate mcids at baseline . linear regression analysis of predictors of change in pedsql total scores * between the baseline and 12-month follow - up visit : search for diabetes in youth study , 20022005 incident cases in the linear regression models , improvements in youth - reported generic core and diabetes module pedsql change scores during 1 year were predicted by lower baseline qol ( = 0.50 and = 0.52 , respectively ; p < 0.0001 ) , male sex ( = 1.66 and = 1.93 , respectively ; p < 0.05 ) , and being privately insured ( = 2.25 and = 2.28 , respectively ; p < 0.01 ) . for parent - reported scores , lower baseline qol ( = 0.47 , p < 0.0001 ) , private insurance ( = 2.46 , p < 0.01 ) , diagnosis of type 1 diabetes ( = 4.36 , p < 0.01 ) , and lower baseline a1c ( = 0.98 , p < lower baseline qol ( = 0.40 , p < 0.0001 ) , lower baseline a1c ( = 0.84 , p < 0.001 ) , and longer duration of diabetes ( = 0.12 , p < 0.01 ) predicted greater improvement in diabetes module change scores . across scales , reporters , and diabetes type , approximately one - third of the sample had pedsql change scores that decreased by 1 mcid , one - third had no significant qol change , and one - third had change scores that increased by 1 mcid . in the logistic regression models predicting a clinically meaningful improvement ( 1 mcid ) in pedsql change scores ( table 4 ) , baseline qol scores significantly predicted an improvement for both youth- and parent - reports on the generic core and diabetes module ( or range 0.920.94 , all p < 0.0001 ) . for youth - report , there were no additional predictors of clinically meaningful improvements on the generic core beyond baseline qol scores , whereas clinically meaningful improvements in diabetes module scores were also predicted by being privately insured ( or 0.68 [ 95% ci 0.480.98 ] , p < 0.05 ) . for parent - report , beyond baseline qol scores , clinically meaningful improvements on the generic core were also predicted by private insurance ( or 0.65 [ 0.450.94 ] , p < 0.05 ) , lower bmi ( 0.86 [ 0.741.00 ] , p < 0.05 ) , and lower a1c ( 0.85 [ 0.770.95 ] , p < 0.01 ) , whereas clinically meaningful improvements on the diabetes module were also predicted by lower a1c ( 0.87 [ 0.790.97 ] , p < 0.05 ) . logistic regression analysis to identify predictors of 1 mcid improvement on pedsql total score between the baseline and 12-month follow - up study visits : the mcids for the pedsql generic core total score established in this study were consistent with published generic core total score mcids in other chronic conditions ( 4.36 for youth - report and 4.50 for parent - report ) ( 13 ) , ranging between 4 and 6 points across reporter and diabetes type . the values for most of the generic core subscales in the current sample of youth with type 1 and type 2 diabetes were higher than the pedsql generic core subscale mcids for youth with other chronic conditions , which are physical health , 6.66 , 6.92 ; emotional functioning , 8.94 , 7.79 ; social functioning , 8.36 , 8.98 ; and school functioning , 9.12 , 9.67 , for youth- and parent - report , respectively ( 13 ) . the only exception was the school functioning subscale , which was lower for both youth- and parent - reports . the higher mcids for most generic core subscales in this sample compared with other conditions suggests that across various domains of functioning , youth with diabetes seem to require more improvement or deterioration in qol to perceive a clinically meaningful difference in their subjective experience than do youth with other chronic conditions . given the complexity of the diabetes treatment regimen , a relatively large amount of change in qol ( 510 points ) may be needed for youth or their parents to perceive any meaningful differences in everyday functioning or activities . this pattern was especially pronounced in youth with type 2 diabetes , who had the highest mcid thresholds for clinically meaningful qol change . youth with type 2 diabetes report poorer qol and encounter multiple physical complaints and social and emotional difficulties ( 9,20 ) . in addition , given the higher zbmi scores among youth with type 2 diabetes compared with type 1 diabetes , there may be a cumulative effect of obesity and type 2 diabetes on some individuals qol ( 21 ) . as such , greater change in qol may be necessary for youth with type 2 diabetes to perceive an improvement or deterioration in their subjective well - being . for the diabetes module , the total score mcids were similar to the generic core total score mcids . although there is no direct point of comparison for the subscale mcids , it is notable that those of the diabetes module are substantially higher than the subscale mcids for the generic core . in particular , the treatment barriers , treatment adherence , and worries subscale mcids tended to be at or above 10 points . the higher mcids for these subscales reflect their lower cronbach s values . although these subscales have not been supported by factor analyses conducted on responses from youth with type 1 diabetes ( 7,10 ) , subscale mcids are reported here for comparison purposes and to illustrate mean score and similarities between parents of youth with type 1 and type 2 diabetes , which had not previously been reported by the search study . for clinicians and clinical trials researchers , fluctuations in qol that do not meet the mcid threshold may not be noticeable by patients or their families yet should be monitored to determine whether more substantial challenges arise and require intervention . the relatively higher mcids for youth with diabetes than for youth with other chronic conditions may translate to a need for interventions that explicitly target various components of general and diabetes - specific qol to achieve a change of sufficient magnitude for patient and parent perception of improvement . interventions to improve qol may benefit from a direct , targeted focus on barriers and facilitators of diabetes management and control . for example , given previous evidence of strong associations between lower qol and more depressive symptoms ( 10 ) , qol interventions that address diabetes - specific emotional concerns may be particularly beneficial . these data support published and ongoing intervention studies that demonstrate benefits of providing psychological support and skills training to youth with diabetes ( 12,23 ) . in addition , efforts to improve qol among youth with type 2 diabetes may achieve greater qol change by addressing diabetes and also weight - related domains of everyday functioning . associations between pedsql scores and a1c suggest that interventions that address and improve qol may ultimately translate to clinical improvements in glycemic control . one hypothesis is that patients who perceive better qol may encounter fewer barriers to diabetes management and may be more motivated to adhere to diabetes treatment recommendations such as self - monitoring . adaptation of established psychological interventions for youth with type 1 diabetes to meet the unique needs of youth with type 2 diabetes is needed ( 23 ) . longitudinal findings demonstrate that a number of demographic and clinical variables predicted increases in pedsql change scores over 1 year . the association between lower pedsql scores at baseline and larger change scores at 12 months likely reflects that participants with poorer initial qol had more opportunity for improvement . the associations of sex , insurance coverage , diabetes type , diabetes duration , and a1c at baseline with 12-month qol improvements are consistent with previous studies examining changes in qol after clinical or medical intervention ( 24,25 ) and help to identify patients who may experience qol fluctuations over time . given different predictors of parent- and youth - reported qol change , interventions will need to be tailored to individuals unique experiences to influence each of their perceptions of qol . because parent - reported change scores were predicted by bmi and a1c , efforts to improve physical well - being and glycemic control may be particularly influential on parental perceptions of their children s qol . however , the finding that children s qol change scores were predicted by sex and insurance status suggests that girls and those with public insurance may be most likely to benefit from additional qol monitoring . through this monitoring , it will be necessary to engage youth in discussion to identify and address the factors that are affecting their qol ratings . the identification of pedsql mcids also allowed us to determine the characteristics that predict who is most likely to experience clinically meaningful improvements in qol . private insurance coverage , lower bmi , and lower a1c at the baseline study visit predicted change that exceeded the threshold of perceived improvement in various qol domains . beyond the auto - correlational effects of baseline qol , these variables may represent protective factors , such as greater access to resources or better health status , that help individuals rebound from periods of lower qol and avoid long - lasting problems that may ultimately affect diabetes management and control . the interpretation of this study s findings should consider the response rates and limitations of the pedsql modules and their analyses . as previously reported , the baseline visit response rate was lower than expected ; older youth , african american youth , and those with type 2 diabetes were less likely to complete a visit ( 26 ) . the pedsql diabetes module was not developed or normed for youth with type 2 diabetes ( 3 ) , so the results of the pedsql diabetes module for youth with type 2 diabetes and their parents may have limited utility . concerns about psychometric properties of the diabetes module subscales among youth with type 1 diabetes have been reported ( 7,10 ) suggesting that use of total scores , and the associated mcids , is more appropriate than subscale scores . in addition , mcids were calculated for all pedsql subscales for consistency , but the lower values for some of these subscales resulted in larger mcids , which should be used with caution . finally , although we used the same approach to calculate mcids as used with the generic core for youth with other chronic conditions ( 13,18,19 ) and evidence demonstrates similarities between mcids established using statistical methods ( e.g. , 1 sem ) and those calculated in other ways ( 17 ) , alternative mcid calculations might have yielded different results . because concurrent measures of qol were not collected , alternative mcid calculations could not be conducted . nonetheless , the establishment of diabetes - specific mcids for the pedsql generic core and diabetes module for parents and youth provide an important reference point for clinicians and researchers interested in the effect of treatments on patient qol . clinical trials researchers may use these mcids to evaluate the degree to which an intervention improves participants qol in a clinically significant way . longitudinal research is needed to determine whether clinically meaningful changes in perceived qol are associated with clinically meaningful changes in indices of diabetes health status , such as glycemic control and risk for acute complications .

objectiveto establish minimal clinically important difference ( mcid ) scores representing the smallest detectable change in quality of life ( qol ) , using the pediatric quality of life inventory ( pedsql ) generic core and diabetes module among youth with diabetes and their parents , and to identify demographic and clinical correlates of qol change over 1 year.research design and methodsparticipants in the search for diabetes in youth study aged > 5 years and parents of youth aged < 18 years completed pedsql surveys at their initial and 12-month study visits . mcids for each pedsql module were calculated using one standard error of measurement . demographic and clinical characteristics associated with qol change were identified through multiple linear and logistic regression analyses.resultsthe sample comprised 5,004 youth ( mean age , 12.5 4.7 years ; mean diabetes duration , 3.4 3.7 years ) . of 100 possible points , pedsql total score mcids for youth with type 1 and type 2 diabetes , respectively , were generic core , 4.88 , 6.27 ( parent ) and 4.72 , 5.41 ( youth ) ; diabetes module , 4.54 , 6.06 ( parent ) and 5.27 , 5.96 ( youth ) . among 1,402 youth with a follow - up visit , lower baseline qol , male sex , private insurance , having type 1 diabetes , longer diabetes duration , and better glycemic control predicted improvements in youth- and parent - reported pedsql total scores over 1 year . clinically meaningful ( 1 mcid ) improvements in total score for at least one pedsql module were predicted by private insurance , lower bmi , and lower a1c at baseline.conclusionsthese diabetes - specific reference points to interpret clinically meaningful change in pedsql scores can be used in clinical care and research for youth with type 1 and type 2 diabetes .

RESEARCH DESIGN AND METHODS Data analysis RESULTS Characteristics of participants MCIDs Predictors of change in QOL scores during 1 year CONCLUSIONS

search for diabetes in youth ( search ) is a multicenter observational study of youth diagnosed with diabetes before age 20 years . of the 20022005 incident cohorts who completed the baseline and 12-month follow - up visits ( n = 1,449 , 57% of incident sample with baseline visits ) , 47 ( 3.2% ) without at least one pedsql module were also excluded , resulting in a final sample of 1,402 participants to identify predictors of qol change over time . youth and their parents reported on youths generic and health - related qol using the pedsql generic core and diabetes module ( 3 ) . thus , participants aged 18 years and older had one score for each module ( self - report ) , participants aged between 5 and 17 years received two scores for each module ( youth and parent report ) , and participants aged younger than 5 years had one score for each module ( parent report only ) . for all versions , items assess the degree of difficulty youth experience with different aspects of everyday living , including physical symptoms and emotional , social , and academic functioning ( generic core ) , as well as treatment adherence and barriers , diabetes - related worries , and communication with others about diabetes ( diabetes module ) . the mean ( sd ) and scale reliability ( cronbach ) values were calculated for each subscale and the total scores for the generic core and the diabetes module . mcids were calculated using a commonly used distribution - based method : one standard error of measurement ( sem ) ( 12,16 ) . the sem and resulting mcids were calculated for the sample of participants and parents for the total and subscale scores of the generic core and diabetes module and were also established separately for each diabetes type . thus , the mcid represents a clinically meaningful or important difference in qol that can be referenced in clinical practice and research . to examine predictors of change in qol , two sets of regression analyses were conducted using data from the subset of participants diagnosed from 20022005 with a 12-month follow - up visit and complete data at both time points ( total n = 1,402 [ n range 1,0721,094 ] across the two modules and two reporters ) . predictors were demographic and clinical variables that have previously demonstrated associations with qol , including age , sex , insurance coverage , race / ethnicity , diabetes type , diabetes duration , z - bmi , and a1c , all of which were assessed at the baseline visit , and models controlled for pedsql scores at baseline . next , to determine the characteristics that predicted clinically meaningful improvement in qol , participants pedsql change scores for 1 year were categorized as positive ( score increase 1 mcid ) , negative ( score decrease 1 mcid ) , or no change . binary logistic regression models were constructed to predict positive qol change of at least 1 mcid compared with negative / no qol change , controlling for all measured demographic and medical variables noted above , time from baseline to follow - up , and baseline pedsql scores . the mean ( sd ) and scale reliability ( cronbach ) values were calculated for each subscale and the total scores for the generic core and the diabetes module . mcids were calculated using a commonly used distribution - based method : one standard error of measurement ( sem ) ( 12,16 ) . the sem and resulting mcids were calculated for the sample of participants and parents for the total and subscale scores of the generic core and diabetes module and were also established separately for each diabetes type . to examine predictors of change in qol , two sets of regression analyses were conducted using data from the subset of participants diagnosed from 20022005 with a 12-month follow - up visit and complete data at both time points ( total n = 1,402 [ n range 1,0721,094 ] across the two modules and two reporters ) . pedsql change scores were calculated as total score at the follow - up visit minus total score at the baseline visit . predictors were demographic and clinical variables that have previously demonstrated associations with qol , including age , sex , insurance coverage , race / ethnicity , diabetes type , diabetes duration , z - bmi , and a1c , all of which were assessed at the baseline visit , and models controlled for pedsql scores at baseline . next , to determine the characteristics that predicted clinically meaningful improvement in qol , participants pedsql change scores for 1 year were categorized as positive ( score increase 1 mcid ) , negative ( score decrease 1 mcid ) , or no change . binary logistic regression models were constructed to predict positive qol change of at least 1 mcid compared with negative / no qol change , controlling for all measured demographic and medical variables noted above , time from baseline to follow - up , and baseline pedsql scores . the study sample was composed of 5,004 children , adolescents , and young adults diagnosed with type 1 or type 2 diabetes in 2002 through 2005 ( n = 4,393 ) or whose diabetes was prevalent in 2001 ( youth were a mean age of 12.5 years ( sd , 4.7 ; range , 222 years ) , 51.3% female , 67.9% non - hispanic white , and 19.3% were publically insured ( medicaid / medicare ) . baseline demographic and clinical characteristics of 5,004 study participants , by diabetes type : search for diabetes in youth study , 20012005 the internal consistency of the pedsql total scores for the sample was excellent for the generic core ( youth report : = 0.87 , parent report : = 0.88 ) and diabetes module ( youth report : = 0.85 , parent report : = 0.88 ) . in most cases , the youth- and parent - reported total and subscale scores were consistently higher for type 1 diabetes than for type 2 diabetes , although the sds were quite large for both groups . mcids for the pedsql generic core and diabetes module at the baseline study visit , by reporter ( youth / parent ) and diabetes type : search for diabetes in youth study , 20012005 the pedsql was completed by 4,041 participant parent pairs for participants aged between 5 and 17 years , 551 participants aged 18 years or older , and 412 parents of participants aged younger than 5 years . the mcid for the generic core total score was 4.83 for youth - report and 5.16 for parent - report ( table 2 ) . for youth with type 1 diabetes , the generic core mcids were 4.72 for youth - report and 4.88 for parent - report . the generic core mcids were higher for those with type 2 diabetes compared the mcids for youth with type 1 diabetes ; mcids among youth with type 2 diabetes were 5.41 for youth - report and 6.27 for parent - report . the mcid for the diabetes module total score was 5.34 for youth - report and 4.64 for parent - report . the diabetes module mcids for participants with type 1 diabetes were 5.27 for youth - report and 4.54 for parent - report . as with the generic core mcids , the diabetes module mcids were higher among youth with type 2 diabetes : 5.96 for youth - report and 6.06 for parent - report . longitudinal analyses were conducted with data from 1,402 youth with at least one parent- or youth - reported score on the generic core or diabetes module at the 12-month follow - up visit ( n range 1,0721,984 across the two modules and two reporters ; table 3 ) . the youth in the longitudinal analysis were younger and had shorter disease duration ( due to the exclusion of the participants whose diabetes was prevalent in 2001 ) , and lower zbmi ( all p < 0.05 ) than youth in the larger cross - sectional sample used to calculate mcids at baseline . linear regression analysis of predictors of change in pedsql total scores * between the baseline and 12-month follow - up visit : search for diabetes in youth study , 20022005 incident cases in the linear regression models , improvements in youth - reported generic core and diabetes module pedsql change scores during 1 year were predicted by lower baseline qol ( = 0.50 and = 0.52 , respectively ; p < 0.0001 ) , male sex ( = 1.66 and = 1.93 , respectively ; p < 0.05 ) , and being privately insured ( = 2.25 and = 2.28 , respectively ; p < 0.01 ) . for parent - reported scores , lower baseline qol ( = 0.47 , p < 0.0001 ) , private insurance ( = 2.46 , p < 0.01 ) , diagnosis of type 1 diabetes ( = 4.36 , p < 0.01 ) , and lower baseline a1c ( = 0.98 , p < 0.001 ) predicted greater improvements in generic core change scores . lower baseline qol ( = 0.40 , p < 0.0001 ) , lower baseline a1c ( = 0.84 , p < 0.001 ) , and longer duration of diabetes ( = 0.12 , p < 0.01 ) predicted greater improvement in diabetes module change scores . in the logistic regression models predicting a clinically meaningful improvement ( 1 mcid ) in pedsql change scores ( table 4 ) , baseline qol scores significantly predicted an improvement for both youth- and parent - reports on the generic core and diabetes module ( or range 0.920.94 , all p < 0.0001 ) . for youth - report , there were no additional predictors of clinically meaningful improvements on the generic core beyond baseline qol scores , whereas clinically meaningful improvements in diabetes module scores were also predicted by being privately insured ( or 0.68 [ 95% ci 0.480.98 ] , p < 0.05 ) . for parent - report , beyond baseline qol scores , clinically meaningful improvements on the generic core were also predicted by private insurance ( or 0.65 [ 0.450.94 ] , p < 0.05 ) , lower bmi ( 0.86 [ 0.741.00 ] , p < 0.05 ) , and lower a1c ( 0.85 [ 0.770.95 ] , p < 0.01 ) , whereas clinically meaningful improvements on the diabetes module were also predicted by lower a1c ( 0.87 [ 0.790.97 ] , p < 0.05 ) . logistic regression analysis to identify predictors of 1 mcid improvement on pedsql total score between the baseline and 12-month follow - up study visits : search for diabetes in youth study , 20022005 incident cases the study sample was composed of 5,004 children , adolescents , and young adults diagnosed with type 1 or type 2 diabetes in 2002 through 2005 ( n = 4,393 ) or whose diabetes was prevalent in 2001 ( youth were a mean age of 12.5 years ( sd , 4.7 ; range , 222 years ) , 51.3% female , 67.9% non - hispanic white , and 19.3% were publically insured ( medicaid / medicare ) . baseline demographic and clinical characteristics of 5,004 study participants , by diabetes type : search for diabetes in youth study , 20012005 the internal consistency of the pedsql total scores for the sample was excellent for the generic core ( youth report : = 0.87 , parent report : = 0.88 ) and diabetes module ( youth report : = 0.85 , parent report : = 0.88 ) . in most cases , the youth- and parent - reported total and subscale scores were consistently higher for type 1 diabetes than for type 2 diabetes , although the sds were quite large for both groups . mcids for the pedsql generic core and diabetes module at the baseline study visit , by reporter ( youth / parent ) and diabetes type : search for diabetes in youth study , 20012005 parent pairs for participants aged between 5 and 17 years , 551 participants aged 18 years or older , and 412 parents of participants aged younger than 5 years . for youth with type 1 diabetes , the generic core mcids were 4.72 for youth - report and 4.88 for parent - report . the generic core mcids were higher for those with type 2 diabetes compared the mcids for youth with type 1 diabetes ; mcids among youth with type 2 diabetes were 5.41 for youth - report and 6.27 for parent - report . the mcid for the diabetes module total score was 5.34 for youth - report and 4.64 for parent - report . the diabetes module mcids for participants with type 1 diabetes were 5.27 for youth - report and 4.54 for parent - report . as with the generic core mcids , the diabetes module mcids were higher among youth with type 2 diabetes : 5.96 for youth - report and 6.06 for parent - report . longitudinal analyses were conducted with data from 1,402 youth with at least one parent- or youth - reported score on the generic core or diabetes module at the 12-month follow - up visit ( n range 1,0721,984 across the two modules and two reporters ; table 3 ) . linear regression analysis of predictors of change in pedsql total scores * between the baseline and 12-month follow - up visit : search for diabetes in youth study , 20022005 incident cases in the linear regression models , improvements in youth - reported generic core and diabetes module pedsql change scores during 1 year were predicted by lower baseline qol ( = 0.50 and = 0.52 , respectively ; p < 0.0001 ) , male sex ( = 1.66 and = 1.93 , respectively ; p < 0.05 ) , and being privately insured ( = 2.25 and = 2.28 , respectively ; p < 0.01 ) . for parent - reported scores , lower baseline qol ( = 0.47 , p < 0.0001 ) , private insurance ( = 2.46 , p < 0.01 ) , diagnosis of type 1 diabetes ( = 4.36 , p < 0.01 ) , and lower baseline a1c ( = 0.98 , p < lower baseline qol ( = 0.40 , p < 0.0001 ) , lower baseline a1c ( = 0.84 , p < 0.001 ) , and longer duration of diabetes ( = 0.12 , p < 0.01 ) predicted greater improvement in diabetes module change scores . in the logistic regression models predicting a clinically meaningful improvement ( 1 mcid ) in pedsql change scores ( table 4 ) , baseline qol scores significantly predicted an improvement for both youth- and parent - reports on the generic core and diabetes module ( or range 0.920.94 , all p < 0.0001 ) . for youth - report , there were no additional predictors of clinically meaningful improvements on the generic core beyond baseline qol scores , whereas clinically meaningful improvements in diabetes module scores were also predicted by being privately insured ( or 0.68 [ 95% ci 0.480.98 ] , p < 0.05 ) . for parent - report , beyond baseline qol scores , clinically meaningful improvements on the generic core were also predicted by private insurance ( or 0.65 [ 0.450.94 ] , p < 0.05 ) , lower bmi ( 0.86 [ 0.741.00 ] , p < 0.05 ) , and lower a1c ( 0.85 [ 0.770.95 ] , p < 0.01 ) , whereas clinically meaningful improvements on the diabetes module were also predicted by lower a1c ( 0.87 [ 0.790.97 ] , p < 0.05 ) . logistic regression analysis to identify predictors of 1 mcid improvement on pedsql total score between the baseline and 12-month follow - up study visits : the mcids for the pedsql generic core total score established in this study were consistent with published generic core total score mcids in other chronic conditions ( 4.36 for youth - report and 4.50 for parent - report ) ( 13 ) , ranging between 4 and 6 points across reporter and diabetes type . the values for most of the generic core subscales in the current sample of youth with type 1 and type 2 diabetes were higher than the pedsql generic core subscale mcids for youth with other chronic conditions , which are physical health , 6.66 , 6.92 ; emotional functioning , 8.94 , 7.79 ; social functioning , 8.36 , 8.98 ; and school functioning , 9.12 , 9.67 , for youth- and parent - report , respectively ( 13 ) . the higher mcids for most generic core subscales in this sample compared with other conditions suggests that across various domains of functioning , youth with diabetes seem to require more improvement or deterioration in qol to perceive a clinically meaningful difference in their subjective experience than do youth with other chronic conditions . this pattern was especially pronounced in youth with type 2 diabetes , who had the highest mcid thresholds for clinically meaningful qol change . in addition , given the higher zbmi scores among youth with type 2 diabetes compared with type 1 diabetes , there may be a cumulative effect of obesity and type 2 diabetes on some individuals qol ( 21 ) . as such , greater change in qol may be necessary for youth with type 2 diabetes to perceive an improvement or deterioration in their subjective well - being . for the diabetes module , the total score mcids were similar to the generic core total score mcids . although these subscales have not been supported by factor analyses conducted on responses from youth with type 1 diabetes ( 7,10 ) , subscale mcids are reported here for comparison purposes and to illustrate mean score and similarities between parents of youth with type 1 and type 2 diabetes , which had not previously been reported by the search study . the relatively higher mcids for youth with diabetes than for youth with other chronic conditions may translate to a need for interventions that explicitly target various components of general and diabetes - specific qol to achieve a change of sufficient magnitude for patient and parent perception of improvement . in addition , efforts to improve qol among youth with type 2 diabetes may achieve greater qol change by addressing diabetes and also weight - related domains of everyday functioning . adaptation of established psychological interventions for youth with type 1 diabetes to meet the unique needs of youth with type 2 diabetes is needed ( 23 ) . longitudinal findings demonstrate that a number of demographic and clinical variables predicted increases in pedsql change scores over 1 year . the associations of sex , insurance coverage , diabetes type , diabetes duration , and a1c at baseline with 12-month qol improvements are consistent with previous studies examining changes in qol after clinical or medical intervention ( 24,25 ) and help to identify patients who may experience qol fluctuations over time . because parent - reported change scores were predicted by bmi and a1c , efforts to improve physical well - being and glycemic control may be particularly influential on parental perceptions of their children s qol . private insurance coverage , lower bmi , and lower a1c at the baseline study visit predicted change that exceeded the threshold of perceived improvement in various qol domains . the pedsql diabetes module was not developed or normed for youth with type 2 diabetes ( 3 ) , so the results of the pedsql diabetes module for youth with type 2 diabetes and their parents may have limited utility . concerns about psychometric properties of the diabetes module subscales among youth with type 1 diabetes have been reported ( 7,10 ) suggesting that use of total scores , and the associated mcids , is more appropriate than subscale scores . nonetheless , the establishment of diabetes - specific mcids for the pedsql generic core and diabetes module for parents and youth provide an important reference point for clinicians and researchers interested in the effect of treatments on patient qol .

thirty - five percent of americans who are aged 20 years and older have type 2 diabetes ( t2d ) . the etiology behind such impressive statistics is complex but can be attributed in part to recent changes in the lifestyle choices of americans , including changes in habitual diet . the what we eat in america ( wweia ) report , based on data obtained from the national health and nutrition examination survey ( nhanes ) , demonstrated that grains ( mainly refined ) have contributed to the bulk of the rise in caloric intake in the us in the last 60 years . increased consumption of added sugars , processed grains , and saturated fat contributed to excess weight gain , which is an influential risk factor in the development of t2d [ 3 , 4 ] . in fact , results from implementation of the diabetes prevention program ( dpp ) illuminated that up to 58% of diabetes can be prevented through lifestyle changes alone , including changes in diet . one underlying pathophysiological process resulting from poor lifestyle habits is inflammation . inflammation is present prior to the development of t2d and cardiovascular disease ( cvd ) , contributing to evidence to support the common soil hypothesis , which is a reference to the common risk factors for the development of these two diseases . cvd is one of the major complications and causes of death in persons with t2d , supporting this hypothesis . the multi - ethnic study of atherosclerosis ( mesa ) showed that higher levels of the inflammatory markers , c - reactive protein ( crp ) and interleukin ( il)-6 , were associated with an increased risk of developing t2d . chronic inflammation in t2d initiates with states of obesity , hyperglycemia , insulin resistance , and the overexpression of proinflammatory proteins like crp and cytokines ( il-1 , il-6 , and tumor necrosis factor - alpha ( tnf- ) ) . these proinflammatory molecules activate the cells of innate immunity , which cause damage to tissues in the vasculature , adipose tissue , and pancreas . acute responses through this proinflammatory pathway are needed for tissue damage repair and can improve function in tissues such as the pancreas , for example ; however , chronic expression can lead to pathological changes which result in disease . these changes clinically manifest as cvd , nephropathy , and symptomatic t2d , as the innate immune system attempts to repair the damaged tissue . moreover , inflammation is stimulated in times of stress , which can be induced by environmental , behavioral , individual , and psychosocial factors and diet - induced hyperglycemia and hypertriglyceridemia . hyperglycemia and hypertriglyceridemia are significant stressors that have also been shown to cause chronic inflammation and contribute to the pathogenesis of t2d . although the published literature is populated with articles and systematic reviews demonstrating the relationship between dietary patterns , individual dietary factors , and incidence of diabetes [ 11 , 12 ] , the primary literature linking inflammation to diet and t2d is minimal . one of the few reported areas is with the mediterranean - style diet , which has been associated with lower inflammatory markers and decreased incidence of t2d [ 12 , 13 ] . although several reviews have investigated the effects of diet for t2d control , few disclosed the association with underlying inflammatory responses . therefore , the aim of this study was to present an integrative review of the published literature on the associations between dietary intake and markers of chronic inflammation in patients with t2d . this review and synthesis of the relevant literature seeks to answer the following research question : what is the current state of the science regarding the association between diet and inflammation in established t2d ? all studies that included a dietary intervention , either exclusively or as part of an intervention , were included . eligibility criteria included diet , inflammatory markers , and t2d evaluated in randomized clinical trial ( rct ) , quasiexperimental , or cross - sectional studies . the following databases were searched : pubmed , cinahl , scopus , and web of science . chronic inflammation , type 2 diabetes , c - reactive protein , interleukins , and tumor necrosis factor ( tnf ) . the search was conducted on articles published between january 2000 ( the year of the first appearance of diet , inflammation , and t2d in the literature ) and may 2012 . studies that focused on prevention of t2d were excluded , as they did not offer data to assist in the exploration of how diet relates to the inflammatory process within the pathogenesis of t2d . other exclusion criteria were studies that focused on supplements alone or exercise without an evaluation of , or comparison to , diet . the search produced 371 studies in pubmed , 265 studies in web of science , and 150 studies in cinahl ( see figure 1 ) . two were eliminated because they included predominantly healthy subjects in a cross - sectional study ; one was eliminated because it included exercise only , one was eliminated due to its focus on prevention of t2d , and two were eliminated because they focused on acute responses to dietary interventions . three additional studies were identified by hand - searching reference lists of the selected articles . a final total of 16 papers were included for critical analysis with synthesis of findings . two studies were cross - sectional [ 14 , 15 ] , two were experimental [ 16 , 17 ] , and twelve were quasiexperimental , as follows : three were crossover experimental designs [ 1820 ] , three were pretest - posttest designs with only one group [ 2123 ] , and six were pretest - posttest designs with two or more comparison groups [ 2429 ] . more than half of the studies were international with one from the middle east , one from canada , three from australia , and six from europe . all subjects in the cross - sectional studies had a diagnosis of t2d , while several experimental studies utilized healthy and/or obese controls . acceptable measures for inflammation included serum evaluations of crp , il-6 , tnf , or a combination of one or more of these with other markers of inflammation . table 1 is an overview of information for broad topics created to aid in abstracting those aspects that were definitive in interpreting the implications of the findings . from this table , it became clear that each study reported inflammatory markers at different time points ( e.g. , some studies did not report baseline values ) , which was informative for the data reduction process . quality assessment ratings were established , and each article was scored based on these criteria ( table 2 ) . tables 3 and 4 contain the results of data abstraction and synthesis , respectively . quality assessment scores were assigned based on 11 questions designed to capture the essence of the study design , methods , and analyses ( table 2 ) . the quality score for each question a score of 0 represented absence of the criteria in question , and a score of 1 designated fulfillment of each criterion . these categories included aims , methods ( 3 questions ) , diet ( 3 questions ) , inflammatory measures , and analysis ( 2 questions ) . there was also one question related to results and conclusions with a rating of 02 , with 2 being of highest quality , which was weighted more heavily due to its importance in interpretation of the study findings . studies were first grouped into experimental ( n = 14 ) and correlational ( n = 2 ) . studies were then further delineated by diet categories of association with inflammation and those with a focus on diabetes control ( glycemic control ) . several experimental studies demonstrated a reduction of systemic inflammation following an intervention of a prescribed diet in patients with t2d . four of the studies with higher quality scores ( 9 ) demonstrated a significant association between diet and inflammation . three of these four contained a low - fat ( 30% of energy from fat ) dietary component [ 18 , 25 , 26 ] . implemented the dietary approaches to stop hypertension ( dash ) diet in an eight - week crossover intervention study . the dash diet is characterized by a high intake of whole grains , fruits , and vegetables , while limiting sodium intake ( < 2400 mg / day ) . after controlling for weight reduction , there was a statistically significant decrease in mean crp in the intervention group compared to the control group ( p = .03 ) . is almost identical in macronutrient composition to the dash diet ( see table 3 ) . after 64 weeks of following this diet with minimal dietary counseling , participants demonstrated a drop in crp levels , that is comparable to that which was found in the azadbakht et al . the control group in this study received a high - protein , low - fat diet , which also caused a decrease in crp , and there was not a significant difference in the mean change in crp between groups ( p = .61 ) . similarly , davis et al . demonstrated that after following an intervention of either a low - fat or low - carbohydrate diet for 24 weeks , crp was only found to be significantly reduced in the low - fat diet arm ( p = .01 ) and not in the low - carbohydrate arm ( p = .94 ) . . showed that after 52 weeks of following a low - glycemic - index , low - fat diet ( ~30% of energy intake from fat ) , crp was reduced from baseline by 29% compared to a high - glycemic - index , low - carbohydrate diet that was also high in monounsaturated fat ( mufa ) ( p < .05 ) . also examined the interaction of diet and time but found no significant effect of time or diet on crp levels ( see table 1 ) . of these four studies , only davis et al . did not report adherence to the prescribed diet . several other studies also demonstrated reductions in crp with dietary interventions , albeit with lower quality scores , and with ranges between 4 and 8 . used a crossover design and randomly assigned participants into one of two groups for 8 weeks . one group began on a low - calorie diet and the other a high - protein low - fat diet for eight weeks , then both groups continued on the high - protein low - fat diet for 44 weeks ( see table 1 ) . mean - crp levels were reduced significantly only in the high - protein , low - fat diet at 52 weeks ( p < .01 ) . although diet diaries were collected throughout the study period , adherence to the diet was not reported . bozzetto et al . conducted a randomized crossover trial with only 12 participants , but the analysis revealed a decreasing mean - crp level with fasting , 3-hour postprandial , and 6-hour postprandial following 4 weeks of a diet high in mufas , compared to the alternate diet which was high in carbohydrates and fiber with a low glycemic index ( p < .05 ) . a study by giannopoulou et al . also demonstrated the anti - inflammatory effects of a diet high in mufas ( see table 1 ) . this study was 14 weeks long , and comparison groups included an exercise - only group and a group that was prescribed exercise as well as the high mufa diet . all three groups demonstrated a significant reduction in mean - crp at final data collection ( p < .01 ) ; however , there was not a significant difference in this reduction between groups . it is interesting to note here that the diet and exercise group did not have a greater decrease in inflammatory markers than the diet - only group . these results indicate that exercise may not add benefit to improving inflammatory markers in patients with t2d . in another longitudinal study , barnard et al . randomized 99 participants to receive either a low - fat vegan diet or a conventional american diabetes association ( ada ) diet for 74 weeks . results showed a significant reduction of crp within groups ( p < .01 for each group ) but a nonsignificant difference between the two diet groups , perhaps due to the similar macronutrient content of the two dietary interventions . following on the premise that increased fat mass in t2d contributes to inflammation , three other studies implemented diets that were low or very low in energy content [ 2123 ] . kozowska et al . implemented a diet with a 20% energy deficit and low protein for eight weeks without a comparison group . this diet did not produce a significant reduction in crp ; however , the 17 participants did exhibit significantly lower tnf- levels ( p = .002 ) following the intervention . additional two studies implemented two weeks of a very - low - calorie diet ( vlcd ) with obese subjects with t2d in highly controlled hospital environments [ 21 , 23 ] . . demonstrated a significant drop in mean - crp after two weeks of following a vlcd diet ( p < .05 ) . . found that 2 weeks of a vlcd significantly decreased mean - crp ( p < .05 ) , as well as decreased il-6 levels ( p < .05 ) . although significant results were found , such diets can not be maintained , and as such the long - term effects are not known . conducted an rct in italy with two groups that were advised to follow a mediterranean - style diet . the mediterranean diet is characterized by three servings of fruits and six servings of vegetables or wild leafy greens per day . fish is consumed 5 to 6 times per week and contributes to the main source of fat , which is monounsaturated and polyunsaturated . the intervention group consumed 4 oz . of red wine per day , while the control group abstained from alcohol . results revealed significantly greater reductions in crp ( p < .01 ) , tnf- ( p < .01 ) , and il-6 ( p < .01 ) in the intervention group compared to the control group after the 52-week study period . in contrast , there were several diets that did not alter inflammatory marker expression significantly . these included the high - carbohydrate / high - fiber / low - glycemic index diet , the low - carbohydrate diet implemented by davis et al . , the low - carbohydrate and dpp - style diet implemented by vetter et al . , and the mediterranean diet . the two cross - sectional studies included in this analysis examined associations between two different aspects of diet and differed significantly in terms of quality in reporting . qi et al . demonstrated a significant association between crp and the dietary intake of interest , showing that with increasing quintiles of cereal fiber intake , crp was significantly lower ( p for trend = .03 ) . this inverse relationship was also present between tnf- and cereal fiber intake ( p for trend = .01 ) . . also demonstrated a significant inverse correlation between alpha ( ) and beta ( ) carotenoids ( foods that contain vitamin a ) and inflammation ( r = .41 and r = .36 , resp . ) . the positive influence of a dietary intervention on glycemic control [ 16 , 20 , 21 , 24 , 27 ] was examined in five studies . measured fasting blood glucose ( fbg ) as a proxy for glycemic control and demonstrated a significant improvement in fbg following the two - week dietary intervention of a very - low - calorie diet ( p < .05 ) . itsiopoulos et al . also demonstrated an improvement in glycemic control following a 24-week crossover intervention with a cretan mediterranean diet as evidenced by a significantly reduced mean glycosylated hemoglobin ( hba1c ) ( p = .012 ) , compared to the control diet which was the participants ' normal eating pattern . both studies also demonstrated improvement in the homeostatic model assessment ( homa ) score , a measure of insulin resistance ( see table 1 ) , although this change was not statistically significant following the mediterranean diet [ 20 , 21 ] . a third study by barnard et al . found that after following a vegan diet for 72 weeks , subjects had significantly improved hba1c levels compared to those who followed a conventional diet as recommended by the ada ( p = .03 ) ( see table 3 ) . a mediterranean diet was also implemented by marfella et al . hba1c levels were not significantly different between the two groups ; however , the mean change in hba1c was greater than in all other studies included in this review ( 1.1% for the intervention group and 1.2% for the control group ) . finally , giannopoulou et al . reported that although none of the intervention groups demonstrated a significant improvement in hba1c , both the diet - only and diet and exercise groups had a significant reduction in fasting glucose levels ( p < .05 for both groups ) . interestingly , only the two interventions with exercise produced a significant reduction in insulin resistance . the great variability between studies decreases the ability to draw definitive conclusions about associations between diet , inflammation , and t2d ; however , particular themes emerged that are worth noting both from this review and in light of previous research . first , of the ten different diets prescribed that were low in fat , both within and across studies , eight of those diets led to significant reductions in inflammatory markers . these included the dash diet , the low - fat dpp - style diet , the low - energy low - protein diet , the vegan and ada diets , the high - protein low - fat diet , and both the high - protein and low - protein diets . however , this review also supported the hypothesis that the high - mufa diet decreased inflammation . previous reports from the whitehall and predimed studies indicated that diets high in mufa and polyunsaturated fatty acids ( pufas ) were correlated with decreased crp [ 32 , 33 ] and il-6 , while diets high in saturated fatty acids were positively correlated with crp . high intake of pufa was also inversely correlated with incidence of t2d in the nurse 's health study . therefore , it was not surprising that the high - mufa diet showed anti - inflammatory effects in the diabetic population [ 16 , 19 , 20 , 29 ] . lacked baseline values for participants ; therefore , it is not appropriate to infer causality for a change in crp over the 4 weeks of the study period , but rather only in the acute postprandial response . unfortunately , glycemic control was not concurrently assessed . dietary patterns , as opposed to specific aspects of diet , have also been shown to correlate with levels of chronic inflammatory markers , but this concept was not adequately examined in the selected studies . for example , the western dietary pattern , characterized by a high consumption of sugary drinks , red meat , and poultry and a low consumption of fruits , vegetables , and fiber , was correlated with higher levels of crp in two large cross - sectional studies conducted in the us [ 34 , 35 ] . the impact of diet on the metabolic milieu of t2d is evident from the results of this integrative review and others . for example , of the two studies that were able to demonstrate a significantly reduced hba1c following the prescribed diet , one failed to report baseline values of glycemic control , and the other only demonstrated a mean reduction of 0.4% . while this result is statistically significant , it might not result in improvement in long - term outcomes of t2d , as demonstrated by findings from the united kingdom prospective diabetes study , which showed that a 1% reduction in hba1c levels can lead to improvements in long - term outcomes and decreased mortality . the implementation of a mediterranean - style diet and a glass of red wine per day produced a mean decrease in hba1c of 1.1% ( 0.06 ) . the control group that did not receive the red wine intervention but did follow a mediterranean diet had a similar decrease in hba1c , as well as crp , tnf- , and il-6 . the reductions in hba1c were not found to be significantly different between intervention and control groups , though the drop in this important glycemic marker is clinically relevant , as it points to decreased incidence of diabetes - related complications and decreased mortality . to further increase their clinical relevance , it is important for studies implementing dietary changes to examine sustainability of the prescribed diet . although both studies of vlcd reported significant decreases in inflammatory markers , such a diet is not sustainable and is likely harmful for the individuals as evidenced by the significant increase in tnf- following the 2-week vlcd implemented by mraz et al . . the lack of long - term followup of the selected patients prevents us from extrapolating from these findings that patients will have sustained results . illuminates that after 52 weeks of the prescribed diets , the group that initially received a low - calorie diet did not show a significant decrease in crp , while the group that was prescribed the high - protein , low - fat diet for all 52 weeks did achieve significant reductions in crp . this highlights that there may not be merit behind the rapid weight - loss from calorie - restricted programs in the aforementioned studies . due to the limited availability of data for analysis on this topic and the suboptimal quality of reporting within the studies , first , most of the studies had small sample sizes , with a range between 12 and 162 ( mean sample size of 53 , and median of 32 ) , limiting the power of the intervention to detect an effect . the two studies with the largest sample sizes implemented the low - glycemic - index diet and mediterranean diet with wine and showed significant reduction in inflammatory marker expression [ 16 , 29 ] . only three studies included in this integrative review conducted a power analysis [ 18 , 24 , 29 ] , and one conducted a retrospective power analysis , which is paramount in substantiating findings or validating the need for further research if the findings were insignificant . second , several studies did not include transparent definitions of the dietary intervention , rendering it difficult to infer specific dietary factors that decrease inflammation from the results . in previous studies with healthy volunteers , decreased inflammation was correlated with several individual dietary factors including fiber , antioxidant vitamins such as vitamin c and carotenoids , and fruit and vegetable intake . for example , in large prospective studies , whole grain intake has been associated with both decreased inflammatory markers and decreased incidence of t2d . of the three experimental studies in the current review that indicated whole grains as an aspect of the diet , only one demonstrated decreased crp levels following the intervention . similarly , the cross - sectional study examining intake of whole grains showed an inverse association between increasing intake of cereal fiber and crp levels . however , one study that implemented a high - carbohydrate , high - fiber diet and another that implemented a cretan - mediterranean diet , both of which are high in fiber , failed to demonstrate a decrease in crp following the intervention [ 19 , 20 ] . a third and final gap in knowledge related to this topic is adherence and change over time . though several studies included in this study were longitudinal , the longest study was 74 weeks , which does not necessarily indicate that benefits obtained will be sustained in the long - term . in which two low - fat diets , one low - protein , and one high - protein were prescribed to 38 participants with regular dietary counseling for the first 12 weeks . at 12 weeks , hba1c had decreased by 9.4% ( p < .001 ) in both groups . results in glycemic control were attenuated , however , at 64 weeks due to the fact that participants had not received regular dietary counseling between weeks 12 and 64 . recidivism such as this will likely be experienced in other long - term studies , underscoring the need for consistent counseling as well as followup in long - term studies that require behavior change . first , the review is limited to the published literature , so it is subject to publication bias . several authors were contacted via email with requests for unpublished research , but none were available . this limited the available literature on the influence of diet on inflammation and glycemic control markedly . there is merit in restriction of the search to this population , given that their inflammatory markers will be elevated relative to the general population and would , therefore , benefit from dietary changes in a more clinically significant way . finally , it should be noted that this integrative review does not address the mechanism of action underlying reduction of systemic inflammation following a dietary intervention . the review by visioli et al . concluded that antioxidants , essential fatty acids , and plant extracts have been shown to reduce systemic inflammation . extrapolating results from this integrative review to make inferences about specific nutrient effects is not possible ; however , it can be inferred that nutrient content of the mediterranean , vegan , and ada diets may contain more of those qualities mentioned in the review . given the limitations of the findings and the inconclusive results reached by this review , the present level of evidence on this topic is level iii , given that most of the studies were quasiexperimental . this paper , therefore , increases the level of evidence by providing an integrative review of the available research . however , disparate methods of reporting findings have rendered synthesis of extant findings difficult and unnecessarily labor - intensive . studies should adhere to the transparent reporting standards outlined by the consolidated standards of reporting trials . adherence to dietary interventions needs to be reported and included in analysis of the findings , possibly by including a measure of adherence as a regression variable . failure to include this critical factor could explain the variability in the findings of this integrative review , for a dose response is a likely influence in the success or failure of an intervention effect . adequately - powered , high quality studies examining the association between dietary intake and inflammation in t2d are also needed . medical nutrition therapy is one aspect of prevention and care of t2d recommended by the ada . although the dpp demonstrated a reduction in the incidence of t2d following the adaptation of lifestyle changes , there have been few studies examining the influence of dietary interventions on inflammation and glycemic control in individuals with established t2d in the us . it is important to conduct these studies domestically because of the diversity of the populations affected by this disease and the increasing rates of t2d . for example , further analysis of the results from the dpp revealed an attenuated benefit from the intervention in non - hispanic black women , highlighting the need for an understanding of the complex sociocultural factors influencing the african american population . studies examining the current dietary practices of individuals diagnosed with t2d are necessary , with a particular focus on differences in culturally - bound beliefs surrounding diet , food , and health . none of the studies included in this review used a theoretical framework to guide the research , which could have contributed to the disparate findings of the included studies . theoretical frameworks guide research questions and facilitate the process of asking a complete question . considering that t2d is both a metabolic and inflammatory disease , it is imperative to design studies that address both of these issues in the methods and the outcome measures . for example , if an intervention of a high mufa diet is implemented , it may be helpful not only to address inflammatory markers in the outcome measures , but also other influences on inflammation that could confound results such as physical activity , socioeconomic status , environmental stressors , and genetic factors as suggested by kang 's biobehavioral model of stress and inflammation . it is unclear from the aforementioned studies how exercise , stress , and genetics contributed to the metabolic and inflammatory outcomes measured , and larger , more rigorous studies are needed that control these variables . finally , based on the results of the aforementioned studies , rcts using participants with t2d are needed for which the nutrition content of the dietary intervention has been optimized for the treatment of t2d . this includes high intake of fruits and vegetables that are high in flavonoids , which gonzlez - castejn and rodriguez - casado report in their review on phytochemicals to have anti - inflammatory and antioxidant activities that may improve the metabolic status of individuals with t2d . the mediterranean diet is an extremely palatable diet rich in fruits , vegetables , fiber , and olive oil which has been shown with and without the addition of alcohol to improve inflammation , prevent the onset of t2d , and improve glycemic control in established t2d [ 16 , 41 , 44 ] . more high - quality rcts implementing this diet are needed to provide the evidence base for modifying clinical practice guidelines in medical nutrition therapy for patients with t2d .

type 2 diabetes ( t2d ) is a growing national health problem affecting 35% of adults 20 years of age in the united states . recently , diabetes has been categorized as an inflammatory disease , sharing many of the adverse outcomes as those reported from cardiovascular disease . medical nutrition therapy is recommended for the treatment of diabetes ; however , these recommendations have not been updated to target the inflammatory component , which can be affected by diet and lifestyle . to assess the current state of evidence for which dietary programs contain the most anti - inflammatory and glycemic control properties for patients with t2d , we conducted an integrative review of the literature . a comprehensive search of the pubmed , cinahl , scopus , and web of science databases from january 2000 to may 2012 yielded 786 articles . the final 16 studies met the selection criteria including randomized control trials , quasiexperimental , or cross - sectional studies that compared varying diets and measured inflammatory markers . the mediterranean and dash diets along with several low - fat diets were associated with lower inflammatory markers . the mediterranean diet demonstrated the most clinically significant reduction in glycosylated hemoglobin ( hba1c ) . information on best dietary guidelines for inflammation and glycemic control in individuals with t2d is lacking . continued research is warranted .

1. Background 2. Problem and Research Aims 3. The Literature Search 4. Search Outcomes 5. Methods 6. Results 7. Discussion

thirty - five percent of americans who are aged 20 years and older have type 2 diabetes ( t2d ) . the etiology behind such impressive statistics is complex but can be attributed in part to recent changes in the lifestyle choices of americans , including changes in habitual diet . the what we eat in america ( wweia ) report , based on data obtained from the national health and nutrition examination survey ( nhanes ) , demonstrated that grains ( mainly refined ) have contributed to the bulk of the rise in caloric intake in the us in the last 60 years . increased consumption of added sugars , processed grains , and saturated fat contributed to excess weight gain , which is an influential risk factor in the development of t2d [ 3 , 4 ] . in fact , results from implementation of the diabetes prevention program ( dpp ) illuminated that up to 58% of diabetes can be prevented through lifestyle changes alone , including changes in diet . inflammation is present prior to the development of t2d and cardiovascular disease ( cvd ) , contributing to evidence to support the common soil hypothesis , which is a reference to the common risk factors for the development of these two diseases . cvd is one of the major complications and causes of death in persons with t2d , supporting this hypothesis . the multi - ethnic study of atherosclerosis ( mesa ) showed that higher levels of the inflammatory markers , c - reactive protein ( crp ) and interleukin ( il)-6 , were associated with an increased risk of developing t2d . these proinflammatory molecules activate the cells of innate immunity , which cause damage to tissues in the vasculature , adipose tissue , and pancreas . acute responses through this proinflammatory pathway are needed for tissue damage repair and can improve function in tissues such as the pancreas , for example ; however , chronic expression can lead to pathological changes which result in disease . these changes clinically manifest as cvd , nephropathy , and symptomatic t2d , as the innate immune system attempts to repair the damaged tissue . moreover , inflammation is stimulated in times of stress , which can be induced by environmental , behavioral , individual , and psychosocial factors and diet - induced hyperglycemia and hypertriglyceridemia . although the published literature is populated with articles and systematic reviews demonstrating the relationship between dietary patterns , individual dietary factors , and incidence of diabetes [ 11 , 12 ] , the primary literature linking inflammation to diet and t2d is minimal . one of the few reported areas is with the mediterranean - style diet , which has been associated with lower inflammatory markers and decreased incidence of t2d [ 12 , 13 ] . therefore , the aim of this study was to present an integrative review of the published literature on the associations between dietary intake and markers of chronic inflammation in patients with t2d . this review and synthesis of the relevant literature seeks to answer the following research question : what is the current state of the science regarding the association between diet and inflammation in established t2d ? eligibility criteria included diet , inflammatory markers , and t2d evaluated in randomized clinical trial ( rct ) , quasiexperimental , or cross - sectional studies . the following databases were searched : pubmed , cinahl , scopus , and web of science . chronic inflammation , type 2 diabetes , c - reactive protein , interleukins , and tumor necrosis factor ( tnf ) . the search was conducted on articles published between january 2000 ( the year of the first appearance of diet , inflammation , and t2d in the literature ) and may 2012 . studies that focused on prevention of t2d were excluded , as they did not offer data to assist in the exploration of how diet relates to the inflammatory process within the pathogenesis of t2d . other exclusion criteria were studies that focused on supplements alone or exercise without an evaluation of , or comparison to , diet . the search produced 371 studies in pubmed , 265 studies in web of science , and 150 studies in cinahl ( see figure 1 ) . two were eliminated because they included predominantly healthy subjects in a cross - sectional study ; one was eliminated because it included exercise only , one was eliminated due to its focus on prevention of t2d , and two were eliminated because they focused on acute responses to dietary interventions . two studies were cross - sectional [ 14 , 15 ] , two were experimental [ 16 , 17 ] , and twelve were quasiexperimental , as follows : three were crossover experimental designs [ 1820 ] , three were pretest - posttest designs with only one group [ 2123 ] , and six were pretest - posttest designs with two or more comparison groups [ 2429 ] . more than half of the studies were international with one from the middle east , one from canada , three from australia , and six from europe . all subjects in the cross - sectional studies had a diagnosis of t2d , while several experimental studies utilized healthy and/or obese controls . acceptable measures for inflammation included serum evaluations of crp , il-6 , tnf , or a combination of one or more of these with other markers of inflammation . , some studies did not report baseline values ) , which was informative for the data reduction process . quality assessment scores were assigned based on 11 questions designed to capture the essence of the study design , methods , and analyses ( table 2 ) . the quality score for each question a score of 0 represented absence of the criteria in question , and a score of 1 designated fulfillment of each criterion . there was also one question related to results and conclusions with a rating of 02 , with 2 being of highest quality , which was weighted more heavily due to its importance in interpretation of the study findings . studies were then further delineated by diet categories of association with inflammation and those with a focus on diabetes control ( glycemic control ) . several experimental studies demonstrated a reduction of systemic inflammation following an intervention of a prescribed diet in patients with t2d . four of the studies with higher quality scores ( 9 ) demonstrated a significant association between diet and inflammation . three of these four contained a low - fat ( 30% of energy from fat ) dietary component [ 18 , 25 , 26 ] . after 64 weeks of following this diet with minimal dietary counseling , participants demonstrated a drop in crp levels , that is comparable to that which was found in the azadbakht et al . the control group in this study received a high - protein , low - fat diet , which also caused a decrease in crp , and there was not a significant difference in the mean change in crp between groups ( p = .61 ) . demonstrated that after following an intervention of either a low - fat or low - carbohydrate diet for 24 weeks , crp was only found to be significantly reduced in the low - fat diet arm ( p = .01 ) and not in the low - carbohydrate arm ( p = .94 ) . showed that after 52 weeks of following a low - glycemic - index , low - fat diet ( ~30% of energy intake from fat ) , crp was reduced from baseline by 29% compared to a high - glycemic - index , low - carbohydrate diet that was also high in monounsaturated fat ( mufa ) ( p < .05 ) . several other studies also demonstrated reductions in crp with dietary interventions , albeit with lower quality scores , and with ranges between 4 and 8 . one group began on a low - calorie diet and the other a high - protein low - fat diet for eight weeks , then both groups continued on the high - protein low - fat diet for 44 weeks ( see table 1 ) . mean - crp levels were reduced significantly only in the high - protein , low - fat diet at 52 weeks ( p < .01 ) . also demonstrated the anti - inflammatory effects of a diet high in mufas ( see table 1 ) . all three groups demonstrated a significant reduction in mean - crp at final data collection ( p < .01 ) ; however , there was not a significant difference in this reduction between groups . it is interesting to note here that the diet and exercise group did not have a greater decrease in inflammatory markers than the diet - only group . these results indicate that exercise may not add benefit to improving inflammatory markers in patients with t2d . randomized 99 participants to receive either a low - fat vegan diet or a conventional american diabetes association ( ada ) diet for 74 weeks . results showed a significant reduction of crp within groups ( p < .01 for each group ) but a nonsignificant difference between the two diet groups , perhaps due to the similar macronutrient content of the two dietary interventions . this diet did not produce a significant reduction in crp ; however , the 17 participants did exhibit significantly lower tnf- levels ( p = .002 ) following the intervention . additional two studies implemented two weeks of a very - low - calorie diet ( vlcd ) with obese subjects with t2d in highly controlled hospital environments [ 21 , 23 ] . the mediterranean diet is characterized by three servings of fruits and six servings of vegetables or wild leafy greens per day . results revealed significantly greater reductions in crp ( p < .01 ) , tnf- ( p < .01 ) , and il-6 ( p < .01 ) in the intervention group compared to the control group after the 52-week study period . , and the mediterranean diet . the two cross - sectional studies included in this analysis examined associations between two different aspects of diet and differed significantly in terms of quality in reporting . measured fasting blood glucose ( fbg ) as a proxy for glycemic control and demonstrated a significant improvement in fbg following the two - week dietary intervention of a very - low - calorie diet ( p < .05 ) . also demonstrated an improvement in glycemic control following a 24-week crossover intervention with a cretan mediterranean diet as evidenced by a significantly reduced mean glycosylated hemoglobin ( hba1c ) ( p = .012 ) , compared to the control diet which was the participants ' normal eating pattern . both studies also demonstrated improvement in the homeostatic model assessment ( homa ) score , a measure of insulin resistance ( see table 1 ) , although this change was not statistically significant following the mediterranean diet [ 20 , 21 ] . hba1c levels were not significantly different between the two groups ; however , the mean change in hba1c was greater than in all other studies included in this review ( 1.1% for the intervention group and 1.2% for the control group ) . reported that although none of the intervention groups demonstrated a significant improvement in hba1c , both the diet - only and diet and exercise groups had a significant reduction in fasting glucose levels ( p < .05 for both groups ) . interestingly , only the two interventions with exercise produced a significant reduction in insulin resistance . the great variability between studies decreases the ability to draw definitive conclusions about associations between diet , inflammation , and t2d ; however , particular themes emerged that are worth noting both from this review and in light of previous research . first , of the ten different diets prescribed that were low in fat , both within and across studies , eight of those diets led to significant reductions in inflammatory markers . these included the dash diet , the low - fat dpp - style diet , the low - energy low - protein diet , the vegan and ada diets , the high - protein low - fat diet , and both the high - protein and low - protein diets . therefore , it was not surprising that the high - mufa diet showed anti - inflammatory effects in the diabetic population [ 16 , 19 , 20 , 29 ] . lacked baseline values for participants ; therefore , it is not appropriate to infer causality for a change in crp over the 4 weeks of the study period , but rather only in the acute postprandial response . dietary patterns , as opposed to specific aspects of diet , have also been shown to correlate with levels of chronic inflammatory markers , but this concept was not adequately examined in the selected studies . for example , the western dietary pattern , characterized by a high consumption of sugary drinks , red meat , and poultry and a low consumption of fruits , vegetables , and fiber , was correlated with higher levels of crp in two large cross - sectional studies conducted in the us [ 34 , 35 ] . the impact of diet on the metabolic milieu of t2d is evident from the results of this integrative review and others . for example , of the two studies that were able to demonstrate a significantly reduced hba1c following the prescribed diet , one failed to report baseline values of glycemic control , and the other only demonstrated a mean reduction of 0.4% . while this result is statistically significant , it might not result in improvement in long - term outcomes of t2d , as demonstrated by findings from the united kingdom prospective diabetes study , which showed that a 1% reduction in hba1c levels can lead to improvements in long - term outcomes and decreased mortality . the control group that did not receive the red wine intervention but did follow a mediterranean diet had a similar decrease in hba1c , as well as crp , tnf- , and il-6 . although both studies of vlcd reported significant decreases in inflammatory markers , such a diet is not sustainable and is likely harmful for the individuals as evidenced by the significant increase in tnf- following the 2-week vlcd implemented by mraz et al . the lack of long - term followup of the selected patients prevents us from extrapolating from these findings that patients will have sustained results . illuminates that after 52 weeks of the prescribed diets , the group that initially received a low - calorie diet did not show a significant decrease in crp , while the group that was prescribed the high - protein , low - fat diet for all 52 weeks did achieve significant reductions in crp . this highlights that there may not be merit behind the rapid weight - loss from calorie - restricted programs in the aforementioned studies . due to the limited availability of data for analysis on this topic and the suboptimal quality of reporting within the studies , first , most of the studies had small sample sizes , with a range between 12 and 162 ( mean sample size of 53 , and median of 32 ) , limiting the power of the intervention to detect an effect . the two studies with the largest sample sizes implemented the low - glycemic - index diet and mediterranean diet with wine and showed significant reduction in inflammatory marker expression [ 16 , 29 ] . only three studies included in this integrative review conducted a power analysis [ 18 , 24 , 29 ] , and one conducted a retrospective power analysis , which is paramount in substantiating findings or validating the need for further research if the findings were insignificant . in previous studies with healthy volunteers , decreased inflammation was correlated with several individual dietary factors including fiber , antioxidant vitamins such as vitamin c and carotenoids , and fruit and vegetable intake . for example , in large prospective studies , whole grain intake has been associated with both decreased inflammatory markers and decreased incidence of t2d . of the three experimental studies in the current review that indicated whole grains as an aspect of the diet , only one demonstrated decreased crp levels following the intervention . similarly , the cross - sectional study examining intake of whole grains showed an inverse association between increasing intake of cereal fiber and crp levels . however , one study that implemented a high - carbohydrate , high - fiber diet and another that implemented a cretan - mediterranean diet , both of which are high in fiber , failed to demonstrate a decrease in crp following the intervention [ 19 , 20 ] . though several studies included in this study were longitudinal , the longest study was 74 weeks , which does not necessarily indicate that benefits obtained will be sustained in the long - term . in which two low - fat diets , one low - protein , and one high - protein were prescribed to 38 participants with regular dietary counseling for the first 12 weeks . results in glycemic control were attenuated , however , at 64 weeks due to the fact that participants had not received regular dietary counseling between weeks 12 and 64 . recidivism such as this will likely be experienced in other long - term studies , underscoring the need for consistent counseling as well as followup in long - term studies that require behavior change . this limited the available literature on the influence of diet on inflammation and glycemic control markedly . there is merit in restriction of the search to this population , given that their inflammatory markers will be elevated relative to the general population and would , therefore , benefit from dietary changes in a more clinically significant way . extrapolating results from this integrative review to make inferences about specific nutrient effects is not possible ; however , it can be inferred that nutrient content of the mediterranean , vegan , and ada diets may contain more of those qualities mentioned in the review . given the limitations of the findings and the inconclusive results reached by this review , the present level of evidence on this topic is level iii , given that most of the studies were quasiexperimental . this paper , therefore , increases the level of evidence by providing an integrative review of the available research . however , disparate methods of reporting findings have rendered synthesis of extant findings difficult and unnecessarily labor - intensive . failure to include this critical factor could explain the variability in the findings of this integrative review , for a dose response is a likely influence in the success or failure of an intervention effect . medical nutrition therapy is one aspect of prevention and care of t2d recommended by the ada . although the dpp demonstrated a reduction in the incidence of t2d following the adaptation of lifestyle changes , there have been few studies examining the influence of dietary interventions on inflammation and glycemic control in individuals with established t2d in the us . it is important to conduct these studies domestically because of the diversity of the populations affected by this disease and the increasing rates of t2d . studies examining the current dietary practices of individuals diagnosed with t2d are necessary , with a particular focus on differences in culturally - bound beliefs surrounding diet , food , and health . none of the studies included in this review used a theoretical framework to guide the research , which could have contributed to the disparate findings of the included studies . considering that t2d is both a metabolic and inflammatory disease , it is imperative to design studies that address both of these issues in the methods and the outcome measures . for example , if an intervention of a high mufa diet is implemented , it may be helpful not only to address inflammatory markers in the outcome measures , but also other influences on inflammation that could confound results such as physical activity , socioeconomic status , environmental stressors , and genetic factors as suggested by kang 's biobehavioral model of stress and inflammation . finally , based on the results of the aforementioned studies , rcts using participants with t2d are needed for which the nutrition content of the dietary intervention has been optimized for the treatment of t2d . this includes high intake of fruits and vegetables that are high in flavonoids , which gonzlez - castejn and rodriguez - casado report in their review on phytochemicals to have anti - inflammatory and antioxidant activities that may improve the metabolic status of individuals with t2d . the mediterranean diet is an extremely palatable diet rich in fruits , vegetables , fiber , and olive oil which has been shown with and without the addition of alcohol to improve inflammation , prevent the onset of t2d , and improve glycemic control in established t2d [ 16 , 41 , 44 ] . more high - quality rcts implementing this diet are needed to provide the evidence base for modifying clinical practice guidelines in medical nutrition therapy for patients with t2d .

type 2 diabetic patients ( n = 23 ) ( with mean duration of diabetes = 29 months ; age > 35 years ) were recruited from the diabetes and pediatric clinics at university of california davis medical center . none of the patients were on thiazolidinediones , ace inhibitors , angiotensin receptor blockers , insulin , or statins , since they interfere with tlr expression and activation ; six type 2 diabetic subjects were on metformin . healthy control subjects ( n = 23 ) , age < 35 years , with normal complete blood count ; no family history of diabetes or other chronic diseases ; normal kidney , liver , and thyroid function ; and fasting plasma glucose < 100 mg / dl were included in the study . healthy control subjects and type 2 diabetic patients were matched for age ( within 510 years ) , sex , and race . subjects with mean a1c over the last year > 10% ; inflammatory disorders ; microvascular and macrovascular complications ; abnormal liver , renal , or thyroid function ; steroid therapy , anti - inflammatory , antihypertensive , or hypolipidemic drugs ; antioxidant supplements in the past 3 months ; pregnancy ; smoking ; abnormal complete blood count ; alcohol consumption > 1 oz / day ; consumption of omega-3 polyunsaturated fatty acid capsules ( > 1 g / day ) ; and chronic high - intensity exercisers were excluded . informed consent was obtained from all the subjects , and the study was approved by the university of california davis institutional review board . after history and physical examination , fasting blood ( 30 ml ) was obtained . all the type 2 diabetic patients had family history of diabetes . of the subjects , complete blood count , lipid and lipoprotein profile , glucose , a1c , c - peptide , and c - reactive protein in all the enrolled subjects were assayed by standard laboratory techniques ( 11 ) . fasting plasma insulin concentrations were measured using an enzyme - linked immunosorbent assay ( elisa ) ( linco research ) . human monocytes were isolated by gradient density centrifugation of peripheral blood using histopaque ( sigma ) and magnetic separation ( miltenyi biotech ) as reported previously ( 3,11 ) . more than 88% of cells were identified as monocytes by cd14 staining , and viability was found to be > 92% . monocytes were examined before and after activation with synthetic lipoprotein pam3cys - ser-(lys ) 4 ( tlr2 ligand ; pam3csk4 , 170 ng / ml ; invitrogen ) and lipopolysaccharide ( lps ) ( tlr4 ligand ; 160 ng / ml ; e. coli 026:b6 ; sigma ) overnight ( 12 h ) , with suitable control subjects ( 3,11 ) . after treatment , cells were incubated with tlr2 , tlr4 , or isotype - matched igg ( ebioscience ) depending on the cell treatment ; 10,000 events were analyzed with the becton - dickinson fluorescence - activated cell sorter bioanalyzer as described previously ( 11 ) after gating for cd14 . intra - assay and interassay coefficient of variation ( cv ) for tlr2 and tlr4 expression was > 8 and > 12% , respectively ( 3,11 ) . serum and cell supernatants were used for measuring interleukin ( il)-1 , il-6 , il-8 , interferon ( ifn)- , monocyte chemoattractant protein ( mcp)-1 , and tumor necrosis factor ( tnf)- with a multiplex assay ( millipore ) as a functional readout of tlr activation . ifn- ( antigenix america ) , hsp60 , hsp70 ( assay designs ) , hmgb1 ( shino - test , japan ) , hyaluronan ( echelon ) , and n-(carboxymethyl ) lysine ( cml ) ( cyclex , japan ) were determined using elisa in the serum of all study subjects . nuclear extracts were used to verify activation of nf-b ( active motif ) in study subjects , indicating increased inflammation , as described previously ( 3,11 ) . intra- and interassay cv for all the assays were determined to be between 7 and 12% ( 21 ) . all the reagents used in the study were tested for endotoxin ( < 100 elisa unit [ eu]/ml ) using a limulus ameobocyte lysate assay ( lonza ) , allowing precise tlr expression measurements devoid of endotoxin ( 3,11 ) . cdna was synthesized using 1 g total rna , and 50 ng was amplified using primer probe sets for tlr1 , tlr2 , tlr4 , tlr6 , md-2 , and 18s ( sa bioscience ) following manufacturer 's cycling parameters . monocyte cell lysates were subjected to electrophoresis and transfer , as reported earlier ( 3,11 ) . blots were probed with human myeloid differentiation factor-88 ( myd88 ) , il receptor associated protein kinase-1 ( irak-1 ) , toll il-1 receptor domain ( tir)-containing adaptor molecule ( ticam-2 ) , ifn regulatory factor-3 ( irf-3 ) , nf-b ( nf-bp65 ; santa cruz biotechnology , santa cruz , ca ) , tir - containing adapter - inducing ifn- ( trif ; abcam ) , and antibodies with respective secondary igg antibodies and developed . besides , cell lysates ( 100 g ) were immunoprecipitated with tlr2 or tlr4 antibody overnight at 4c as reported previously ( 3 ) and blotted with tlr6 , hmgb1 , hsp60 ( santa cruz ) , and hyaluronan ( ray biotech ) antibodies as depicted in fig . 2f and g. densitometric ratios were determined as reported earlier from four independent assays ( 3,11 ) . statistical analyses were performed using sas software ( sas institute , cary , nc ) . parametric data were analyzed using paired , two - tailed t tests and nonparametric data using wilcoxon 's signed - rank tests . level of significance was set at p < 0.05 . human monocytes were isolated by gradient density centrifugation of peripheral blood using histopaque ( sigma ) and magnetic separation ( miltenyi biotech ) as reported previously ( 3,11 ) . more than 88% of cells were identified as monocytes by cd14 staining , and viability was found to be > 92% . monocytes were examined before and after activation with synthetic lipoprotein pam3cys - ser-(lys ) 4 ( tlr2 ligand ; pam3csk4 , 170 ng / ml ; invitrogen ) and lipopolysaccharide ( lps ) ( tlr4 ligand ; 160 ng / ml ; e. coli 026:b6 ; sigma ) overnight ( 12 h ) , with suitable control subjects ( 3,11 ) . after treatment , cells were incubated with tlr2 , tlr4 , or isotype - matched igg ( ebioscience ) depending on the cell treatment ; 10,000 events were analyzed with the becton - dickinson fluorescence - activated cell sorter bioanalyzer as described previously ( 11 ) after gating for cd14 . intra - assay and interassay coefficient of variation ( cv ) for tlr2 and tlr4 expression was > 8 and > 12% , respectively ( 3,11 ) . serum and cell supernatants were used for measuring interleukin ( il)-1 , il-6 , il-8 , interferon ( ifn)- , monocyte chemoattractant protein ( mcp)-1 , and tumor necrosis factor ( tnf)- with a multiplex assay ( millipore ) as a functional readout of tlr activation . ifn- ( antigenix america ) , hsp60 , hsp70 ( assay designs ) , hmgb1 ( shino - test , japan ) , hyaluronan ( echelon ) , and n-(carboxymethyl ) lysine ( cml ) ( cyclex , japan ) were determined using elisa in the serum of all study subjects . nuclear extracts were used to verify activation of nf-b ( active motif ) in study subjects , indicating increased inflammation , as described previously ( 3,11 ) . intra- and interassay cv for all the assays were determined to be between 7 and 12% ( 21 ) . all the reagents used in the study were tested for endotoxin ( < 100 elisa unit [ eu]/ml ) using a limulus ameobocyte lysate assay ( lonza ) , allowing precise tlr expression measurements devoid of endotoxin ( 3,11 ) . cdna was synthesized using 1 g total rna , and 50 ng was amplified using primer probe sets for tlr1 , tlr2 , tlr4 , tlr6 , md-2 , and 18s ( sa bioscience ) following manufacturer 's cycling parameters . monocyte cell lysates were subjected to electrophoresis and transfer , as reported earlier ( 3,11 ) . blots were probed with human myeloid differentiation factor-88 ( myd88 ) , il receptor associated protein kinase-1 ( irak-1 ) , toll il-1 receptor domain ( tir)-containing adaptor molecule ( ticam-2 ) , ifn regulatory factor-3 ( irf-3 ) , nf-b ( nf-bp65 ; santa cruz biotechnology , santa cruz , ca ) , tir - containing adapter - inducing ifn- ( trif ; abcam ) , and antibodies with respective secondary igg antibodies and developed . in all assays , -actin and total nonphosphorylated proteins were used as internal control . besides , cell lysates ( 100 g ) were immunoprecipitated with tlr2 or tlr4 antibody overnight at 4c as reported previously ( 3 ) and blotted with tlr6 , hmgb1 , hsp60 ( santa cruz ) , and hyaluronan ( ray biotech ) antibodies as depicted in fig . 2f and g. densitometric ratios were determined as reported earlier from four independent assays ( 3,11 ) . statistical analyses were performed using sas software ( sas institute , cary , nc ) . parametric data were analyzed using paired , two - tailed t tests and nonparametric data using wilcoxon 's signed - rank tests . subject characteristics are detailed in table 1 . there were no significant differences in age and lipid profile between control and type 2 diabetes groups . levels of glucose , a1c , ffas , and homa - ir were significantly higher in type 2 diabetic subjects than in control subjects . c - peptide and advanced glycation end product and cml levels were significantly increased in type 2 diabetic subjects compared with control subjects . c - reactive protein , a prototypic atherosclerotic marker , is increased in type 2 diabetic subjects compared with control subjects . we first determined the levels of tlr2 and tlr4 expression by flow cytometric analysis in control and type 2 diabetic subjects . monocyte surface expression of tlr2 and tlr4 was significantly elevated in type 2 diabetes compared with control in both the resting and activated state ( pam3csk4 or lps ) ( fig . next , to determine if the increase in monocyte tlr2 and tlr4 expression in type 2 diabetes resulted from increased mrna expression , we investigated the tlr2 and tlr4 mrna levels by real - time rt - pcr . figure 1b and e depict the corresponding increased tlr2 and tlr4 mrna expression in type 2 diabetes compared with control subjects . we further confirmed the increased monocyte tlr2 and tlr4 protein content using western blot assay ( fig . we measured downstream functional readouts ( il-1 , il-6 , il-8 , and tnf- ) of tlr2/4 ligation in resting and activated monocytes ( fig . 1 g ) . there was a significant upregulation of proinflammatory cytokines in type 2 diabetic patients compared with control subjects in both the basal and activated state . a : tlr2 surface protein expression was measured in monocytes after pam3csk4 ( pam ) challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . b : monocyte tlr2 mrna expression ratios in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods c : representative western blot depicting the tlr2 protein expression in pooled resting monocytes from three control subjects and three type 2 diabetic subjects . d : tlr4 surface protein expression was measured in monocytes after lps challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . e : monocyte tlr4 mrna expression ratios in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods f : representative western blot depicting the tlr4 protein expression in pooled resting monocytes from three control and three type 2 diabetic subjects . g : release of cytokines in resting and activated monocyte cell culture supernatants from control and type 2 diabetic subjects using multiplex assay as described in research design and methods . * p < 0.001 vs. untreated c ; * * p < 0.005 vs. control ( healthy control ) . c , untreated ; lps , lipopolysaccharide , tlr4 ligand ; pam , pam3csk4 , synthetic tlr2 ligand . identification of tlr2 and tlr4 ligands and cofactors is a key element in tlr activation and is unclear in type 2 diabetic subjects . beyond the established microbial ligands of tlr2 and tlr4 , several molecules of endogenous origin have been suggested to act as tlr2 and tlr4 ligands , notably hmgb1 , hsp60 , hsp70 , and hyaluronan fragments . accordingly , we measured concentration of endotoxin , hmgb1 , hyaluronan , hsp60 , and hsp70 in the serum of type 2 diabetic subjects and control subjects using elisas . figure 2 shows the significant increased levels of these ligands in type 2 diabetic subjects . in addition , coimmunoprecipitation with tlr2 and tlr4 and blotting for hmgb1 , hsp60 , and hyaluronan showed strong association with both tlr2 and tlr4 in type 2 diabetes compared with control subjects ( fig . 2f and g ) . a : endotoxin concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using limulus ameobocyte lysate assay as described in research design and methods . b : hmgb1 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . c : hyaluronan concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . d : hsp60 concentration in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects was measured using elisa as described in research design and methods . e : hsp70 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . representative western blot showing enhanced expression of tlr6 , hmgb1 , hyaluronan , and hsp60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with tlr2 antibody as detailed in research design and methods . representative western blot showing enhanced expression of hmgb1 , hyaluronan , and hsp60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with tlr4 antibody as detailed in research design and methods . tlr downstream signaling proteins myd88 , pirak-1 , trif , irf-3 , tecam-2 , and nf-b p65 were performed using specific antibodies to the respective ( phospho ) proteins , as described in research design and methods using -actin as a loading and internal control for myd88 , trif , irf-3 , tecam-2 , and nf-b p65 and irak for pirak-1 . i : the dna binding activity of nuclear nf-b p65 in control ( n = 23 ) and type 2 diabetes ( n = 23 ) monocytes was assessed by elisa as detailed in research design and methods in the basal state . j : serum concentration of cytokines / chemokines in study subjects were measured using multiplex assays as described in research design and methods . k : serum concentration of cytokines / chemokines in study subjects were measured using multiplex assays as described in research design and methods . it has been previously shown that tlr2 dimerizes with tlr1 or tlr6 and results in receptor activation and downstream signaling upon saturated fatty acid challenge . to determine whether tlr1 or tlr6 is required for the activation of tlr2 , we measured tlr1 and tlr6 mrna expression in monocytes of type 2 diabetes and control subjects . tlr6 mrna levels significantly increased in type 2 diabetes ( 3.8 0.4 vs. 1.3 0.2 mrna/18s ratio ; p < 0.05 ) compared with control subjects , whereas tlr1 mrna levels remained unchanged ( control : 1.5 0.4 , type 2 diabetes : 1.9 0.5 mrna/18s ratio ) , and coimmunoprecipitation showed an increased tlr2/tlr6 association in type 2 diabetes ( fig . further , md-2 mrna expression was also significantly increased ( control 1.2 0.2 vs. 2.9 0.3 mrna/18s ratio ) in type 2 diabetes monocytes . these data are in line with our earlier observation that under hyperglycemic conditions , tlr2 heterodimerizes with tlr6 in monocytes ( 3 ) . tlr2 and tlr4 both use myd88 and activate nf-b , common downstream signaling components for all tlrs except tlr3 ( 3,9 ) . therefore , activation of myd88-dependent and -independent pathways was used to interpret the activation of tlr2 and tlr4 . phosphorylation of irak-1 , myd88 , trif , tecam-2 , irf-3 protein expression in cytoplasmic extract , and p65 protein levels in nuclear extracts of monocytes were significantly increased in type 2 diabetes compared with control subjects , with no change in total protein and -actin levels ( fig . 2h ) , suggesting activation of tlr - mediated signaling cascade ( both myd88-dependent and -independent ) in type 2 diabetes . 2h ) . to further investigate tlr2- and tlr4-mediated inflammation in type 2 diabetes , we measured nf-b activity in the nuclear extracts of type 2 diabetes and control subject monocytes . type 2 diabetes showed increased nf-b p65-dependent dna binding activity compared with control subjects ( p < 0.01 ) ( fig . we measured il-1 , il-6 , il-8 , mcp-1 , ifn- , and tnf- serum concentration in type 2 diabetes and control subjects as a functional readout of tlr activation . there was a significant increase in all the proinflammatory mediators in type 2 diabetic subjects compared with control subjects ( fig . 2j and k ) . additionally , there was a significant correlation between bmi and tlr2 expression ( r = 0.5 ; p < 0.05 ) and tlr4 expression ( r = 0.43 ; p < 0.01 ) and significant correlation between tlr expression and glucose ( tlr2 : r = 0.7 ; tlr4 , r = 0.64 , p < 0.001 ) , ffa levels ( tlr2 : r = 0.66 ; tlr4 , r = 0.66 , p < 0.05 ) , homa - ir ( tlr2 : r = 0.64 ; tlr4 , r = 0.54 , p < 0.005 ) , and a1c ( tlr2 : r = 0.66 ; tlr4 , r = 0.62 , p < 0.001 ) . in addition , hsp60 , hsp70 , hmgb1 , and endotoxin levels significantly correlated with tlr2 expression ( r = 0.43 , r = 0.64 , r = 0.48 , and r = 0.16 ; p < 0.05 ) and tlr4 expression ( r = 0.43 , r = 0.65 , r = 0.41 , and r = 0.21 ; p < 0.001 ) , respectively . there was a significant correlation between tlr2 expression and il-1 , tnf- ( r = 0.5 , r = 0.5 ; p < 0.001 ) , mcp-1 , and nf-b ( r = 0.42 , r = 0.35 ; p < 0.05 ) and between tlr4 and il-1 , tnf- ( r = 0 . 56 , r = 0.55 , p < 0.001 ) , ifn- , and nf-b ( r = 0.46 , r = 0.4 , p < 0.05 ) . because bmi was significantly different between control and type 2 diabetes , additional statistical analyses using bmi as a covariant were performed . tlr2 , tlr4 ( with and without pam3csk4 or lps ) , nf-b , endogenous ligands , cml , and cytokines were significantly higher in type 2 diabetes compared with control subjects ( p < 0.001 ) , whereas il-1 ( p = 0.09 ) , ifn- ( p = 0.16 ) , and endotoxin ( p = 0.11 ) were no longer significant . subject characteristics are detailed in table 1 . there were no significant differences in age and lipid profile between control and type 2 diabetes groups . levels of glucose , a1c , ffas , and homa - ir were significantly higher in type 2 diabetic subjects than in control subjects . c - peptide and advanced glycation end product and cml levels were significantly increased in type 2 diabetic subjects compared with control subjects . c - reactive protein , a prototypic atherosclerotic marker , is increased in type 2 diabetic subjects compared with control subjects . we first determined the levels of tlr2 and tlr4 expression by flow cytometric analysis in control and type 2 diabetic subjects . monocyte surface expression of tlr2 and tlr4 was significantly elevated in type 2 diabetes compared with control in both the resting and activated state ( pam3csk4 or lps ) ( fig . next , to determine if the increase in monocyte tlr2 and tlr4 expression in type 2 diabetes resulted from increased mrna expression , we investigated the tlr2 and tlr4 mrna levels by real - time rt - pcr . figure 1b and e depict the corresponding increased tlr2 and tlr4 mrna expression in type 2 diabetes compared with control subjects . we further confirmed the increased monocyte tlr2 and tlr4 protein content using western blot assay ( fig . we measured downstream functional readouts ( il-1 , il-6 , il-8 , and tnf- ) of tlr2/4 ligation in resting and activated monocytes ( fig . a : tlr2 surface protein expression was measured in monocytes after pam3csk4 ( pam ) challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . b : monocyte tlr2 mrna expression ratios in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods . c : representative western blot depicting the tlr2 protein expression in pooled resting monocytes from three control subjects and three type 2 diabetic subjects . d : tlr4 surface protein expression was measured in monocytes after lps challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . e : monocyte tlr4 mrna expression ratios in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods . f : representative western blot depicting the tlr4 protein expression in pooled resting monocytes from three control and three type 2 diabetic subjects . g : release of cytokines in resting and activated monocyte cell culture supernatants from control and type 2 diabetic subjects using multiplex assay as described in research design and methods . * p < 0.001 vs. untreated c ; * * p < 0.005 vs. control ( healthy control ) . c , untreated ; lps , lipopolysaccharide , tlr4 ligand ; pam , pam3csk4 , synthetic tlr2 ligand . identification of tlr2 and tlr4 ligands and cofactors is a key element in tlr activation and is unclear in type 2 diabetic subjects . beyond the established microbial ligands of tlr2 and tlr4 , several molecules of endogenous origin have been suggested to act as tlr2 and tlr4 ligands , notably hmgb1 , hsp60 , hsp70 , and hyaluronan fragments . accordingly , we measured concentration of endotoxin , hmgb1 , hyaluronan , hsp60 , and hsp70 in the serum of type 2 diabetic subjects and control subjects using elisas . figure 2 shows the significant increased levels of these ligands in type 2 diabetic subjects . in addition , coimmunoprecipitation with tlr2 and tlr4 and blotting for hmgb1 , hsp60 , and hyaluronan showed strong association with both tlr2 and tlr4 in type 2 diabetes compared with control subjects ( fig . 2f and g ) . a : endotoxin concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using limulus ameobocyte lysate assay as described in research design and methods . b : hmgb1 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . c : hyaluronan concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . d : hsp60 concentration in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects was measured using elisa as described in research design and methods . e : hsp70 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . representative western blot showing enhanced expression of tlr6 , hmgb1 , hyaluronan , and hsp60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with tlr2 antibody as detailed in research design and methods . representative western blot showing enhanced expression of hmgb1 , hyaluronan , and hsp60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with tlr4 antibody as detailed in research design and methods . tlr downstream signaling proteins myd88 , pirak-1 , trif , irf-3 , tecam-2 , and nf-b p65 were performed using specific antibodies to the respective ( phospho ) proteins , as described in research design and methods using -actin as a loading and internal control for myd88 , trif , irf-3 , tecam-2 , and nf-b p65 and irak for pirak-1 . i : the dna binding activity of nuclear nf-b p65 in control ( n = 23 ) and type 2 diabetes ( n = 23 ) monocytes was assessed by elisa as detailed in research design and methods in the basal state . j : serum concentration of cytokines / chemokines in study subjects were measured using multiplex assays as described in research design and methods . k : serum concentration of cytokines / chemokines in study subjects were measured using multiplex assays as described in research design and methods . it has been previously shown that tlr2 dimerizes with tlr1 or tlr6 and results in receptor activation and downstream signaling upon saturated fatty acid challenge . to determine whether tlr1 or tlr6 is required for the activation of tlr2 , we measured tlr1 and tlr6 mrna expression in monocytes of type 2 diabetes and control subjects . tlr6 mrna levels significantly increased in type 2 diabetes ( 3.8 0.4 vs. 1.3 0.2 mrna/18s ratio ; p < 0.05 ) compared with control subjects , whereas tlr1 mrna levels remained unchanged ( control : 1.5 0.4 , type 2 diabetes : 1.9 0.5 mrna/18s ratio ) , and coimmunoprecipitation showed an increased tlr2/tlr6 association in type 2 diabetes ( fig . further , md-2 mrna expression was also significantly increased ( control 1.2 0.2 vs. 2.9 0.3 mrna/18s ratio ) in type 2 diabetes monocytes . these data are in line with our earlier observation that under hyperglycemic conditions , tlr2 heterodimerizes with tlr6 in monocytes ( 3 ) . tlr2 and tlr4 both use myd88 and activate nf-b , common downstream signaling components for all tlrs except tlr3 ( 3,9 ) . therefore , activation of myd88-dependent and -independent pathways was used to interpret the activation of tlr2 and tlr4 . phosphorylation of irak-1 , myd88 , trif , tecam-2 , irf-3 protein expression in cytoplasmic extract , and p65 protein levels in nuclear extracts of monocytes were significantly increased in type 2 diabetes compared with control subjects , with no change in total protein and -actin levels ( fig . 2h ) , suggesting activation of tlr - mediated signaling cascade ( both myd88-dependent and -independent ) in type 2 diabetes . to further investigate tlr2- and tlr4-mediated inflammation in type 2 diabetes , we measured nf-b activity in the nuclear extracts of type 2 diabetes and control subject monocytes . type 2 diabetes showed increased nf-b p65-dependent dna binding activity compared with control subjects ( p < 0.01 ) ( fig . we measured il-1 , il-6 , il-8 , mcp-1 , ifn- , and tnf- serum concentration in type 2 diabetes and control subjects as a functional readout of tlr activation . there was a significant increase in all the proinflammatory mediators in type 2 diabetic subjects compared with control subjects ( fig . additionally , there was a significant correlation between bmi and tlr2 expression ( r = 0.5 ; p < 0.05 ) and tlr4 expression ( r = 0.43 ; p < 0.01 ) and significant correlation between tlr expression and glucose ( tlr2 : r = 0.7 ; tlr4 , r = 0.64 , p < 0.001 ) , ffa levels ( tlr2 : r = 0.66 ; tlr4 , r = 0.66 , p < 0.05 ) , homa - ir ( tlr2 : r = 0.64 ; tlr4 , r = 0.54 , p < 0.005 ) , and a1c ( tlr2 : r = 0.66 ; tlr4 , r = 0.62 , p < 0.001 ) . in addition , hsp60 , hsp70 , hmgb1 , and endotoxin levels significantly correlated with tlr2 expression ( r = 0.43 , r = 0.64 , r = 0.48 , and r = 0.16 ; p < 0.05 ) and tlr4 expression ( r = 0.43 , r = 0.65 , r = 0.41 , and r = 0.21 ; p < 0.001 ) , respectively . there was a significant correlation between tlr2 expression and il-1 , tnf- ( r = 0.5 , r = 0.5 ; p < 0.001 ) , mcp-1 , and nf-b ( r = 0.42 , r = 0.35 ; p < 0.05 ) and between tlr4 and il-1 , tnf- ( r = 0 . 56 , r = 0.55 , p < 0.001 ) , ifn- , and nf-b ( r = 0.46 , r = 0.4 , p < 0.05 ) . because bmi was significantly different between control and type 2 diabetes , additional statistical analyses using bmi as a covariant were performed . tlr2 , tlr4 ( with and without pam3csk4 or lps ) , nf-b , endogenous ligands , cml , and cytokines were significantly higher in type 2 diabetes compared with control subjects ( p < 0.001 ) , whereas il-1 ( p = 0.09 ) , ifn- ( p = 0.16 ) , and endotoxin ( p = 0.11 ) were no longer significant . the interactions among inflammation , hyperglycemia , insulin resistance , and type 2 diabetes have clear implications for atherosclerosis via the innate immune system . besides being activators of inflammation under hyperglycemia and insulin resistance ( 3,57 ) , both tlr2 and tlr4 are critical in atherosclerosis ( 7 ) . in this study , we provided key evidence for increased monocyte tlr2 and tlr4 expression , activation , cofactor expression , ligands , and downstream signaling contributing to systemic inflammation seen in type 2 diabetic subjects . our observations significantly add to the emerging role of tlrs in atherosclerosis and diabetes and are consistent with other studies in this context ( 3,7,11 ) . increased tlr2 and tlr4 expression is demonstrated in atherosclerotic plaque macrophages and in animal models of atherosclerosis . tlr2/4 knockout mice on c57bl6 , apoe , and ldlr background and myd88 knockout mice show reduced lesion size , lipid content , and macrophage infiltration in plaques and reduced inflammation ( 22,23 ) . in two recent studies , we showed increased tlr2 and tlr4 expression , tlr ligands , intracellular signaling , and tlr - mediated inflammation in monocytes with significant correlation to a1c levels in type 1 diabetic patients ( 11,21 ) . moreover , tlr2/4 expression and activation is increased in human monocytes under hyperglycemia conditions ( 3 ) . ( 15 ) have shown abnormal tlr4 expression in skeletal muscle of a small number of insulin - resistant subjects , with little information on the tlr4-myd88 signaling , levels of tlr4 cofactors ( cd14/md-2 ) , tlr4 ligand ( endotoxin ) , and its correlation to tlr4 expression . moreover , the in vitro experiments in this article with myotubes and ffas lacked appropriate endotoxin controls , and it is not clear if the study subjects were on any cholesterol - lowering medications , adding complexity to the conclusions . ( 16 ) showed increased tlr2 expression in the adipose tissue of type 2 diabetes with strong correlates to endotoxin levels , with no change in tlr4 expression . this study is inconclusive in terms of the lack of tlr4 expression , even under high endotoxin levels , smaller sample size , and minimal patient medication details . du et al . ( 24 ) in a descriptive study showed that monocytes from lada , ( latent autoimmune diabetes in adults ) type 2 diabetic patients on sulfonylurea therapy have significantly higher tlr4 and cd14 expression than healthy control subjects , with no mechanistic details . in this context , our study fills in all the above gaps sequentially : first , we provide data on both tlr2 and tlr4 mrna / protein levels and critical downstream signaling events that follow their ligation in type 2 diabetes ; second , we determined cofactors required for tlr2/4 activation ; third , we demonstrated ligands in use for tlr2/4 activation ; fourth , notably , we and others have shown that statins , thiazolidinediones , and angiotensin receptor blockers downregulate tlrs and our type 2 diabetic patients are not on any of these drugs ; fifth , our tlr data showed significant correlation with major clinical estimates of type 2 diabetes , namely adjusted bmi , glucose , homa - ir , ffas , ligands , cytokines ; and finally , data are shown for both myd88-dependent and -independent signaling pathways , which may be acting collectively . in addition to the well - characterized microbial ligands , several molecules of the host origin ( endogenous ) have been proposed to act as ligands for tlr2 and tlr4 , including hmgb1 , hsps , and hyaluronan ( 25 ) , and were not examined in type 2 diabetes . it induces activation of intracellular signaling pathways via tlr2 , tlr4 , and the receptor for advanced glycation end products ( rages ) , thus acting as an extracellular alarmin . hsp60 and hsp70 induce the production of proinflammatory cytokines via activation of tlr2 and tlr4 . the inflammatory effects of recombinant human hsp60 were shown to be tlr4 dependent , suggesting that hsp60 may be a tlr4 ligand . low molecular weight degradation products of hyaluronan elicit proinflammatory responses in murine alveolar macrophages in rheumatoid arthritis models and other chronic inflammatory conditions . endotoxin is the most important ligand required for tlr4 activation ( 14 ) , and its levels are significantly increased in type 2 diabetic patients . in the present study , we show that type 2 diabetic patients have high circulating levels of hmgb1 , hsp60 , hsp70 , and hyaluronan , which could trigger tlr2 and tlr4 activation , leading to a proinflammatory state by the activation of tlr2/4 , synergistically with glucose , ffas , and endotoxin . our coimmunoprecipitation studies further suggest an association between tlr2 , tlr4 , hmgb1 , and hsp60 . dimerization is a major event in the functional activation of tlrs and results in cytokine production ( 3 ) . tlr2 activity requires heterodimerization with tlr1 or tlr6 to recognize ligands . using luciferase reporter assays and real - time rt - pcr , we showed that high glucose induces tlr2 and tlr6 heterodimerization , resulting in nf-b activation and cytokine production ( 3 ) . , we demonstrate that both tlr6 and md-2 mrna expression is increased in type 2 diabetic patients , suggesting their requirement for tlr ligation in type 2 diabetic patients . tlrs mainly signal through the adapter protein myd88 via activation of nf-b , resulting in the increased transcription of inflammation - related genes , such as those encoding indicated cytokines ( 3,9,11 ) . in addition , a myd88-independent pathway involving trif is essential to tlr3 and tlr4 signaling and induces ifn-. in two recent studies , we showed that activation of the myd88 pathway is increased in monocytes exposed to high glucose and in type 1 diabetic patients ( 3,9,11 ) . in the present study , we examined myd88-dependent and -independent tlr activation , by determining irf-3 and ifn- levels as a biological indicator of myd88-independent activation . taken together , the novel findings of this comprehensive study suggest that there is significant elevation of tlr2 and tlr4 protein , mrna , endogenous ligands , and cofactors in type 2 diabetic patients , which , in concert with hyperglycemia , contributes to the increase in tlr2 and tlr4 signaling that results in the proinflammatory state of type 2 diabetes . in future studies , we will address the mechanism of synergistic effects of hyperglycemia , ffas , and endogenous ligands on tlr2 and tlr4 expression and signaling .

objectiveindividuals with type 2 diabetes have a myriad of metabolic aberrations including increased inflammation , increasing their cardiovascular risk . toll - like receptors ( tlrs ) and their ligands play a key role in insulin resistance and atherosclerosis . however , there is a paucity of data examining the expression and activity of tlrs in type 2 diabetes . thus , in the present study , we examined tlr2 and tlr4 mrna and protein expression , their ligands , and signaling in monocytes of recently diagnosed type 2 diabetic patients.research design and methodstlr mrna , protein expression , tlr ligands , and tlr signaling were measured in freshly isolated monocytes from healthy human control subjects ( n = 23 ) and type 2 diabetic subjects ( n = 23 ) using real - time rt - pcr , western blot , and flow cytometric assays.resultstype 2 diabetic subjects had significantly increased tlr2 , tlr4 mrna , and protein in monocytes compared with control subjects ( p < 0.05 ) . increased tlr2 and tlr4 expression correlated with bmi , homeostasis model assessment insulin resistance ( homa - ir ) , glucose , a1c , n-(carboxymethyl ) lysine ( cml ) , and free fatty acid ( ffa ) . ligands of tlr2 and tlr4 , namely , hsp60 , hsp70 , hmgb1 , endotoxin , and hyaluronan levels , were elevated in type 2 diabetic subjects and positively correlated with tlr2 and tlr4 . type 2 diabetic subjects showed increased myd88 , phosphorylated irak-1 , trif , ticam-1 , irf-3 , and nf-b p65 expression in monocytes compared with control subjects . furthermore , tlr - myd88-nf-b signaling resulted in elevated levels of cytokines ( p < 0.05 ) , but increased interleukin ( il)-1 , interferon ( ifn)- , and endotoxin were not significant when adjusted for bmi.conclusionsin this comprehensive study , we make the novel observation that tlr2 and tlr4 expression and their ligands , signaling , and functional activation are increased in recently diagnosed type 2 diabetes and contribute to the proinflammatory state .

RESEARCH DESIGN AND METHODS Human monocytes Real-time RT-PCR Western blots and coimmunoprecipitation Statistical analysis RESULTS Baseline characteristics of the study subjects TLR2 and TLR4 mRNA and protein expression in type 2 diabetes monocytes TLR2 and TLR4 ligand and cofactor expression in type 2 diabetes TLR2 and TLR4 activation results in MyD88-dependent and -independent signaling in type 2 diabetes monocytes Increased TLR2 and TLR4 expression results in increased inflammation mediated by NF-B Increased TLR expression correlates with BMI, glucose, HOMA-IR, and inflammation CONCLUSIONS

healthy control subjects ( n = 23 ) , age < 35 years , with normal complete blood count ; no family history of diabetes or other chronic diseases ; normal kidney , liver , and thyroid function ; and fasting plasma glucose < 100 mg / dl were included in the study . ifn- ( antigenix america ) , hsp60 , hsp70 ( assay designs ) , hmgb1 ( shino - test , japan ) , hyaluronan ( echelon ) , and n-(carboxymethyl ) lysine ( cml ) ( cyclex , japan ) were determined using elisa in the serum of all study subjects . ifn- ( antigenix america ) , hsp60 , hsp70 ( assay designs ) , hmgb1 ( shino - test , japan ) , hyaluronan ( echelon ) , and n-(carboxymethyl ) lysine ( cml ) ( cyclex , japan ) were determined using elisa in the serum of all study subjects . levels of glucose , a1c , ffas , and homa - ir were significantly higher in type 2 diabetic subjects than in control subjects . c - peptide and advanced glycation end product and cml levels were significantly increased in type 2 diabetic subjects compared with control subjects . we first determined the levels of tlr2 and tlr4 expression by flow cytometric analysis in control and type 2 diabetic subjects . monocyte surface expression of tlr2 and tlr4 was significantly elevated in type 2 diabetes compared with control in both the resting and activated state ( pam3csk4 or lps ) ( fig . next , to determine if the increase in monocyte tlr2 and tlr4 expression in type 2 diabetes resulted from increased mrna expression , we investigated the tlr2 and tlr4 mrna levels by real - time rt - pcr . figure 1b and e depict the corresponding increased tlr2 and tlr4 mrna expression in type 2 diabetes compared with control subjects . a : tlr2 surface protein expression was measured in monocytes after pam3csk4 ( pam ) challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . b : monocyte tlr2 mrna expression ratios in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods c : representative western blot depicting the tlr2 protein expression in pooled resting monocytes from three control subjects and three type 2 diabetic subjects . d : tlr4 surface protein expression was measured in monocytes after lps challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . e : monocyte tlr4 mrna expression ratios in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods f : representative western blot depicting the tlr4 protein expression in pooled resting monocytes from three control and three type 2 diabetic subjects . identification of tlr2 and tlr4 ligands and cofactors is a key element in tlr activation and is unclear in type 2 diabetic subjects . beyond the established microbial ligands of tlr2 and tlr4 , several molecules of endogenous origin have been suggested to act as tlr2 and tlr4 ligands , notably hmgb1 , hsp60 , hsp70 , and hyaluronan fragments . accordingly , we measured concentration of endotoxin , hmgb1 , hyaluronan , hsp60 , and hsp70 in the serum of type 2 diabetic subjects and control subjects using elisas . in addition , coimmunoprecipitation with tlr2 and tlr4 and blotting for hmgb1 , hsp60 , and hyaluronan showed strong association with both tlr2 and tlr4 in type 2 diabetes compared with control subjects ( fig . a : endotoxin concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using limulus ameobocyte lysate assay as described in research design and methods . b : hmgb1 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . c : hyaluronan concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . d : hsp60 concentration in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects was measured using elisa as described in research design and methods . e : hsp70 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . representative western blot showing enhanced expression of tlr6 , hmgb1 , hyaluronan , and hsp60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with tlr2 antibody as detailed in research design and methods . tlr downstream signaling proteins myd88 , pirak-1 , trif , irf-3 , tecam-2 , and nf-b p65 were performed using specific antibodies to the respective ( phospho ) proteins , as described in research design and methods using -actin as a loading and internal control for myd88 , trif , irf-3 , tecam-2 , and nf-b p65 and irak for pirak-1 . i : the dna binding activity of nuclear nf-b p65 in control ( n = 23 ) and type 2 diabetes ( n = 23 ) monocytes was assessed by elisa as detailed in research design and methods in the basal state . to determine whether tlr1 or tlr6 is required for the activation of tlr2 , we measured tlr1 and tlr6 mrna expression in monocytes of type 2 diabetes and control subjects . tlr6 mrna levels significantly increased in type 2 diabetes ( 3.8 0.4 vs. 1.3 0.2 mrna/18s ratio ; p < 0.05 ) compared with control subjects , whereas tlr1 mrna levels remained unchanged ( control : 1.5 0.4 , type 2 diabetes : 1.9 0.5 mrna/18s ratio ) , and coimmunoprecipitation showed an increased tlr2/tlr6 association in type 2 diabetes ( fig . phosphorylation of irak-1 , myd88 , trif , tecam-2 , irf-3 protein expression in cytoplasmic extract , and p65 protein levels in nuclear extracts of monocytes were significantly increased in type 2 diabetes compared with control subjects , with no change in total protein and -actin levels ( fig . type 2 diabetes showed increased nf-b p65-dependent dna binding activity compared with control subjects ( p < 0.01 ) ( fig . there was a significant increase in all the proinflammatory mediators in type 2 diabetic subjects compared with control subjects ( fig . additionally , there was a significant correlation between bmi and tlr2 expression ( r = 0.5 ; p < 0.05 ) and tlr4 expression ( r = 0.43 ; p < 0.01 ) and significant correlation between tlr expression and glucose ( tlr2 : r = 0.7 ; tlr4 , r = 0.64 , p < 0.001 ) , ffa levels ( tlr2 : r = 0.66 ; tlr4 , r = 0.66 , p < 0.05 ) , homa - ir ( tlr2 : r = 0.64 ; tlr4 , r = 0.54 , p < 0.005 ) , and a1c ( tlr2 : r = 0.66 ; tlr4 , r = 0.62 , p < 0.001 ) . in addition , hsp60 , hsp70 , hmgb1 , and endotoxin levels significantly correlated with tlr2 expression ( r = 0.43 , r = 0.64 , r = 0.48 , and r = 0.16 ; p < 0.05 ) and tlr4 expression ( r = 0.43 , r = 0.65 , r = 0.41 , and r = 0.21 ; p < 0.001 ) , respectively . there was a significant correlation between tlr2 expression and il-1 , tnf- ( r = 0.5 , r = 0.5 ; p < 0.001 ) , mcp-1 , and nf-b ( r = 0.42 , r = 0.35 ; p < 0.05 ) and between tlr4 and il-1 , tnf- ( r = 0 . tlr2 , tlr4 ( with and without pam3csk4 or lps ) , nf-b , endogenous ligands , cml , and cytokines were significantly higher in type 2 diabetes compared with control subjects ( p < 0.001 ) , whereas il-1 ( p = 0.09 ) , ifn- ( p = 0.16 ) , and endotoxin ( p = 0.11 ) were no longer significant . levels of glucose , a1c , ffas , and homa - ir were significantly higher in type 2 diabetic subjects than in control subjects . we first determined the levels of tlr2 and tlr4 expression by flow cytometric analysis in control and type 2 diabetic subjects . monocyte surface expression of tlr2 and tlr4 was significantly elevated in type 2 diabetes compared with control in both the resting and activated state ( pam3csk4 or lps ) ( fig . next , to determine if the increase in monocyte tlr2 and tlr4 expression in type 2 diabetes resulted from increased mrna expression , we investigated the tlr2 and tlr4 mrna levels by real - time rt - pcr . figure 1b and e depict the corresponding increased tlr2 and tlr4 mrna expression in type 2 diabetes compared with control subjects . a : tlr2 surface protein expression was measured in monocytes after pam3csk4 ( pam ) challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . b : monocyte tlr2 mrna expression ratios in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods . c : representative western blot depicting the tlr2 protein expression in pooled resting monocytes from three control subjects and three type 2 diabetic subjects . d : tlr4 surface protein expression was measured in monocytes after lps challenge in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by flow cytometry as described in research design and methods . e : monocyte tlr4 mrna expression ratios in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects by real - time rt - pcr as described in research design and methods . identification of tlr2 and tlr4 ligands and cofactors is a key element in tlr activation and is unclear in type 2 diabetic subjects . beyond the established microbial ligands of tlr2 and tlr4 , several molecules of endogenous origin have been suggested to act as tlr2 and tlr4 ligands , notably hmgb1 , hsp60 , hsp70 , and hyaluronan fragments . accordingly , we measured concentration of endotoxin , hmgb1 , hyaluronan , hsp60 , and hsp70 in the serum of type 2 diabetic subjects and control subjects using elisas . in addition , coimmunoprecipitation with tlr2 and tlr4 and blotting for hmgb1 , hsp60 , and hyaluronan showed strong association with both tlr2 and tlr4 in type 2 diabetes compared with control subjects ( fig . a : endotoxin concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using limulus ameobocyte lysate assay as described in research design and methods . b : hmgb1 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . c : hyaluronan concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . d : hsp60 concentration in control ( n = 23 ) and type 2 diabetes ( n = 23 ) subjects was measured using elisa as described in research design and methods . e : hsp70 concentration in control ( n = 23 ) and type 2 diabetic ( n = 23 ) subjects was measured using elisa as described in research design and methods . representative western blot showing enhanced expression of tlr6 , hmgb1 , hyaluronan , and hsp60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with tlr2 antibody as detailed in research design and methods . tlr downstream signaling proteins myd88 , pirak-1 , trif , irf-3 , tecam-2 , and nf-b p65 were performed using specific antibodies to the respective ( phospho ) proteins , as described in research design and methods using -actin as a loading and internal control for myd88 , trif , irf-3 , tecam-2 , and nf-b p65 and irak for pirak-1 . i : the dna binding activity of nuclear nf-b p65 in control ( n = 23 ) and type 2 diabetes ( n = 23 ) monocytes was assessed by elisa as detailed in research design and methods in the basal state . to determine whether tlr1 or tlr6 is required for the activation of tlr2 , we measured tlr1 and tlr6 mrna expression in monocytes of type 2 diabetes and control subjects . tlr6 mrna levels significantly increased in type 2 diabetes ( 3.8 0.4 vs. 1.3 0.2 mrna/18s ratio ; p < 0.05 ) compared with control subjects , whereas tlr1 mrna levels remained unchanged ( control : 1.5 0.4 , type 2 diabetes : 1.9 0.5 mrna/18s ratio ) , and coimmunoprecipitation showed an increased tlr2/tlr6 association in type 2 diabetes ( fig . phosphorylation of irak-1 , myd88 , trif , tecam-2 , irf-3 protein expression in cytoplasmic extract , and p65 protein levels in nuclear extracts of monocytes were significantly increased in type 2 diabetes compared with control subjects , with no change in total protein and -actin levels ( fig . type 2 diabetes showed increased nf-b p65-dependent dna binding activity compared with control subjects ( p < 0.01 ) ( fig . additionally , there was a significant correlation between bmi and tlr2 expression ( r = 0.5 ; p < 0.05 ) and tlr4 expression ( r = 0.43 ; p < 0.01 ) and significant correlation between tlr expression and glucose ( tlr2 : r = 0.7 ; tlr4 , r = 0.64 , p < 0.001 ) , ffa levels ( tlr2 : r = 0.66 ; tlr4 , r = 0.66 , p < 0.05 ) , homa - ir ( tlr2 : r = 0.64 ; tlr4 , r = 0.54 , p < 0.005 ) , and a1c ( tlr2 : r = 0.66 ; tlr4 , r = 0.62 , p < 0.001 ) . in addition , hsp60 , hsp70 , hmgb1 , and endotoxin levels significantly correlated with tlr2 expression ( r = 0.43 , r = 0.64 , r = 0.48 , and r = 0.16 ; p < 0.05 ) and tlr4 expression ( r = 0.43 , r = 0.65 , r = 0.41 , and r = 0.21 ; p < 0.001 ) , respectively . tlr2 , tlr4 ( with and without pam3csk4 or lps ) , nf-b , endogenous ligands , cml , and cytokines were significantly higher in type 2 diabetes compared with control subjects ( p < 0.001 ) , whereas il-1 ( p = 0.09 ) , ifn- ( p = 0.16 ) , and endotoxin ( p = 0.11 ) were no longer significant . in this study , we provided key evidence for increased monocyte tlr2 and tlr4 expression , activation , cofactor expression , ligands , and downstream signaling contributing to systemic inflammation seen in type 2 diabetic subjects . in two recent studies , we showed increased tlr2 and tlr4 expression , tlr ligands , intracellular signaling , and tlr - mediated inflammation in monocytes with significant correlation to a1c levels in type 1 diabetic patients ( 11,21 ) . in this context , our study fills in all the above gaps sequentially : first , we provide data on both tlr2 and tlr4 mrna / protein levels and critical downstream signaling events that follow their ligation in type 2 diabetes ; second , we determined cofactors required for tlr2/4 activation ; third , we demonstrated ligands in use for tlr2/4 activation ; fourth , notably , we and others have shown that statins , thiazolidinediones , and angiotensin receptor blockers downregulate tlrs and our type 2 diabetic patients are not on any of these drugs ; fifth , our tlr data showed significant correlation with major clinical estimates of type 2 diabetes , namely adjusted bmi , glucose , homa - ir , ffas , ligands , cytokines ; and finally , data are shown for both myd88-dependent and -independent signaling pathways , which may be acting collectively . in addition to the well - characterized microbial ligands , several molecules of the host origin ( endogenous ) have been proposed to act as ligands for tlr2 and tlr4 , including hmgb1 , hsps , and hyaluronan ( 25 ) , and were not examined in type 2 diabetes . in the present study , we show that type 2 diabetic patients have high circulating levels of hmgb1 , hsp60 , hsp70 , and hyaluronan , which could trigger tlr2 and tlr4 activation , leading to a proinflammatory state by the activation of tlr2/4 , synergistically with glucose , ffas , and endotoxin . taken together , the novel findings of this comprehensive study suggest that there is significant elevation of tlr2 and tlr4 protein , mrna , endogenous ligands , and cofactors in type 2 diabetic patients , which , in concert with hyperglycemia , contributes to the increase in tlr2 and tlr4 signaling that results in the proinflammatory state of type 2 diabetes .

quality of life ( ql ) may be defined as an individual s perception of their well - being . health - related ql reflects the impact of a healthy state on a person s ability to lead a fulfilling life , and covers the individual s satisfaction in physical , functional , psychological , and social domains.1 studies2,3 have shown that ql worsens after brain damage ( vascular lesions , head injury , infections ) . changes in ql in the poststroke period do not seem to be age-4 or sex - dependent , but rather are due to the onset of depression5 and a low level of reacquisition in motor functions , above all in the upper limbs.6 ql improves within the family environment.7 aphasia is the loss of or radical change in voluntary speech as a consequence of damage to the left cerebral hemisphere , and generally causes significant changes in a patient s self - image and family , social , and work relationships.8 the impairment in language skills due to aphasia causes problems in verbal expression , auditory comprehension , reading , and writing . aphasia is a prominent cause of limitation on communication activities , such as using the phone or writing a letter . aphasia will have relatively little direct impact upon the performance of domestic activities of daily living , but it will particularly affect complex social activities , such as work and participating in community activities and leisure activities involving other people . studies have documented high levels of depression9 and social exclusion,10 and low levels of leisure and other social activities,11 social contacts,12 and ql13,14 among patients with aphasia ( pwa ) . significant correlations have been found between degree of aphasia and the social , emotional , mobility , and total scores of ql , measured with the nottingham health profile.15 in a population - based study , lam and wodchis16 found that aphasia exhibited the largest negative influence on ql , followed by cancer and alzheimer s disease . pwa reported significantly worse ql than nonaphasic patients , since aphasia influenced independence , social relationships , and access to their environment . le dorze and brassard17 have described the consequences of aphasia by analyzing the personal accounts of aphasic individuals , relatives , or friends . these consequences were classified into one of three categories : disabilities , handicaps , and coping behaviors . in the first group , the handicaps included changes in situations of communication , in interpersonal relationships , loss of autonomy , and restriction of activities . coping behaviors , however , were adopted by pwa and their relatives to adjust the undesired effects of the various disabilities and handicaps they experienced . although a number of stroke - specific ql scales have been developed , most exclude stroke survivors with aphasia , subjects most prone to social isolation and exclusion . this is due to the difficulties faced in proposing a questionnaire to subjects with severe comprehension and expression deficits . in regard to the studies on the impact of aphasia on patient s life , there are two paradigms . from a qualitative perspective , ethnographic methods , like participant and nonparticipant observations and analysis of artifacts such as diaries , can reflect the everyday experiences of pwa and their social inclusion , but they do not suggest how patients feel their ql to be . from a quantitative perspective , many authors18,19 have evaluated the ql of patients with severe aphasia , asking proxy respondents to report on their partners health - related quality of life ( hrql ) . also in this case , evaluating one s emotional distress , aphasia severity , communication and activity limitations , other medical problems , and social aspects all influenced ql . the extent of aphasia ( severity , language impairment , communication disability ) was associated with or predictive of lower hrql in seven of the eight reviewed studies . hilari et al20 evaluated the ql in patients with and without aphasia , testing a stroke - specific hrql scale ( the stroke and aphasia quality of life scale [ saqol-39 ] ) in a generic stroke sample that included patients both with and without aphasia . the saqol-39 generic stroke scale measures hrql after stroke in three domains : physical , psychosocial , and communication . this scale demonstrates good internal consistency , test retest reliability and validity , and adequate responsiveness to changes in a sample of patients with acute and chronic stroke . the information obtained from ql measures can be useful to identify patients problems , determine treatment priorities , manage interventions , and monitor disease periods . the first aim of this observational study was to conduct a preliminary evaluation of the psychometric properties ( validity , reliability ) of a ql questionnaire for aphasics ( qlqa ) , in which we focused particularly on difficulties in interpersonal relationships , on loss of independence , and on abilities in daily life as a result of language disorders . moreover , in the aphasic group , we studied how qlqa was sensitive to the severity and type of aphasia and to the time from onset . finally , we tried to identify the specific role of linguistic deficits on ql , minimizing the effects of motor impairment . the qlqa scores of pwa with hemiparesis were compared to those of a control group affected by a right - brain injury or a neurological peripheral disease and motor impairment , but with no linguistic or communicative problems . a total of 164 consecutive patients with neurological central disease admitted to our neurorehabilitation unit for cognitive and physical rehabilitation from 2011 to 2012 were examined . eighteen of them were excluded for bilateral or multiple lesions identified by neurodiagnostic scans ( computerized axial tomography or magnetic resonance imaging ) . the experimental group consisted of 146 pwa and right hemiparesis , all cared for by a caregiver . inclusion criteria were aphasia resulting from unilateral left - hemisphere stroke , unknown prestroke history of severe cognitive decline , or mental health problems . no patients presented with prestroke neuropsychological deficits , psychiatric disorders , history of alcohol or drug abuse , head injury , or tumoral lesions . patients were excluded if they did not speak italian premorbidly a control group consisted of 37 subjects with right - hemisphere cerebral damage , hemiparesis , and varying degrees of unilateral spatial neglect . written consent all the patients underwent an initial screening to evaluate the presence and type of linguistic disorders and the severity of motor impairment . all aphasic patients were evaluated by the aachener aphasia test ( aat)21 to analyze language deficits and to diagnose type of aphasia . for this study , the patients underwent the following series of tests , administered by a speech therapist : the subtest of spontaneous speech of the aat,21 structured in six parts ( communicative behavior , articulation and prosody , automatic language , semantics , phonology , syntax ) , with a score range from 0 to 5 the token test22 to assess verbal comprehension with 36 items raven s coloured progressive matrices,23,24 which provides a nonverbal measure of intellectual abilities ; the patient must logically complete a given visual spatial pattern , choosing from a set of six alternatives a scale of functional independence the functional independence measure25,26 ( fim ) divided into scores for motor fim ( 13 items ) and cognitive fim ( five items ) another scale of functional independence the functional assessment measure ( fam)2729 cognitive subscale a compound of fourteen items evaluating comprehension , expression , reading , writing , speech intelligibility , social interaction , emotional status , adjustment to limitation , employability , problem solving , memory , orientation , attention span , and safety judgment hrql questionnaires give outcome measures that evaluate the impact of health on a person s ability to lead a fulfilling life , and generally incorporate the individual s perceptions of physical , mental / emotional , family , and social functioning . although a number of stroke - specific ql scales have been developed , measures that ecologically identify both the effects of linguistic communicative disability and the quality of everyday life are still needed for pwa . they could be useful to bridge the gap between linguistic rehabilitation and the patient s real use of residual means of communication or ability to perform the daily life activities in which language is involved . in this way , they could link the individual rehabilitation training to outcomes of increased functional autonomy in social and communicative environments , according to the recent world health organization international classification of functioning , disability and health ( icf).30 one example of these measures is the functional outcome questionnaire for aphasia,31 used in the us . it consists of 32 items , and is focused on the efficacy of the aphasic s verbal and nonverbal communicative abilities . this scale assesses the ability to communicate basic needs and new information and the ability to make routine requests ; it also investigates the issues of communication and comprehension . the saqol-3932 scale , recently translated into italian and also into spanish,33,34 is a more complete ql assessment ( it includes measurement of general , physical , and psychosocial health and vitality ) , even if there are few linguistic items compared to the total number ( nine of 39 ) . our qlqa has the aim of addressing a lack of ql measures in the italian language , including , as with previous ones , measurement of several domains , such as physical , psychological , communication , and social participation . engell et al35 developed a pictorial procedure for rating ql to minimize the influence of aphasia . they transformed an existing ql inventory the modified german version of the sickness impact profile36 into a picture - based representation . the authors selected the aachen quality of life inventory ( alqi),37 a german - language adaptation of the sickness impact profile . the alqi items were transformed into a pictorial version to maximize aphasic patients understanding of the verbal statements , as well as to permit them to give nonverbal responses . this procedure allowed for self - rating of aphasic patients in parallel to a proxy rating by caregivers . there was in fact also a written version for the caregivers that corresponded item by item to the patients pictorial version . the limited use of these assessments has negative effects on the course of clinical welfare . in italy , stroke rehabilitation still focuses more on the improvement of physical autonomy rather than on a global increase in neuropsychological functions , which are often affected . rehabilitation therapy should aim not only to regain communicative linguistic abilities , but also to help in adapting to the disability and to encourage social integration and personal well - being , thus improving ql . our ql questionnaire was developed by means of the aforementioned conceptual considerations and analysis of literature studies and of the previous ql and functional questionnaires for aphasic patients , in order to point out the aphasic disease , handicap and coping behavior in everyday life . the questionnaire consists of 37 questions that are important for daily well - being , chosen from a larger repertoire by a mixed group of pwa and their caregivers and members of the italian aphasia association of our region ( puglia ) . we consequently based the qlqa more on the percentage of residual abilities performed by the individuals with aphasia rather than on their perceived ql , which was difficult to investigate , considering their communicative problems . express the disability , mainly as relationship problems and loss of autonomy caused by linguistic deficits ; it consists in a majority of questions regarding language - related activities and abilities consider verbal comprehension difficulties include items of residual functional disability due to motor deficits include items on psychological problems that occur both in the poststroke phase and later , partly as a result of brain damage but also the social and individual aftereffects of aphasia highlight relational and nonrelational residual problems due to language deficits . the questions , formulated as easily and succinctly as possible , assess the ability to perform basic functions and convey health problems , the psychological changes due to the disability and the ability to socialize , the ability of linguistic and contextual comprehension , and expression in routine daily activities . the items are scored on a 5-point scale , with 0= the individual is able to successfully perform the behavior 0% of the time , 1= the individual is able to successfully perform the behavior 25% of the time , 2= the individual is able to successfully perform the behavior 50% of the time , 3= the individual is able to successfully perform the behavior 75% of the time , and 4= the individual is able to successfully perform the behavior 100% of the time . the qlqa was administered by a speech therapist using verbal or pragmatic means ( gestures and drawings ) when the patient showed comprehension deficits , in the presence of the caregiver . when there was no agreement in the answers , we considered those of the caregiver more reliable , so excluding the influence of the patients reduced consciousness of their own limits or anxiety / depression state . demographic and clinical variables of aphasic sample and control subjects were evaluated with descriptive statistics ( means , standard deviations , etc),using student s t - test for analyzing the differences between two samples . due to the ordinal nature of the scale , differences in the qlqa scores and in other tests of both groups were analyzed using nonparametric tests ( mann whitney u ) . we used standard psychometric methods3840 to evaluate internal consistency , test retest reliability , and construct validity ( convergent and discriminant ) of qlqa in 146 aphasic patients . a principal - component analysis ( pca , a type of factor analysis ) was used to determine the number and the type of domains underlying the ql of aphasic patients and reduce the number of items in the qlqa to those best measuring it . in order to evaluate convergent and discriminant validity , a multiple correlation among the scores of the qlqa subtests ( communication , autonomy , and psychological condition ) , resulting from pca , and the fim and fam scores and the correct linguistic tests scores were calculated by spearman s correlation coefficient . internal consistency was calculated using cronbach s -test and test retest reliability using intraclass correlation coefficient ( icc ) . data analyses were carried out with spss 18.0 for windows ( ibm , armonk , ny , usa ) . a total of 164 consecutive patients with neurological central disease admitted to our neurorehabilitation unit for cognitive and physical rehabilitation from 2011 to 2012 were examined . eighteen of them were excluded for bilateral or multiple lesions identified by neurodiagnostic scans ( computerized axial tomography or magnetic resonance imaging ) . the experimental group consisted of 146 pwa and right hemiparesis , all cared for by a caregiver . inclusion criteria were aphasia resulting from unilateral left - hemisphere stroke , unknown prestroke history of severe cognitive decline , or mental health problems . no patients presented with prestroke neuropsychological deficits , psychiatric disorders , history of alcohol or drug abuse , head injury , or tumoral lesions . patients were excluded if they did not speak italian premorbidly a control group consisted of 37 subjects with right - hemisphere cerebral damage , hemiparesis , and varying degrees of unilateral spatial neglect . written consent all the patients underwent an initial screening to evaluate the presence and type of linguistic disorders and the severity of motor impairment . all aphasic patients were evaluated by the aachener aphasia test ( aat)21 to analyze language deficits and to diagnose type of aphasia . for this study , the patients underwent the following series of tests , administered by a speech therapist : the subtest of spontaneous speech of the aat,21 structured in six parts ( communicative behavior , articulation and prosody , automatic language , semantics , phonology , syntax ) , with a score range from 0 to 5 the token test22 to assess verbal comprehension with 36 items raven s coloured progressive matrices,23,24 which provides a nonverbal measure of intellectual abilities ; the patient must logically complete a given visual spatial pattern , choosing from a set of six alternatives a scale of functional independence the functional independence measure25,26 ( fim ) divided into scores for motor fim ( 13 items ) and cognitive fim ( five items ) another scale of functional independence the functional assessment measure ( fam)2729 cognitive subscale a compound of fourteen items evaluating comprehension , expression , reading , writing , speech intelligibility , social interaction , emotional status , adjustment to limitation , employability , problem solving , memory , orientation , attention span , and safety judgment hrql questionnaires give outcome measures that evaluate the impact of health on a person s ability to lead a fulfilling life , and generally incorporate the individual s perceptions of physical , mental / emotional , family , and social functioning . although a number of stroke - specific ql scales have been developed , measures that ecologically identify both the effects of linguistic communicative disability and the quality of everyday life are still needed for pwa . they could be useful to bridge the gap between linguistic rehabilitation and the patient s real use of residual means of communication or ability to perform the daily life activities in which language is involved . in this way , they could link the individual rehabilitation training to outcomes of increased functional autonomy in social and communicative environments , according to the recent world health organization international classification of functioning , disability and health ( icf).30 one example of these measures is the functional outcome questionnaire for aphasia,31 used in the us . it consists of 32 items , and is focused on the efficacy of the aphasic s verbal and nonverbal communicative abilities . this scale assesses the ability to communicate basic needs and new information and the ability to make routine requests ; it also investigates the issues of communication and comprehension . the saqol-3932 scale , recently translated into italian and also into spanish,33,34 is a more complete ql assessment ( it includes measurement of general , physical , and psychosocial health and vitality ) , even if there are few linguistic items compared to the total number ( nine of 39 ) . our qlqa has the aim of addressing a lack of ql measures in the italian language , including , as with previous ones , measurement of several domains , such as physical , psychological , communication , and social participation . engell et al35 developed a pictorial procedure for rating ql to minimize the influence of aphasia . they transformed an existing ql inventory the modified german version of the sickness impact profile36 into a picture - based representation . the authors selected the aachen quality of life inventory ( alqi),37 a german - language adaptation of the sickness impact profile . the alqi items were transformed into a pictorial version to maximize aphasic patients understanding of the verbal statements , as well as to permit them to give nonverbal responses . this procedure allowed for self - rating of aphasic patients in parallel to a proxy rating by caregivers . there was in fact also a written version for the caregivers that corresponded item by item to the patients pictorial version . the limited use of these assessments has negative effects on the course of clinical welfare . in italy , stroke rehabilitation still focuses more on the improvement of physical autonomy rather than on a global increase in neuropsychological functions , which are often affected . rehabilitation therapy should aim not only to regain communicative linguistic abilities , but also to help in adapting to the disability and to encourage social integration and personal well - being , thus improving ql . our ql questionnaire was developed by means of the aforementioned conceptual considerations and analysis of literature studies and of the previous ql and functional questionnaires for aphasic patients , in order to point out the aphasic disease , handicap and coping behavior in everyday life . the questionnaire consists of 37 questions that are important for daily well - being , chosen from a larger repertoire by a mixed group of pwa and their caregivers and members of the italian aphasia association of our region ( puglia ) . we consequently based the qlqa more on the percentage of residual abilities performed by the individuals with aphasia rather than on their perceived ql , which was difficult to investigate , considering their communicative problems . express the disability , mainly as relationship problems and loss of autonomy caused by linguistic deficits ; it consists in a majority of questions regarding language - related activities and abilities consider verbal comprehension difficulties include items of residual functional disability due to motor deficits include items on psychological problems that occur both in the poststroke phase and later , partly as a result of brain damage but also the social and individual aftereffects of aphasia highlight relational and nonrelational residual problems due to language deficits . the questions , formulated as easily and succinctly as possible , assess the ability to perform basic functions and convey health problems , the psychological changes due to the disability and the ability to socialize , the ability of linguistic and contextual comprehension , and expression in routine daily activities . the items are scored on a 5-point scale , with 0= the individual is able to successfully perform the behavior 0% of the time , 1= the individual is able to successfully perform the behavior 25% of the time , 2= the individual is able to successfully perform the behavior 50% of the time , 3= the individual is able to successfully perform the behavior 75% of the time , and 4= the individual is able to successfully perform the behavior 100% of the time . the qlqa was administered by a speech therapist using verbal or pragmatic means ( gestures and drawings ) when the patient showed comprehension deficits , in the presence of the caregiver . when there was no agreement in the answers , we considered those of the caregiver more reliable , so excluding the influence of the patients reduced consciousness of their own limits or anxiety / depression state . demographic and clinical variables of aphasic sample and control subjects were evaluated with descriptive statistics ( means , standard deviations , etc),using student s t - test for analyzing the differences between two samples . due to the ordinal nature of the scale , differences in the qlqa scores and in other tests of both groups were analyzed using nonparametric tests ( mann whitney u ) . we used standard psychometric methods3840 to evaluate internal consistency , test retest reliability , and construct validity ( convergent and discriminant ) of qlqa in 146 aphasic patients . a principal - component analysis ( pca , a type of factor analysis ) was used to determine the number and the type of domains underlying the ql of aphasic patients and reduce the number of items in the qlqa to those best measuring it . in order to evaluate convergent and discriminant validity , a multiple correlation among the scores of the qlqa subtests ( communication , autonomy , and psychological condition ) , resulting from pca , and the fim and fam scores and the correct linguistic tests scores were calculated by spearman s correlation coefficient . internal consistency was calculated using cronbach s -test and test retest reliability using intraclass correlation coefficient ( icc ) . data analyses were carried out with spss 18.0 for windows ( ibm , armonk , ny , usa ) . table 1 shows the demographic and clinical variables of the entire sample of aphasic patients . the 146 aphasic subjects ( 67 female and 79 male ) had a mean age of 68.4 years , a mean education of 6.96 years , and a mean poststroke period before assessment of 399.16 days . most patients ( 80.2% ) were married and had a caregiver , 17 aphasic subjects were widowed but had a child as a caregiver , while twelve patients were single . the language assessment by aat classified 102 patients as nonfluent pwa ( 61 with global aphasia , 39 with broca s aphasia , and two with transcortical motor aphasia ) , and 38 patients with a fluent form of aphasia ( 25 with wernicke s aphasia , twelve amnesic , one with a transcortical sensorial aphasia ) . olkin measure ( kmo ) verified the sampling adequacy for the analyses ( kmo = 0.924 ) , and all kmo values for individual items were > 0.6 , which is well above the acceptable limit of 0.5 . bartlett s test of sphericity =4,674.195 , p<0.001 indicated that correlations between items were sufficiently large for pca . the final model explained 59.5% of the variance and included three factors : the first factor , named communication , comprises 22 items evaluating the patient s ability to express and understand in real life and pragmatic situations ; the second factor , named psychological condition , includes six items evaluating the impact of language deficits on emotional status ; and the third factor , named autonomy , includes nine items assessing the independence of the subject in activities of daily life . we assessed the qlqa reliability , ie , the internal consistency and test retest reliability . to assess the extent to which qlqa items measure the format and homogeneity of the scales , we calculated the internal consistency using cronbach s -coefficients ( table 3 ) for the whole scale and for each of the three subscales . the internal consistency of the qlqa was high ( =0.96 , criteria for acceptability = cronbach s >0.70 ) . the qlqa subscales showed similarly high reliability , ranging from 0.79 for psychological condition to 0.97 for communication subscale and 0.89 for autonomy subscale . the test retest reliability , defined as the stability of an instrument over time , was evaluated by administering this test at a 3-day interval to a small sample of 14 ( 10% of complete sample ) aphasic patients with similar characteristics to those of the overall sample in terms of age , sex , marital status , and aphasia . retest reliability for both overall ( icc = 0.98 ) and subscale scores ( communication icc = 0.95 , autonomy icc = 0.85 , psychological condition icc = 0.65 ) . iccs < 0.40 were seen as indicating poor agreement , 0.400.75 fair - to - good agreement , and 0.761.00 excellent agreement.39 qlqa total , communication , and autonomy subscales had excellent reliability . the lower test retest reliability of the psychological condition subscale can be lined up with a physiological variation of the patient s mood state . the convergent validity was calculated by correlating the qlqa subscales and total scores with tests that assessed the same or similar constructs . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . all these scales in fact evaluated the patients autonomy both for motor and in cognitive aspects . high correlations were found also between qlqa total and communication subscale and aat scores ( respectively , r=0.58 , r=0.63 ) . a significant correlation was found with fam emotional status and social interaction subscales and qlqa psychological condition subscale . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . low correlations were found between the qlqa total and subscale scores and raven s coloured progressive matrices score . for the qlqa psychological condition subscale , we found low correlation coefficients , showing the specificity of this subscale . subjects with aphasia ( n=146 ) were divided into two groups based on the severity of language disorders , according to aat assessment . a total of 112 patients had severe aphasia , and 34 had mild language disorders . whitney test to compare the qlqa , linguistic tests , and fim scores of severe and mild patients . severe patients , apart from having a more severe linguistic deficit , were significantly more impaired in motor and cognitive autonomy , based on fim data ( p<0.001 ) . significant differences were found in communicative , autonomy , and total qlqa scores ( p<0.001 ) : eighty - four aphasic patients were assessed within 3 months from stroke , and 62 patients were evaluated in a chronic stage of disease ( after 3 months ) . the two groups were different for age ( p<0.01 ) and time from stroke ( p<0.001 ) , and were comparable for educational level ( table 6 ) . linguistic and motor conditions were significantly different between the groups . no difference was found in raven s test score . qlqa patients evaluated in the acute stage showed greater impairment in communication , autonomy , and total score . finally , we evaluated the specific role of linguistic deficits on ql in two groups of patients with and without aphasia in the acute stage of disease . since the time from stroke has an influence on ql of patients , we chose to compare the qlqa scores of 84 acute pwa with hemiplegia to those of a control group of 37 subjects with motor impairment and without aphasic problems , assessed within 3 months from stroke . the two groups were compared in the following series of tests : raven s coloured progressive matrices the control patients had a mean age of 64.79 years , a mean education of 7.69 years , and a mean time from stroke of 114.19 days . pwa and controls were similar for age , educational level , sex , and motor autonomy . significant differences were found in time from stroke ( p<0.001 ) , cognitive ( p<0.001 ) and total fim scores ( p<0.001 ) , and token test ( p<0.001 ) . statistically significant differences between pwa and control subjects were found regarding qlqa total and subscale scores ( communication , autonomy , and psychological condition ) . we assessed the qlqa reliability , ie , the internal consistency and test retest reliability . to assess the extent to which qlqa items measure the format and homogeneity of the scales , we calculated the internal consistency using cronbach s -coefficients ( table 3 ) for the whole scale and for each of the three subscales . the internal consistency of the qlqa was high ( =0.96 , criteria for acceptability = cronbach s >0.70 ) . the qlqa subscales showed similarly high reliability , ranging from 0.79 for psychological condition to 0.97 for communication subscale and 0.89 for autonomy subscale . the test retest reliability , defined as the stability of an instrument over time , was evaluated by administering this test at a 3-day interval to a small sample of 14 ( 10% of complete sample ) aphasic patients with similar characteristics to those of the overall sample in terms of age , sex , marital status , and aphasia . retest reliability for both overall ( icc = 0.98 ) and subscale scores ( communication icc = 0.95 , autonomy icc = 0.85 , psychological condition icc = 0.65 ) . iccs < 0.40 were seen as indicating poor agreement , 0.400.75 fair - to - good agreement , and 0.761.00 excellent agreement.39 qlqa total , communication , and autonomy subscales had excellent reliability . the lower test retest reliability of the psychological condition subscale can be lined up with a physiological variation of the patient s mood state . the convergent validity was calculated by correlating the qlqa subscales and total scores with tests that assessed the same or similar constructs . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . all these scales in fact evaluated the patients autonomy both for motor and in cognitive aspects . high correlations were found also between qlqa total and communication subscale and aat scores ( respectively , r=0.58 , r=0.63 ) . a significant correlation was found with fam emotional status and social interaction subscales and qlqa psychological condition subscale . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . low correlations were found between the qlqa total and subscale scores and raven s coloured progressive matrices score . for the qlqa psychological condition subscale , we found low correlation coefficients , showing the specificity of this subscale . the convergent validity was calculated by correlating the qlqa subscales and total scores with tests that assessed the same or similar constructs . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . all these scales in fact evaluated the patients autonomy both for motor and in cognitive aspects . high correlations were found also between qlqa total and communication subscale and aat scores ( respectively , r=0.58 , r=0.63 ) . a significant correlation was found with fam emotional status and social interaction subscales and qlqa psychological condition subscale . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . low correlations were found between the qlqa total and subscale scores and raven s coloured progressive matrices score . for the qlqa psychological condition subscale , we found low correlation coefficients , showing the specificity of this subscale . subjects with aphasia ( n=146 ) were divided into two groups based on the severity of language disorders , according to aat assessment . a total of 112 patients had severe aphasia , and 34 had mild language disorders . whitney test to compare the qlqa , linguistic tests , and fim scores of severe and mild patients . severe patients , apart from having a more severe linguistic deficit , were significantly more impaired in motor and cognitive autonomy , based on fim data ( p<0.001 ) . significant differences were found in communicative , autonomy , and total qlqa scores ( p<0.001 ) : mild pwa in fact had a better ql than severe patients . eighty - four aphasic patients were assessed within 3 months from stroke , and 62 patients were evaluated in a chronic stage of disease ( after 3 months ) . the two groups were different for age ( p<0.01 ) and time from stroke ( p<0.001 ) , and were comparable for educational level ( table 6 ) . qlqa patients evaluated in the acute stage showed greater impairment in communication , autonomy , and total score . finally , we evaluated the specific role of linguistic deficits on ql in two groups of patients with and without aphasia in the acute stage of disease . since the time from stroke has an influence on ql of patients , we chose to compare the qlqa scores of 84 acute pwa with hemiplegia to those of a control group of 37 subjects with motor impairment and without aphasic problems , assessed within 3 months from stroke . the two groups were compared in the following series of tests : raven s coloured progressive matrices the control patients had a mean age of 64.79 years , a mean education of 7.69 years , and a mean time from stroke of 114.19 days . pwa and controls were similar for age , educational level , sex , and motor autonomy . significant differences were found in time from stroke ( p<0.001 ) , cognitive ( p<0.001 ) and total fim scores ( p<0.001 ) , and token test ( p<0.001 ) . statistically significant differences between pwa and control subjects were found regarding qlqa total and subscale scores ( communication , autonomy , and psychological condition ) . the results of this preliminary psychometric examination suggest that the qlqa is a reliable and valid measure of ql in people with aphasia . the internal consistency was found to be high . the data of convergent and discriminant validity are not univocal : the correlation coefficients are not very high , and sometimes there were correlations also with different measures . this could be due to the ecological value of the qlqa : as in daily life , qlqa comprehension is influenced and improved by pragmatic aids , unlike token test performances , causing the absence of any relationship between pwa subjective estimation of their comprehension difficulties and the token test results.41 moreover , also other measures used for this study , such as fam , evaluate the patient s functional autonomy in multiple cognitive domains that do not leave out of consideration the use of language . this could explain , for example , the correlations that we found between qlqa total , subscales , and fam subscales . the qlqa differs from other ql questionnaires due to the greater importance given to the reduction of autonomy caused by aphasia , independently of motor deficits , as shown by the differences between pwa and controls . our results showed that the time from stroke and the severity of aphasia influence the patient s functional and communicative autonomy , but not the psychological condition . the mild and the chronic aphasic patients had a better quality of life than severe and acute ones , underlining the fact the passing of time helps patients with language disorders to adapt themselves to the new condition . in the literature , other studies,20,42 reported low - to - moderate improvements in ql after stroke . the physical and communication domains present greater improvement between acute stroke and 3 months , while psychosocial well - being takes longer to improve poststroke . regarding severity of language disease , engell et al35 found a correlation between the alqi ratings and the performance scores of the aat . total , physical , and psychosocial scores were significantly correlated with communicative and systematic failures in spontaneous language , but not with articulation disorders . moreover subjects with aphasia , independently of severity and time , tend towards social isolation and demand that other family members manage personal and family problems . our results agree with many studies in the literature that underline that the frequency of depression in aphasic patients is higher than in other stroke survivors.9,43 this test was given to pwa in the presence of a caregiver and with the aid of a speech therapist to overcome comprehension / expression deficits and avoid the problem of missing data . this could be a valid method when using these questionnaires in future , since severe pwa have until now been excluded from similar evaluations . other studies18 suggested that proxy respondents can provide reliable information on the hrql of pwa at the chronic stage of disease , even if self - report is more valid than any proxy report . however , using proxies may be a useful way to obtain information on the hrql of patients with severe aphasia . with qlqa , the differences among emotional , social , and communicative aspects of pwa and patients with motor deficits clearly emerge . this has an important effect on rehabilitation : on the one hand , it justifies the equal importance of language and motor rehabilitation ; on the other , it explains the reasons for a greater incidence of stress among pwa and their family members.44 hrql scales are essential in stroke assessment and outcome measurement . with patients subjective evaluation of their functioning and well - being , the speech - language pathologists and related professionals get a more holistic picture of how stroke and aphasia has affected patients lives , and they can make more informed decisions on what needs to be targeted in intervention . lastly , these findings suggest that people with stroke , and particularly those with aphasia , need long - term service provision that takes into account their affected mood , through community - based interventions , eg , participation in personally relevant meaningful activities . the use of qlqa in the assessment of people with aphasia focuses on the frequency of activities of life , depending on the correct use of language . it may point out gains in linguistic and communicative behaviors , due to rehabilitation training and/or personal ability of coping with disease limitation , and it shows daily personal aspects of discomfort , which are rarely evidenced by the patients , while leading them to isolation and passivity . timely interventions by speech - language pathologists and occupational therapists , psychological support to patients and caregivers , and changes in communicative environments are needed to promote socialization , according to the conceptual models of the icf . as aphasia causes communication impairment and altered relationships , for aphasic people the aim of rehabilitation should be to improve either the disability ( making language and communication more efficient ) or the possibilities of coping behaviors , intervening in the social sphere , and reinforcing self - esteem and the desire to return to personal autonomy . a limitation of this study lies in the comparison of the qlqa with measures of functional autonomy . it could be interesting also to compare the qlqa psychological condition subscale with scales of mood state , in order to increase the construct validity of our questionnaire . as is common with new measures , further research is needed to confirm its psychometric properties in independent samples . moreover , it is necessary to evaluate the test retest reliability in a larger sample of pwa . our next objective is to evaluate the ql of aphasic patients through a multicentric longitudinal study , in order to assess how ql changes with the evolution of language disease . qlqa is a valid measure of ql in pwa , contributing to a better distinction between severe and mild aphasia and also to the variations in ql depending on the time interval from stroke . improvement in the severity of language deficits also causes an improvement in ql , except for the psychological condition . this underlines the important role of aphasia in social isolation and emotional distress of patients . the regular use of ql scales in the assessment of pwa highlights their disability , handicap , and ability to cope . qlqa would be a useful tool in planning rehabilitation with a view to achieving greater functional autonomy in social and communicative environments .

backgroundquality of life ( ql ) can be defined as the individual s perception of their own well - being . aphasia is the most important potential consequence of stroke and has a profound effect on a patient s life , causing emotional distress , depression , and social isolation , due to loss of language functions.aimsto draw up a ql questionnaire for aphasics ( qlqa ) focusing particularly on difficulties in interpersonal relationships and on the loss of independence as a result of language disorders . we reported the results of a psychometric evaluation of this measure . moreover , we experimentally focused on the differences in qlqa between patients affected only by neurological motor impairment and hemiparetic patients with aphasia ( pwa ) in order to verify the specific role of aphasia on ql . we also explored if the qlqa is sensitive to the severity of aphasia and to the time elapsing from the stroke.methodsa total of 146 consecutive pwa and 37 control subjects were enrolled to evaluate the reliability ( internal consistency and test retest reliability ) and validity of the qlqa , using standard psychometric methods . patients were divided into acute ( within 3 months since stroke ) and chronic ( beyond 3 months ) groups , and into mild and severe according to the severity of aphasia . the experimental group of only acute pwa was compared to control subjects , with right hemispherical lesion and without aphasia in qlqa total and partial scores.resultsthe qlqa had good internal consistency and test retest reliability . acute and chronic pwa and mild and severe ones differed in qlqa total , communication , and autonomy subscales . no differences were found in psychological condition . between aphasic and control patients , significant differences were found in all qlqa subscales.conclusionthe qlqa is a valid measure of ql in pwa , contributing to a better distinction between severe and mild aphasia , and it is sensitive also to the variations in ql depending on the time interval from stroke .

Introduction Materials and methods Subjects Materials Quality of life questionnaire for aphasics Psychometric analysis Results Reliability Validity Convergent and discriminant validity Sensitivity to the severity of aphasia Sensitivity to time from stroke Differences between PWA and control subjects Discussion Conclusion

quality of life ( ql ) may be defined as an individual s perception of their well - being . health - related ql reflects the impact of a healthy state on a person s ability to lead a fulfilling life , and covers the individual s satisfaction in physical , functional , psychological , and social domains.1 studies2,3 have shown that ql worsens after brain damage ( vascular lesions , head injury , infections ) . changes in ql in the poststroke period do not seem to be age-4 or sex - dependent , but rather are due to the onset of depression5 and a low level of reacquisition in motor functions , above all in the upper limbs.6 ql improves within the family environment.7 aphasia is the loss of or radical change in voluntary speech as a consequence of damage to the left cerebral hemisphere , and generally causes significant changes in a patient s self - image and family , social , and work relationships.8 the impairment in language skills due to aphasia causes problems in verbal expression , auditory comprehension , reading , and writing . studies have documented high levels of depression9 and social exclusion,10 and low levels of leisure and other social activities,11 social contacts,12 and ql13,14 among patients with aphasia ( pwa ) . significant correlations have been found between degree of aphasia and the social , emotional , mobility , and total scores of ql , measured with the nottingham health profile.15 in a population - based study , lam and wodchis16 found that aphasia exhibited the largest negative influence on ql , followed by cancer and alzheimer s disease . in the first group , the handicaps included changes in situations of communication , in interpersonal relationships , loss of autonomy , and restriction of activities . in regard to the studies on the impact of aphasia on patient s life , there are two paradigms . also in this case , evaluating one s emotional distress , aphasia severity , communication and activity limitations , other medical problems , and social aspects all influenced ql . hilari et al20 evaluated the ql in patients with and without aphasia , testing a stroke - specific hrql scale ( the stroke and aphasia quality of life scale [ saqol-39 ] ) in a generic stroke sample that included patients both with and without aphasia . this scale demonstrates good internal consistency , test retest reliability and validity , and adequate responsiveness to changes in a sample of patients with acute and chronic stroke . the first aim of this observational study was to conduct a preliminary evaluation of the psychometric properties ( validity , reliability ) of a ql questionnaire for aphasics ( qlqa ) , in which we focused particularly on difficulties in interpersonal relationships , on loss of independence , and on abilities in daily life as a result of language disorders . moreover , in the aphasic group , we studied how qlqa was sensitive to the severity and type of aphasia and to the time from onset . finally , we tried to identify the specific role of linguistic deficits on ql , minimizing the effects of motor impairment . written consent all the patients underwent an initial screening to evaluate the presence and type of linguistic disorders and the severity of motor impairment . for this study , the patients underwent the following series of tests , administered by a speech therapist : the subtest of spontaneous speech of the aat,21 structured in six parts ( communicative behavior , articulation and prosody , automatic language , semantics , phonology , syntax ) , with a score range from 0 to 5 the token test22 to assess verbal comprehension with 36 items raven s coloured progressive matrices,23,24 which provides a nonverbal measure of intellectual abilities ; the patient must logically complete a given visual spatial pattern , choosing from a set of six alternatives a scale of functional independence the functional independence measure25,26 ( fim ) divided into scores for motor fim ( 13 items ) and cognitive fim ( five items ) another scale of functional independence the functional assessment measure ( fam)2729 cognitive subscale a compound of fourteen items evaluating comprehension , expression , reading , writing , speech intelligibility , social interaction , emotional status , adjustment to limitation , employability , problem solving , memory , orientation , attention span , and safety judgment hrql questionnaires give outcome measures that evaluate the impact of health on a person s ability to lead a fulfilling life , and generally incorporate the individual s perceptions of physical , mental / emotional , family , and social functioning . in this way , they could link the individual rehabilitation training to outcomes of increased functional autonomy in social and communicative environments , according to the recent world health organization international classification of functioning , disability and health ( icf).30 one example of these measures is the functional outcome questionnaire for aphasia,31 used in the us . our ql questionnaire was developed by means of the aforementioned conceptual considerations and analysis of literature studies and of the previous ql and functional questionnaires for aphasic patients , in order to point out the aphasic disease , handicap and coping behavior in everyday life . the questionnaire consists of 37 questions that are important for daily well - being , chosen from a larger repertoire by a mixed group of pwa and their caregivers and members of the italian aphasia association of our region ( puglia ) . express the disability , mainly as relationship problems and loss of autonomy caused by linguistic deficits ; it consists in a majority of questions regarding language - related activities and abilities consider verbal comprehension difficulties include items of residual functional disability due to motor deficits include items on psychological problems that occur both in the poststroke phase and later , partly as a result of brain damage but also the social and individual aftereffects of aphasia highlight relational and nonrelational residual problems due to language deficits . the items are scored on a 5-point scale , with 0= the individual is able to successfully perform the behavior 0% of the time , 1= the individual is able to successfully perform the behavior 25% of the time , 2= the individual is able to successfully perform the behavior 50% of the time , 3= the individual is able to successfully perform the behavior 75% of the time , and 4= the individual is able to successfully perform the behavior 100% of the time . due to the ordinal nature of the scale , differences in the qlqa scores and in other tests of both groups were analyzed using nonparametric tests ( mann whitney u ) . we used standard psychometric methods3840 to evaluate internal consistency , test retest reliability , and construct validity ( convergent and discriminant ) of qlqa in 146 aphasic patients . in order to evaluate convergent and discriminant validity , a multiple correlation among the scores of the qlqa subtests ( communication , autonomy , and psychological condition ) , resulting from pca , and the fim and fam scores and the correct linguistic tests scores were calculated by spearman s correlation coefficient . for this study , the patients underwent the following series of tests , administered by a speech therapist : the subtest of spontaneous speech of the aat,21 structured in six parts ( communicative behavior , articulation and prosody , automatic language , semantics , phonology , syntax ) , with a score range from 0 to 5 the token test22 to assess verbal comprehension with 36 items raven s coloured progressive matrices,23,24 which provides a nonverbal measure of intellectual abilities ; the patient must logically complete a given visual spatial pattern , choosing from a set of six alternatives a scale of functional independence the functional independence measure25,26 ( fim ) divided into scores for motor fim ( 13 items ) and cognitive fim ( five items ) another scale of functional independence the functional assessment measure ( fam)2729 cognitive subscale a compound of fourteen items evaluating comprehension , expression , reading , writing , speech intelligibility , social interaction , emotional status , adjustment to limitation , employability , problem solving , memory , orientation , attention span , and safety judgment hrql questionnaires give outcome measures that evaluate the impact of health on a person s ability to lead a fulfilling life , and generally incorporate the individual s perceptions of physical , mental / emotional , family , and social functioning . in this way , they could link the individual rehabilitation training to outcomes of increased functional autonomy in social and communicative environments , according to the recent world health organization international classification of functioning , disability and health ( icf).30 one example of these measures is the functional outcome questionnaire for aphasia,31 used in the us . our qlqa has the aim of addressing a lack of ql measures in the italian language , including , as with previous ones , measurement of several domains , such as physical , psychological , communication , and social participation . rehabilitation therapy should aim not only to regain communicative linguistic abilities , but also to help in adapting to the disability and to encourage social integration and personal well - being , thus improving ql . our ql questionnaire was developed by means of the aforementioned conceptual considerations and analysis of literature studies and of the previous ql and functional questionnaires for aphasic patients , in order to point out the aphasic disease , handicap and coping behavior in everyday life . express the disability , mainly as relationship problems and loss of autonomy caused by linguistic deficits ; it consists in a majority of questions regarding language - related activities and abilities consider verbal comprehension difficulties include items of residual functional disability due to motor deficits include items on psychological problems that occur both in the poststroke phase and later , partly as a result of brain damage but also the social and individual aftereffects of aphasia highlight relational and nonrelational residual problems due to language deficits . the items are scored on a 5-point scale , with 0= the individual is able to successfully perform the behavior 0% of the time , 1= the individual is able to successfully perform the behavior 25% of the time , 2= the individual is able to successfully perform the behavior 50% of the time , 3= the individual is able to successfully perform the behavior 75% of the time , and 4= the individual is able to successfully perform the behavior 100% of the time . due to the ordinal nature of the scale , differences in the qlqa scores and in other tests of both groups were analyzed using nonparametric tests ( mann whitney u ) . we used standard psychometric methods3840 to evaluate internal consistency , test retest reliability , and construct validity ( convergent and discriminant ) of qlqa in 146 aphasic patients . in order to evaluate convergent and discriminant validity , a multiple correlation among the scores of the qlqa subtests ( communication , autonomy , and psychological condition ) , resulting from pca , and the fim and fam scores and the correct linguistic tests scores were calculated by spearman s correlation coefficient . the language assessment by aat classified 102 patients as nonfluent pwa ( 61 with global aphasia , 39 with broca s aphasia , and two with transcortical motor aphasia ) , and 38 patients with a fluent form of aphasia ( 25 with wernicke s aphasia , twelve amnesic , one with a transcortical sensorial aphasia ) . we assessed the qlqa reliability , ie , the internal consistency and test retest reliability . the test retest reliability , defined as the stability of an instrument over time , was evaluated by administering this test at a 3-day interval to a small sample of 14 ( 10% of complete sample ) aphasic patients with similar characteristics to those of the overall sample in terms of age , sex , marital status , and aphasia . iccs < 0.40 were seen as indicating poor agreement , 0.400.75 fair - to - good agreement , and 0.761.00 excellent agreement.39 qlqa total , communication , and autonomy subscales had excellent reliability . the lower test retest reliability of the psychological condition subscale can be lined up with a physiological variation of the patient s mood state . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . subjects with aphasia ( n=146 ) were divided into two groups based on the severity of language disorders , according to aat assessment . whitney test to compare the qlqa , linguistic tests , and fim scores of severe and mild patients . significant differences were found in communicative , autonomy , and total qlqa scores ( p<0.001 ) : eighty - four aphasic patients were assessed within 3 months from stroke , and 62 patients were evaluated in a chronic stage of disease ( after 3 months ) . finally , we evaluated the specific role of linguistic deficits on ql in two groups of patients with and without aphasia in the acute stage of disease . since the time from stroke has an influence on ql of patients , we chose to compare the qlqa scores of 84 acute pwa with hemiplegia to those of a control group of 37 subjects with motor impairment and without aphasic problems , assessed within 3 months from stroke . significant differences were found in time from stroke ( p<0.001 ) , cognitive ( p<0.001 ) and total fim scores ( p<0.001 ) , and token test ( p<0.001 ) . statistically significant differences between pwa and control subjects were found regarding qlqa total and subscale scores ( communication , autonomy , and psychological condition ) . we assessed the qlqa reliability , ie , the internal consistency and test retest reliability . the test retest reliability , defined as the stability of an instrument over time , was evaluated by administering this test at a 3-day interval to a small sample of 14 ( 10% of complete sample ) aphasic patients with similar characteristics to those of the overall sample in terms of age , sex , marital status , and aphasia . iccs < 0.40 were seen as indicating poor agreement , 0.400.75 fair - to - good agreement , and 0.761.00 excellent agreement.39 qlqa total , communication , and autonomy subscales had excellent reliability . the lower test retest reliability of the psychological condition subscale can be lined up with a physiological variation of the patient s mood state . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . high correlation coefficients were found between qlqa total , the communication and autonomy subscales , and fim ( respectively , r=0.69 , r=0.62 , r=0.67 ) and fam scores ( respectively , r=0.75 , r=0.73 , r=0.64 ) . to evaluate the discriminant validity , we calculated the correlation coefficients between the qlqa total and subscale scores and the scores of tests assessing different functions . subjects with aphasia ( n=146 ) were divided into two groups based on the severity of language disorders , according to aat assessment . whitney test to compare the qlqa , linguistic tests , and fim scores of severe and mild patients . significant differences were found in communicative , autonomy , and total qlqa scores ( p<0.001 ) : mild pwa in fact had a better ql than severe patients . eighty - four aphasic patients were assessed within 3 months from stroke , and 62 patients were evaluated in a chronic stage of disease ( after 3 months ) . finally , we evaluated the specific role of linguistic deficits on ql in two groups of patients with and without aphasia in the acute stage of disease . since the time from stroke has an influence on ql of patients , we chose to compare the qlqa scores of 84 acute pwa with hemiplegia to those of a control group of 37 subjects with motor impairment and without aphasic problems , assessed within 3 months from stroke . significant differences were found in time from stroke ( p<0.001 ) , cognitive ( p<0.001 ) and total fim scores ( p<0.001 ) , and token test ( p<0.001 ) . statistically significant differences between pwa and control subjects were found regarding qlqa total and subscale scores ( communication , autonomy , and psychological condition ) . the results of this preliminary psychometric examination suggest that the qlqa is a reliable and valid measure of ql in people with aphasia . this could be due to the ecological value of the qlqa : as in daily life , qlqa comprehension is influenced and improved by pragmatic aids , unlike token test performances , causing the absence of any relationship between pwa subjective estimation of their comprehension difficulties and the token test results.41 moreover , also other measures used for this study , such as fam , evaluate the patient s functional autonomy in multiple cognitive domains that do not leave out of consideration the use of language . the qlqa differs from other ql questionnaires due to the greater importance given to the reduction of autonomy caused by aphasia , independently of motor deficits , as shown by the differences between pwa and controls . our results showed that the time from stroke and the severity of aphasia influence the patient s functional and communicative autonomy , but not the psychological condition . the mild and the chronic aphasic patients had a better quality of life than severe and acute ones , underlining the fact the passing of time helps patients with language disorders to adapt themselves to the new condition . with qlqa , the differences among emotional , social , and communicative aspects of pwa and patients with motor deficits clearly emerge . with patients subjective evaluation of their functioning and well - being , the speech - language pathologists and related professionals get a more holistic picture of how stroke and aphasia has affected patients lives , and they can make more informed decisions on what needs to be targeted in intervention . the use of qlqa in the assessment of people with aphasia focuses on the frequency of activities of life , depending on the correct use of language . it may point out gains in linguistic and communicative behaviors , due to rehabilitation training and/or personal ability of coping with disease limitation , and it shows daily personal aspects of discomfort , which are rarely evidenced by the patients , while leading them to isolation and passivity . it could be interesting also to compare the qlqa psychological condition subscale with scales of mood state , in order to increase the construct validity of our questionnaire . moreover , it is necessary to evaluate the test retest reliability in a larger sample of pwa . our next objective is to evaluate the ql of aphasic patients through a multicentric longitudinal study , in order to assess how ql changes with the evolution of language disease . qlqa is a valid measure of ql in pwa , contributing to a better distinction between severe and mild aphasia and also to the variations in ql depending on the time interval from stroke . improvement in the severity of language deficits also causes an improvement in ql , except for the psychological condition . this underlines the important role of aphasia in social isolation and emotional distress of patients .

according to the measuring the information society report 2014 by the international telecommunication union , nearly 3,000 million people use the internet , which is more than 40% of the world 's population ( 7,200 million ) . there are almost as many mobile - cellular subscriptions ( 6.9 billion ) as people on earth , more than three quarters of them ( 5.4 billion ) in the developing world , and more than half ( 3.6 billion ) in the asia - pacific region . a study published by the lancet described " whatsappitis " as the wrist pain a young women had as a result of sending messages on their phones . the widespread use of mobile phones has resulted in higher access to the internet through these devices . in 2014 , the growth is driven by the availability of cheaper smartphones and the variety of types of plans on offer in the market . in south korea , internet penetration is very high ; 82.1% of the population goes online according to the korea internet & security agency . this percentage includes people older than three years old that who have used the internet at least once in the last month . the rising penetration rate of smartphones all over the world opens new possibilities in healthcare . accessing information , gathering health data and contacting health professionals are some of the promising opportunities that these devices offer . this is not just a small trend , but mobile health ( mhealth ) has widened acceptance by the public . it is estimated that there are 100,000 mhealth applications , but not all of them have the necessary quality . there is a call by the sector to establish quality control mechanisms to guarantee patients ' safety . the european commission recently published a report that gathers the views and actions suggested by stakeholders of the field following a public consultation . according to the european citizen 's digital health literacy report published in september 2014 , 59% of european citizens used the internet to look for health information in 2014 . fifty - five percent looked for general information , 54% looked for information on a specific condition , 23% looked for specific information on a treatment , and 10% used the net to get a second opinion after visiting their doctor . between 82% and 87% of people ( depending on the type of information searched ) who searched for health - related information used searches engines . the second source of information ( 47%-48% ) was specific and dedicated websites ( blogs and forums ) , while between 33% and 38% looked for information from official health websites , such as those of the ministry of health or the world health organization ( who ) . online newspapers or magazines ( 20%-26% ) and social networks ( 16%-23% ) come next , followed by health mobile apps ( 13%-17% ) and patient organization websites ( 13%-17% ) . for example , the leading social network in south korea is youtube , followed by facebook . in nigeria ( africa ) , more and more hospitals , medical professionals , and health authorities have opened accounts in youtube , facebook , and twitter to reach their potential clients . according to a study published by the journal of medical internet research , nearly 95% of us hospitals are on facebook , while only 50% have a twitter account . in western europe , the use of social media by hospitals is also growing but there are significant differences between countries . a study published in the journal of medical internet research shows how its use remains small , except for the netherlands and the united kingdom . nevertheless , it grew from 2009 to 2011 in networks such as facebook ( from 10% to 67% ) , linkedin ( from 20% to 31% ) , and youtube ( from 2% to 19% ) . many concepts explain the use of health 2.0 in healthcare : health 2.0 , medicine 2.0 , health social media , ehealth , mhealth . the study " definition of health 2.0 and medicine 2.0 : a systematic review " shows that this is a developing area , and there is not a consensus on its definition . some of the concepts that include health 2.0 are apomediation , participation , openness , and collaboration . gunther eysenbach , a health policy and ehealth professor at the university of toronto , describes the fact that when a user accesses information on the internet , he / she cuts out the gatekeepers ( like the primary care doctor ) and goes directly to the relevant source of information . in the last years , patients have become the main actors of health 2.0 , leading a true revolution in healthcare management . e - patients ( # epatients ) achieve better health outcomes thanks to being connected and sharing experiences in social networks ; previously , their health depended solely on their doctors ' capacity . epatients like dave debronkart ( @epatientdave ) , jan geissler ( @jangeissler ) , and andrew schorr ( @ andrewschorr ) overcame their cancer because they went online and connected with peer patients who told them about new treatments or clinical essays for their conditions . in addition to patients ' activism , health 2.0 also includes the online activity of medical professionals ( peer - to - peer ) , electronic health records , elearning , online medical publications ( peer review 2.0 ) , and science 2.0 . according to the european citizen 's digital health literacy report published in september 2014 , 59% of european citizens used the internet to look for health information in 2014 . fifty - five percent looked for general information , 54% looked for information on a specific condition , 23% looked for specific information on a treatment , and 10% used the net to get a second opinion after visiting their doctor . between 82% and 87% of people ( depending on the type of information searched ) who searched for health - related information used searches engines . the second source of information ( 47%-48% ) was specific and dedicated websites ( blogs and forums ) , while between 33% and 38% looked for information from official health websites , such as those of the ministry of health or the world health organization ( who ) . online newspapers or magazines ( 20%-26% ) and social networks ( 16%-23% ) come next , followed by health mobile apps ( 13%-17% ) and patient organization websites ( 13%-17% ) . for example , the leading social network in south korea is youtube , followed by facebook . in nigeria ( africa ) , more and more hospitals , medical professionals , and health authorities have opened accounts in youtube , facebook , and twitter to reach their potential clients . according to a study published by the journal of medical internet research , nearly 95% of us hospitals are on facebook , while only 50% have a twitter account . in western europe , the use of social media by hospitals is also growing but there are significant differences between countries . a study published in the journal of medical internet research shows how its use remains small , except for the netherlands and the united kingdom . nevertheless , it grew from 2009 to 2011 in networks such as facebook ( from 10% to 67% ) , linkedin ( from 20% to 31% ) , and youtube ( from 2% to 19% ) . many concepts explain the use of health 2.0 in healthcare : health 2.0 , medicine 2.0 , health social media , ehealth , mhealth . the study " definition of health 2.0 and medicine 2.0 : a systematic review " shows that this is a developing area , and there is not a consensus on its definition . some of the concepts that include health 2.0 are apomediation , participation , openness , and collaboration . gunther eysenbach , a health policy and ehealth professor at the university of toronto , describes the fact that when a user accesses information on the internet , he / she cuts out the gatekeepers ( like the primary care doctor ) and goes directly to the relevant source of information . in the last years , patients have become the main actors of health 2.0 , leading a true revolution in healthcare management . e - patients ( # epatients ) achieve better health outcomes thanks to being connected and sharing experiences in social networks ; previously , their health depended solely on their doctors ' capacity . epatients like dave debronkart ( @epatientdave ) , jan geissler ( @jangeissler ) , and andrew schorr ( @ andrewschorr ) overcame their cancer because they went online and connected with peer patients who told them about new treatments or clinical essays for their conditions . in addition to patients ' activism , health 2.0 also includes the online activity of medical professionals ( peer - to - peer ) , electronic health records , elearning , online medical publications ( peer review 2.0 ) , and science 2.0 . in this section we will explain how health 2.0 is shaping the health domain using four study cases . first of all , we will explain the role of health 2.0 in diabetes management as a prime example of a chronic condition . then , we will focus on the case of an eating disorder , specifically , pro - anorexia ( the promotion of anorexia as a lifestyle ) . the third case study will focus on the use of social media for vaccination communication . online communities of patients are very common in diabetes . in these networks people affected with diabetes these networks can be extremely large with dozens of thousands members and fairly complex networks . chomutare et al . studied the dynamics of online communities of diabetes patients , and they found that the most centric members ( those with more trust within the communities ) tend to have more years of experience with disease . many of these patients have become experts in their disease and provide a great amount of valuable information for their peers . shows that the most experienced patients - those who have been living longer with the disease - tend to be leaders in the community . similarly , a study published in the bmj found that patients with cancer could be also effective in controlling the quality of health information , thus highlighting the importance of experienced patients in the control of online health information . by contrast , seals of quality that started to proliferate a few years ago do not guarantee the veracity of the information published , as stated in a study by the american cancer society about quality criteria in online information about breast cancer . online communities of patients can also collaborate with researchers using social media . in tuanalyze , a joint initiative between tudiabetes and boston children 's hospital , diabetes patients can monitor , evaluate , and share their health data , while they are participating in a research on diabetes and social media ( figure 1 ) . their data is analysed to determine a correlation between people 's participation in health social networking and level of diabetes management . there is a wide range of mobile applications to help diabetes patients ( 347 million people ) manage their condition . these tools allow them to monitor their glucose level , weight , and other data ; share them ; and get motivated to lead a healthy life . although there is not enough clinical evidence about their efficacy , mobile health tools many interesting initiatives have been created to help patients control their condition through their mobile phones , such as diabetes diary and big blue test . according to a study by research2guidance , there are more than 1,100 apps ( ios and android ) to manage diabetes . despite the vast number of applications , their quality has been put into question in many studies ( e.g. , see chomutare et al . ) . researchers have pointed out that most applications do not offer enough educational features and lack social media tools . there have been studies on cancer apps too , and the results show that they also need significant improvement . the use of wearable devices that monitor physical activity and gather vital data has also grown in recent years , and they are popular among patients with diabetes . a few studies about the medical applications , calibration , and validation of wearable devices have been done . studies have shown that the development of wearable systems targeting specific clinical applications has potential in healthcare ( figure 2 ) . this new wide range of tools to store health data raises major questions regarding their integration into the healthcare system , the need for mass storage systems , and the privacy of users ' data . the big challenge is to find a way to access this knowledge while respecting people 's privacy . it is striking that such a serious health problem as anorexia has an army of activists that defend this disease as a lifestyle . pro - anorexia activists ( the so - called pro - ana ) are very visible in social networks , especially youtube and flickr , where image is predominant . they have even created mobile applications that defend their ' lifestyle ' ( thinspo , currently available in google play ) . on youtube , one can easily stumble across pro - anorexia videos when looking for health information on eating disorders . the study shows that pro - anorexia videos are better rated and more highly favoured than videos with serious information about eating disorders . anorexia activists use the same tags as reliable health videos , and their websites sometimes rank higher in youtube or google . this happens because of the way searching engine algorithms are designed , according to researchers . a study shows the high level of interaction between proanorexia and pro - recovery communities in social networks ( figure 3 ) . findings suggest that clinicians need to be aware of pro - anorexia contents online and focus on new intervention methods , possibly tailored to individual characteristics . over decades nevertheless , there is still a strong anti - vaccination movement ( it has existed since vaccines were invented more than 200 years ago ) , which in recent years , has moved its campaign to social networks . their activists are very good at manipulating information and spreading rumours that question vaccine efficacy and safety . " with ever increasing access to internet - based information , an unsubstantiated rumour about vaccines can rapidly circle the globe and undermine immunization services , sparking outbreaks of disease and untold deaths " stated who , unicef , and world bank in 2010 . various studies have analysed the influence of the antivaccination movement in social networks and in vaccination decisions or on the public trust in vaccines . some anti - vaccination websites , such as the national vaccine information center , look absolutely credible and can easily trick users into questioning vaccines safety . research has shown that anti - vaccination contents are more popular in social networks than validated vaccination contents . most videos ( news clips or consumer generated content ) were negative in tone , disapproving of the vaccine . another study analysed vaccination search and concluded that previous attitudes towards vaccination will influence the results of a search ; trusted health websites often use different vocabulary than those with concerns about vaccination ; search behaviour needs to be considered in online public health interventions . on the other hand , the study showed that there is a correlation between the sentiments expressed in social networks towards a new vaccine and the vaccination rates in a region . recently , the vaccine confidence project was created to monitor people 's trust in vaccination through social networks , given the importance that these tools have in vaccination decisions . the london school of hygiene and tropical medicine leads the project with the support of prestigious health organizations , such as the bill & melinda gates foundation and the world health organization . the recent ebola outbreak in western africa is another good example of the power of social networks in influencing people 's behaviour . the news of the epidemic created a climate of global nervousness with rumours and misinformation quickly spreading through social networks . the outbreak was first detected by healthmap , a website that delivers real - time data on infectious diseases for health institutions and governments . the role that social networks have played in the spread of rumours about ebola has been analysed in several studies . the british medical journal published an article that evaluated tweets regarding the outbreak emitted from african countries . social networks also helped spread rumours about fake treatments ; some of them even making the general news . similarly , fake news stories were also spread by sms and whatsapp messages . at the same time , mobile technologies can be a useful tool to control and contain epidemics , as some experts pointed out during the ebola crisis . sms is an instantaneous , interactive , and cheap tool that can reach a great number of people . through mobile phones these networks can be extremely large with dozens of thousands members and fairly complex networks . studied the dynamics of online communities of diabetes patients , and they found that the most centric members ( those with more trust within the communities ) tend to have more years of experience with disease . many of these patients have become experts in their disease and provide a great amount of valuable information for their peers . shows that the most experienced patients - those who have been living longer with the disease - tend to be leaders in the community . similarly , a study published in the bmj found that patients with cancer could be also effective in controlling the quality of health information , thus highlighting the importance of experienced patients in the control of online health information . by contrast , seals of quality that started to proliferate a few years ago do not guarantee the veracity of the information published , as stated in a study by the american cancer society about quality criteria in online information about breast cancer . online communities of patients can also collaborate with researchers using social media . in tuanalyze , a joint initiative between tudiabetes and boston children 's hospital , diabetes patients can monitor , evaluate , and share their health data , while they are participating in a research on diabetes and social media ( figure 1 ) . their data is analysed to determine a correlation between people 's participation in health social networking and level of diabetes management . there is a wide range of mobile applications to help diabetes patients ( 347 million people ) manage their condition . these tools allow them to monitor their glucose level , weight , and other data ; share them ; and get motivated to lead a healthy life . although there is not enough clinical evidence about their efficacy , mobile health tools many interesting initiatives have been created to help patients control their condition through their mobile phones , such as diabetes diary and big blue test . according to a study by research2guidance , there are more than 1,100 apps ( ios and android ) to manage diabetes . despite the vast number of applications , their quality has been put into question in many studies ( e.g. , see chomutare et al . ) . researchers have pointed out that most applications do not offer enough educational features and lack social media tools . there have been studies on cancer apps too , and the results show that they also need significant improvement . the use of wearable devices that monitor physical activity and gather vital data has also grown in recent years , and they are popular among patients with diabetes . a few studies about the medical applications , calibration , and validation of wearable devices have been done . studies have shown that the development of wearable systems targeting specific clinical applications has potential in healthcare ( figure 2 ) . this new wide range of tools to store health data raises major questions regarding their integration into the healthcare system , the need for mass storage systems , and the privacy of users ' data . the big challenge is to find a way to access this knowledge while respecting people 's privacy . these networks can be extremely large with dozens of thousands members and fairly complex networks . studied the dynamics of online communities of diabetes patients , and they found that the most centric members ( those with more trust within the communities ) tend to have more years of experience with disease . many of these patients have become experts in their disease and provide a great amount of valuable information for their peers . shows that the most experienced patients - those who have been living longer with the disease - tend to be leaders in the community . similarly , a study published in the bmj found that patients with cancer could be also effective in controlling the quality of health information , thus highlighting the importance of experienced patients in the control of online health information . by contrast , seals of quality that started to proliferate a few years ago do not guarantee the veracity of the information published , as stated in a study by the american cancer society about quality criteria in online information about breast cancer . online communities of patients can also collaborate with researchers using social media . in tuanalyze , a joint initiative between tudiabetes and boston children 's hospital , diabetes patients can monitor , evaluate , and share their health data , while they are participating in a research on diabetes and social media ( figure 1 ) . their data is analysed to determine a correlation between people 's participation in health social networking and level of diabetes management . there is a wide range of mobile applications to help diabetes patients ( 347 million people ) manage their condition . these tools allow them to monitor their glucose level , weight , and other data ; share them ; and get motivated to lead a healthy life . although there is not enough clinical evidence about their efficacy , mobile health tools many interesting initiatives have been created to help patients control their condition through their mobile phones , such as diabetes diary and big blue test . according to a study by research2guidance , there are more than 1,100 apps ( ios and android ) to manage diabetes . despite the vast number of applications , their quality has been put into question in many studies ( e.g. , see chomutare et al . ) . researchers have pointed out that most applications do not offer enough educational features and lack social media tools . there have been studies on cancer apps too , and the results show that they also need significant improvement . the use of wearable devices that monitor physical activity and gather vital data has also grown in recent years , and they are popular among patients with diabetes . a few studies about the medical applications , calibration , and validation of wearable devices have been done . studies have shown that the development of wearable systems targeting specific clinical applications has potential in healthcare ( figure 2 ) . this new wide range of tools to store health data raises major questions regarding their integration into the healthcare system , the need for mass storage systems , and the privacy of users ' data . the big challenge is to find a way to access this knowledge while respecting people 's privacy . it is striking that such a serious health problem as anorexia has an army of activists that defend this disease as a lifestyle . pro - anorexia activists ( the so - called pro - ana ) are very visible in social networks , especially youtube and flickr , where image is predominant . they have even created mobile applications that defend their ' lifestyle ' ( thinspo , currently available in google play ) . on youtube , one can easily stumble across pro - anorexia videos when looking for health information on eating disorders . the study shows that pro - anorexia videos are better rated and more highly favoured than videos with serious information about eating disorders . anorexia activists use the same tags as reliable health videos , and their websites sometimes rank higher in youtube or google . this happens because of the way searching engine algorithms are designed , according to researchers . a study shows the high level of interaction between proanorexia and pro - recovery communities in social networks ( figure 3 ) . findings suggest that clinicians need to be aware of pro - anorexia contents online and focus on new intervention methods , possibly tailored to individual characteristics . over decades , vaccines have saved millions of lives and have helped to eradicate numerous diseases . nevertheless , there is still a strong anti - vaccination movement ( it has existed since vaccines were invented more than 200 years ago ) , which in recent years , has moved its campaign to social networks . their activists are very good at manipulating information and spreading rumours that question vaccine efficacy and safety . " with ever increasing access to internet - based information , an unsubstantiated rumour about vaccines can rapidly circle the globe and undermine immunization services , sparking outbreaks of disease and untold deaths " stated who , unicef , and world bank in 2010 . various studies have analysed the influence of the antivaccination movement in social networks and in vaccination decisions or on the public trust in vaccines . some anti - vaccination websites , such as the national vaccine information center , look absolutely credible and can easily trick users into questioning vaccines safety . research has shown that anti - vaccination contents are more popular in social networks than validated vaccination contents . most videos ( news clips or consumer generated content ) were negative in tone , disapproving of the vaccine . another study analysed vaccination search and concluded that previous attitudes towards vaccination will influence the results of a search ; trusted health websites often use different vocabulary than those with concerns about vaccination ; search behaviour needs to be considered in online public health interventions . on the other hand , the study showed that there is a correlation between the sentiments expressed in social networks towards a new vaccine and the vaccination rates in a region . recently , the vaccine confidence project was created to monitor people 's trust in vaccination through social networks , given the importance that these tools have in vaccination decisions . the london school of hygiene and tropical medicine leads the project with the support of prestigious health organizations , such as the bill & melinda gates foundation and the world health organization . the recent ebola outbreak in western africa is another good example of the power of social networks in influencing people 's behaviour . the news of the epidemic created a climate of global nervousness with rumours and misinformation quickly spreading through social networks . the outbreak was first detected by healthmap , a website that delivers real - time data on infectious diseases for health institutions and governments . the role that social networks have played in the spread of rumours about ebola has been analysed in several studies . the british medical journal published an article that evaluated tweets regarding the outbreak emitted from african countries . the researchers concluded that most of the messages had false information and that ' bad ' tweets were more retweeted than the ones with truthful information . social networks also helped spread rumours about fake treatments ; some of them even making the general news . similarly , fake news stories were also spread by sms and whatsapp messages . at the same time , mobile technologies can be a useful tool to control and contain epidemics , as some experts pointed out during the ebola crisis . sms is an instantaneous , interactive , and cheap tool that can reach a great number of people . through mobile phones as we have seen , the internet is rapidly expanding and is being used for health purposes by a great number of people . sometimes they help spread rumours and fake information and can undermine public health policies . given the lack of knowledge on the use of health social media , there is a need for complex multidisciplinary research ( mathematical , computer science , sociology , health , communication , etc . ) to help us understand how to use social networks to support public health . a better understanding of social media will give health authorities new tools to help decision - making at global , national , local , and corporate levels . for example , epidemiology is gradually becoming a discipline in which digital sources of information are becoming more important , and this has led to the creation of the sub - discipline of digital epidemiology . there is an unprecedented amount of data that can be used in public health due the potential combination of data acquired from mobile phones , electronic health records , social media and other sources . moreover , new analytics tools need to be developed to analyse those sources of data in a way that can benefit healthcare professionals and authorities .

objectivesthe use of internet in the health domain is becoming a major worldwide trend . millions of citizens are searching online health information and also publishing content about their health . patients are engaging with other patients in online communities using different types of social media . the boundaries between mobile health , social media , wearable , games , and big data are becoming blurrier due the integration of all those technologies . in this paper we provide an overview of the major research challenges with the area of health social media.methodswe use several study cases to exemplify the current trends and highlight future research challenges.resultsinternet is exploding and is being used for health purposes by a great deal of the population . social networks have a powerful influence in health decisions . given the lack of knowledge on the use of health social media , there is a need for complex multidisciplinary research to help us understand how to use social networks in favour of public health . a bigger understanding of social media will give health authorities new tools to help decision - making at global , national , local , and corporate level.conclusionsthere is an unprecedented amount of data that can be used in public health due the potential combination of data acquired from mobile phones , electronic health records , social media , and other sources . to identify meaningful information from those data sources it is not trial . moreover , new analytics tools will need to be developed to analyse those sources of data in a way that it can benefit healthcare professionals and authorities .

Introduction 1. Use of the Internet to Search for Health-Related Information 2. Social Media: Same Networks Everywhere 3. e-Patients, Prime Actors in Health 2.0 Study Cases in Health 2.0: Diabetes, Anorexia, Vaccination, and Ebola 1. Diabetes and Social Media 1) Expert e-Patients in diabetes 2) The boom of mobile applications for diabetes 2. Anorexia: the Power of Misinformation 3. Anti-vaccination, a Dangerous Movement on the Net 4. Ebola: How Panic Spreads through Social Media Conclusion

according to the measuring the information society report 2014 by the international telecommunication union , nearly 3,000 million people use the internet , which is more than 40% of the world 's population ( 7,200 million ) . there are almost as many mobile - cellular subscriptions ( 6.9 billion ) as people on earth , more than three quarters of them ( 5.4 billion ) in the developing world , and more than half ( 3.6 billion ) in the asia - pacific region . the widespread use of mobile phones has resulted in higher access to the internet through these devices . in 2014 , the growth is driven by the availability of cheaper smartphones and the variety of types of plans on offer in the market . in south korea , internet penetration is very high ; 82.1% of the population goes online according to the korea internet & security agency . this percentage includes people older than three years old that who have used the internet at least once in the last month . this is not just a small trend , but mobile health ( mhealth ) has widened acceptance by the public . there is a call by the sector to establish quality control mechanisms to guarantee patients ' safety . according to the european citizen 's digital health literacy report published in september 2014 , 59% of european citizens used the internet to look for health information in 2014 . fifty - five percent looked for general information , 54% looked for information on a specific condition , 23% looked for specific information on a treatment , and 10% used the net to get a second opinion after visiting their doctor . between 82% and 87% of people ( depending on the type of information searched ) who searched for health - related information used searches engines . the second source of information ( 47%-48% ) was specific and dedicated websites ( blogs and forums ) , while between 33% and 38% looked for information from official health websites , such as those of the ministry of health or the world health organization ( who ) . in nigeria ( africa ) , more and more hospitals , medical professionals , and health authorities have opened accounts in youtube , facebook , and twitter to reach their potential clients . according to a study published by the journal of medical internet research , nearly 95% of us hospitals are on facebook , while only 50% have a twitter account . in western europe , the use of social media by hospitals is also growing but there are significant differences between countries . a study published in the journal of medical internet research shows how its use remains small , except for the netherlands and the united kingdom . many concepts explain the use of health 2.0 in healthcare : health 2.0 , medicine 2.0 , health social media , ehealth , mhealth . the study " definition of health 2.0 and medicine 2.0 : a systematic review " shows that this is a developing area , and there is not a consensus on its definition . some of the concepts that include health 2.0 are apomediation , participation , openness , and collaboration . in the last years , patients have become the main actors of health 2.0 , leading a true revolution in healthcare management . e - patients ( # epatients ) achieve better health outcomes thanks to being connected and sharing experiences in social networks ; previously , their health depended solely on their doctors ' capacity . in addition to patients ' activism , health 2.0 also includes the online activity of medical professionals ( peer - to - peer ) , electronic health records , elearning , online medical publications ( peer review 2.0 ) , and science 2.0 . according to the european citizen 's digital health literacy report published in september 2014 , 59% of european citizens used the internet to look for health information in 2014 . between 82% and 87% of people ( depending on the type of information searched ) who searched for health - related information used searches engines . the second source of information ( 47%-48% ) was specific and dedicated websites ( blogs and forums ) , while between 33% and 38% looked for information from official health websites , such as those of the ministry of health or the world health organization ( who ) . online newspapers or magazines ( 20%-26% ) and social networks ( 16%-23% ) come next , followed by health mobile apps ( 13%-17% ) and patient organization websites ( 13%-17% ) . in nigeria ( africa ) , more and more hospitals , medical professionals , and health authorities have opened accounts in youtube , facebook , and twitter to reach their potential clients . in western europe , the use of social media by hospitals is also growing but there are significant differences between countries . many concepts explain the use of health 2.0 in healthcare : health 2.0 , medicine 2.0 , health social media , ehealth , mhealth . the study " definition of health 2.0 and medicine 2.0 : a systematic review " shows that this is a developing area , and there is not a consensus on its definition . some of the concepts that include health 2.0 are apomediation , participation , openness , and collaboration . gunther eysenbach , a health policy and ehealth professor at the university of toronto , describes the fact that when a user accesses information on the internet , he / she cuts out the gatekeepers ( like the primary care doctor ) and goes directly to the relevant source of information . in the last years , patients have become the main actors of health 2.0 , leading a true revolution in healthcare management . e - patients ( # epatients ) achieve better health outcomes thanks to being connected and sharing experiences in social networks ; previously , their health depended solely on their doctors ' capacity . epatients like dave debronkart ( @epatientdave ) , jan geissler ( @jangeissler ) , and andrew schorr ( @ andrewschorr ) overcame their cancer because they went online and connected with peer patients who told them about new treatments or clinical essays for their conditions . in addition to patients ' activism , health 2.0 also includes the online activity of medical professionals ( peer - to - peer ) , electronic health records , elearning , online medical publications ( peer review 2.0 ) , and science 2.0 . in this section we will explain how health 2.0 is shaping the health domain using four study cases . first of all , we will explain the role of health 2.0 in diabetes management as a prime example of a chronic condition . the third case study will focus on the use of social media for vaccination communication . online communities of patients are very common in diabetes . studied the dynamics of online communities of diabetes patients , and they found that the most centric members ( those with more trust within the communities ) tend to have more years of experience with disease . many of these patients have become experts in their disease and provide a great amount of valuable information for their peers . shows that the most experienced patients - those who have been living longer with the disease - tend to be leaders in the community . similarly , a study published in the bmj found that patients with cancer could be also effective in controlling the quality of health information , thus highlighting the importance of experienced patients in the control of online health information . by contrast , seals of quality that started to proliferate a few years ago do not guarantee the veracity of the information published , as stated in a study by the american cancer society about quality criteria in online information about breast cancer . online communities of patients can also collaborate with researchers using social media . in tuanalyze , a joint initiative between tudiabetes and boston children 's hospital , diabetes patients can monitor , evaluate , and share their health data , while they are participating in a research on diabetes and social media ( figure 1 ) . their data is analysed to determine a correlation between people 's participation in health social networking and level of diabetes management . there is a wide range of mobile applications to help diabetes patients ( 347 million people ) manage their condition . these tools allow them to monitor their glucose level , weight , and other data ; share them ; and get motivated to lead a healthy life . although there is not enough clinical evidence about their efficacy , mobile health tools many interesting initiatives have been created to help patients control their condition through their mobile phones , such as diabetes diary and big blue test . the use of wearable devices that monitor physical activity and gather vital data has also grown in recent years , and they are popular among patients with diabetes . this new wide range of tools to store health data raises major questions regarding their integration into the healthcare system , the need for mass storage systems , and the privacy of users ' data . the big challenge is to find a way to access this knowledge while respecting people 's privacy . it is striking that such a serious health problem as anorexia has an army of activists that defend this disease as a lifestyle . pro - anorexia activists ( the so - called pro - ana ) are very visible in social networks , especially youtube and flickr , where image is predominant . on youtube , one can easily stumble across pro - anorexia videos when looking for health information on eating disorders . anorexia activists use the same tags as reliable health videos , and their websites sometimes rank higher in youtube or google . this happens because of the way searching engine algorithms are designed , according to researchers . a study shows the high level of interaction between proanorexia and pro - recovery communities in social networks ( figure 3 ) . findings suggest that clinicians need to be aware of pro - anorexia contents online and focus on new intervention methods , possibly tailored to individual characteristics . over decades nevertheless , there is still a strong anti - vaccination movement ( it has existed since vaccines were invented more than 200 years ago ) , which in recent years , has moved its campaign to social networks . with ever increasing access to internet - based information , an unsubstantiated rumour about vaccines can rapidly circle the globe and undermine immunization services , sparking outbreaks of disease and untold deaths " stated who , unicef , and world bank in 2010 . various studies have analysed the influence of the antivaccination movement in social networks and in vaccination decisions or on the public trust in vaccines . research has shown that anti - vaccination contents are more popular in social networks than validated vaccination contents . another study analysed vaccination search and concluded that previous attitudes towards vaccination will influence the results of a search ; trusted health websites often use different vocabulary than those with concerns about vaccination ; search behaviour needs to be considered in online public health interventions . on the other hand , the study showed that there is a correlation between the sentiments expressed in social networks towards a new vaccine and the vaccination rates in a region . recently , the vaccine confidence project was created to monitor people 's trust in vaccination through social networks , given the importance that these tools have in vaccination decisions . the london school of hygiene and tropical medicine leads the project with the support of prestigious health organizations , such as the bill & melinda gates foundation and the world health organization . the recent ebola outbreak in western africa is another good example of the power of social networks in influencing people 's behaviour . the news of the epidemic created a climate of global nervousness with rumours and misinformation quickly spreading through social networks . the outbreak was first detected by healthmap , a website that delivers real - time data on infectious diseases for health institutions and governments . the role that social networks have played in the spread of rumours about ebola has been analysed in several studies . social networks also helped spread rumours about fake treatments ; some of them even making the general news . sms is an instantaneous , interactive , and cheap tool that can reach a great number of people . through mobile phones these networks can be extremely large with dozens of thousands members and fairly complex networks . studied the dynamics of online communities of diabetes patients , and they found that the most centric members ( those with more trust within the communities ) tend to have more years of experience with disease . many of these patients have become experts in their disease and provide a great amount of valuable information for their peers . shows that the most experienced patients - those who have been living longer with the disease - tend to be leaders in the community . similarly , a study published in the bmj found that patients with cancer could be also effective in controlling the quality of health information , thus highlighting the importance of experienced patients in the control of online health information . by contrast , seals of quality that started to proliferate a few years ago do not guarantee the veracity of the information published , as stated in a study by the american cancer society about quality criteria in online information about breast cancer . online communities of patients can also collaborate with researchers using social media . in tuanalyze , a joint initiative between tudiabetes and boston children 's hospital , diabetes patients can monitor , evaluate , and share their health data , while they are participating in a research on diabetes and social media ( figure 1 ) . their data is analysed to determine a correlation between people 's participation in health social networking and level of diabetes management . there is a wide range of mobile applications to help diabetes patients ( 347 million people ) manage their condition . these tools allow them to monitor their glucose level , weight , and other data ; share them ; and get motivated to lead a healthy life . although there is not enough clinical evidence about their efficacy , mobile health tools many interesting initiatives have been created to help patients control their condition through their mobile phones , such as diabetes diary and big blue test . researchers have pointed out that most applications do not offer enough educational features and lack social media tools . the use of wearable devices that monitor physical activity and gather vital data has also grown in recent years , and they are popular among patients with diabetes . this new wide range of tools to store health data raises major questions regarding their integration into the healthcare system , the need for mass storage systems , and the privacy of users ' data . the big challenge is to find a way to access this knowledge while respecting people 's privacy . studied the dynamics of online communities of diabetes patients , and they found that the most centric members ( those with more trust within the communities ) tend to have more years of experience with disease . many of these patients have become experts in their disease and provide a great amount of valuable information for their peers . shows that the most experienced patients - those who have been living longer with the disease - tend to be leaders in the community . similarly , a study published in the bmj found that patients with cancer could be also effective in controlling the quality of health information , thus highlighting the importance of experienced patients in the control of online health information . by contrast , seals of quality that started to proliferate a few years ago do not guarantee the veracity of the information published , as stated in a study by the american cancer society about quality criteria in online information about breast cancer . online communities of patients can also collaborate with researchers using social media . in tuanalyze , a joint initiative between tudiabetes and boston children 's hospital , diabetes patients can monitor , evaluate , and share their health data , while they are participating in a research on diabetes and social media ( figure 1 ) . their data is analysed to determine a correlation between people 's participation in health social networking and level of diabetes management . there is a wide range of mobile applications to help diabetes patients ( 347 million people ) manage their condition . these tools allow them to monitor their glucose level , weight , and other data ; share them ; and get motivated to lead a healthy life . although there is not enough clinical evidence about their efficacy , mobile health tools many interesting initiatives have been created to help patients control their condition through their mobile phones , such as diabetes diary and big blue test . researchers have pointed out that most applications do not offer enough educational features and lack social media tools . the use of wearable devices that monitor physical activity and gather vital data has also grown in recent years , and they are popular among patients with diabetes . a few studies about the medical applications , calibration , and validation of wearable devices have been done . this new wide range of tools to store health data raises major questions regarding their integration into the healthcare system , the need for mass storage systems , and the privacy of users ' data . on youtube , one can easily stumble across pro - anorexia videos when looking for health information on eating disorders . this happens because of the way searching engine algorithms are designed , according to researchers . findings suggest that clinicians need to be aware of pro - anorexia contents online and focus on new intervention methods , possibly tailored to individual characteristics . nevertheless , there is still a strong anti - vaccination movement ( it has existed since vaccines were invented more than 200 years ago ) , which in recent years , has moved its campaign to social networks . with ever increasing access to internet - based information , an unsubstantiated rumour about vaccines can rapidly circle the globe and undermine immunization services , sparking outbreaks of disease and untold deaths " stated who , unicef , and world bank in 2010 . various studies have analysed the influence of the antivaccination movement in social networks and in vaccination decisions or on the public trust in vaccines . research has shown that anti - vaccination contents are more popular in social networks than validated vaccination contents . another study analysed vaccination search and concluded that previous attitudes towards vaccination will influence the results of a search ; trusted health websites often use different vocabulary than those with concerns about vaccination ; search behaviour needs to be considered in online public health interventions . on the other hand , the study showed that there is a correlation between the sentiments expressed in social networks towards a new vaccine and the vaccination rates in a region . recently , the vaccine confidence project was created to monitor people 's trust in vaccination through social networks , given the importance that these tools have in vaccination decisions . the london school of hygiene and tropical medicine leads the project with the support of prestigious health organizations , such as the bill & melinda gates foundation and the world health organization . the recent ebola outbreak in western africa is another good example of the power of social networks in influencing people 's behaviour . the news of the epidemic created a climate of global nervousness with rumours and misinformation quickly spreading through social networks . the outbreak was first detected by healthmap , a website that delivers real - time data on infectious diseases for health institutions and governments . the role that social networks have played in the spread of rumours about ebola has been analysed in several studies . the researchers concluded that most of the messages had false information and that ' bad ' tweets were more retweeted than the ones with truthful information . sms is an instantaneous , interactive , and cheap tool that can reach a great number of people . through mobile phones as we have seen , the internet is rapidly expanding and is being used for health purposes by a great number of people . sometimes they help spread rumours and fake information and can undermine public health policies . given the lack of knowledge on the use of health social media , there is a need for complex multidisciplinary research ( mathematical , computer science , sociology , health , communication , etc . ) to help us understand how to use social networks to support public health . a better understanding of social media will give health authorities new tools to help decision - making at global , national , local , and corporate levels . for example , epidemiology is gradually becoming a discipline in which digital sources of information are becoming more important , and this has led to the creation of the sub - discipline of digital epidemiology . there is an unprecedented amount of data that can be used in public health due the potential combination of data acquired from mobile phones , electronic health records , social media and other sources . moreover , new analytics tools need to be developed to analyse those sources of data in a way that can benefit healthcare professionals and authorities .

acinetobacter baumannii is a gram - negative , aerobic , non - motile , non - fastidious strictly aerobic and glucose - non - fermenting bacterium that has coccobacillary morphology on non - selective agar and is classified as an opportunistic pathogen in hospitals . it was experimentally shown that the majority of a. baumannii strains survive longer than escherichia coli on dry surfaces , and some strains survive for more than four months . these characteristics are beneficial for the organism to survive in hospital environments and cause infection . a. baumannii has been recognized as the most important cause of nosocomial infections in immunocompromised patients particularly those in the intensive care units ( icus ) ( 1 ) , and is ranked second after pseudomonas aeruginosa among gramnegative nosocomial pathogens ( 2 ) . a. baumannii is the causative agent of several types of infections including bloodstream infections , ventilator - associated pneumonia , skin and soft tissue infections , meningitis and urinary tract infections ( 3 ) . this bacterium can survive on different medical equipments and even on healthy human skin ( 4 ) . a. baumannii is resistant to most available antibiotics and there are increasing reports of multidrug - resistant a. baumannii ( mdrab ) outbreaks in clinical settings worldwide ( 5 ) . multidrug - resistant a. baumannii strains are resistant to at least three different classes of antimicrobial agents mainly beta - lactams , aminoglycosides , carbapenems and fluoroquinolones ( 6 ) . aminoglycosides show various characteristics that make them useful for antimicrobial therapy . the bactericidal activity of aminoglycosides depend more on their concentration than on the duration of bacterial exposure to inhibitory concentrations of the antibiotics . the potential of aminoglycosides to kill bacteria depends on the concentration of the antibiotic , and increases with increasing concentrations . in addition , aminoglycosides continue to kill bacteria even after the aminoglycoside is detectable , exhibiting an important postantibiotic effect . these drugs attack the bacteria in a two - step process ; firstly , uptake of aminoglycosides into the bacteria takes place , which is an important process for their biological activity , and secondly , inside the bacterial cell the aminoglycoside binds to the ribosome and inhibits protein synthesis . however , in the recent years different resistance mechanisms have emerged against these antimicrobial agents . extended spectrum beta lactamases ( esbls ) are a class of group a beta lactamases which hydrolyze first , second and third generation cephalosporines but are inhibited by beta - lactamase inhibitors like clavulanic acid ( 7 - 9 ) . the most prevalent resistance mechanisms to carbapenem antibiotics in a. baumannii is production of oxatype lactamases ( 10 , 11 ) , and resistance to quinolones is related to alterations in the target enzymes , gyra and parc . aminoglycosides have long been used for the treatment of acinetobacter infections and still are an important alternative for therapy of infections caused by mdr strains . however , resistance to aminoglycosides has increased in the recent years in these bacteria ( 12 ) . the major mechanism of aminoglycoside resistance in clinical isolates of gram - negative bacteria is enzymatic modification of amino- or hydroxyl - groups of the aminoglycosides . enzymatic modification of aminoglycosides results in reduced or abolished binding of the aminoglycoside molecule to the ribosome . previous studies indicated that there are several mechanisms of resistance to aminoglycosides in the acinetobacter spp . the most prevalent resistance mechanism is attributed to enzymatic inactivation by acetyltransferases ( aac ) , nucleotidyl transferases ( ant ) and ( phosphotransferases ) aph ( 13 ) . many of the aminoglycoside modifying enzymes ( ame ) results in clinical resistance , but in general only the aphs and aacs produce high levels of resistance . the aim of this study was to evaluate the prevalence of aminoglycoside resistance and frequency of aacc1 , apha6 , aada1 and aadb aminoglycoside resistance genes among a. baumannii strains isolated from patients referred to imam reza hospital of tabriz city . in this study a total of 103 clinical a. baumannii strains were collected from hospitalized patients at imam reza hospital of tabriz university of medical sciences . the isolates were from different clinical samples including tracheal secretion , bronchial lavage , blood , wound , sputum and urine . the samples were transferred to the laboratory of the department of microbiology in the medicine faculty and all isolates were identified by using standard biochemical tests such as , gram staining , oxidase test , catalase test , motility , citrate utilization , oxidative / fermentative glucose ( o / f ) test and growth ability at 44c ( 15 ) . species identification was confirmed by detection of blaoxa-51-like genes , as described previously ( 16 ) . the antimicrobial resistance of the isolates was determined by using the standard disk diffusion method according to the clinical laboratory standard institute ( clsi ) guidelines ( 2011 ) . disk diffusion is a semi quantitative method used to examine bacterial susceptibility to specific antibiotics . it allows categorization of bacterial isolates as susceptible , intermediate or resistant to a variety of antimicrobial agents . the antimicrobial agents used in this study consisted of cefotaxime ( 30 g ) , cefixime ( 5 g ) , ceftizoxime ( 30 g ) , ceftazidime ( 30 g ) , ceftriaxone ( 30 g ) , cephalexin ( 30 g ) , cephalothin ( 30 g ) , tetracycline ( 30 g ) , ciprofloxacin ( 5 g ) , cotrimoxazole ( 25 g ) , ticarcillin ( 75 mg ) , polymyxin b ( 300 g ) , tobramycin ( 10 g ) , chloramphenicol ( 30 g ) , norfloxacin ( 10 g ) , ofloxacin ( 5 g ) , amikacin ( 30 g ) , gentamicin ( 10 g ) , ampicillin ( 10 g ) , carbenicillin ( 100 g ) , rifampin ( 5 g ) and colistin ( 110 g ) ( mast , merseyside , uk ) . the isolates were cultured on mueller hinton agar plates ( merck , germany ) inoculated with a bacterial suspension equal to 0.5 mcfarland and incubated at 37c for 1824 hours . the diameter of the zone of growth inhibition was measured using the clsi guidelines ( 16 ) . all a. baumannii isolates were grown for 18 hours at 37c in macconkey agar ( difco bd bioscience ) and dna were extracted by sodium dodecyl sulfate ( sds)-proteinase k phenol chloroform method as described previously ( 17 ) . briefly , 4 - 5 fresh colonies were resuspended in 300 l of tris - edta ( te ) buffer containing sds ( 1% ) and proteinase k ( 10 g / ml ) , and incubated at 40c for 3 hours followed by phenol - chloroform extraction and ethanol precipitation . the genes encoding aminoglycoside - modifying enzymes , including phosphotransferases aph ( 3')-via ( apha6 ) , acetyltransferases aac ( 3)-ia ( aacc1 ) , nucleotidyl transferases ant ( 2'')-ia ( aadb ) and ant(3 " ) -ia ( aada1 ) , were detected by pcr . polymerase chain reaction ( pcr ) is a rapid method for in vitro amplification of dna . pcr reactions were performed in a final volume of 25 l ( 24 l pcr master mix plus 1 l of template dna ) . pcr amplification was carried out in a thermal cycler with the following parameters : initial denaturation at 95c for 3 minutes , followed by 35 cycles of 60 seconds , denaturation at 94c , 60 seconds for annealing at primers annealing temperatures ( table 1 ) and 45 seconds for extension at 72c with a final extension at 72c for 5 minutes ( table 2 ) . the presence and sizes of amplicons were analyzed by electrophoresis on 1.2 % agarose gel in a tris - acetate - edta ( tae ) buffer at 90 volts alongside a 1 kb dna ladder ( fermentas , lithuania ) . electrophoresis was performed for one hour and finally the gels were stained with ethidium bromide and visualized using the gel documentation system . in this study a total of 103 clinical a. baumannii strains were collected from hospitalized patients at imam reza hospital of tabriz university of medical sciences . the isolates were from different clinical samples including tracheal secretion , bronchial lavage , blood , wound , sputum and urine . the samples were transferred to the laboratory of the department of microbiology in the medicine faculty and all isolates were identified by using standard biochemical tests such as , gram staining , oxidase test , catalase test , motility , citrate utilization , oxidative / fermentative glucose ( o / f ) test and growth ability at 44c ( 15 ) . species identification was confirmed by detection of blaoxa-51-like genes , as described previously ( 16 ) . the antimicrobial resistance of the isolates was determined by using the standard disk diffusion method according to the clinical laboratory standard institute ( clsi ) guidelines ( 2011 ) . disk diffusion is a semi quantitative method used to examine bacterial susceptibility to specific antibiotics . it allows categorization of bacterial isolates as susceptible , intermediate or resistant to a variety of antimicrobial agents . the antimicrobial agents used in this study consisted of cefotaxime ( 30 g ) , cefixime ( 5 g ) , ceftizoxime ( 30 g ) , ceftazidime ( 30 g ) , ceftriaxone ( 30 g ) , cephalexin ( 30 g ) , cephalothin ( 30 g ) , tetracycline ( 30 g ) , ciprofloxacin ( 5 g ) , cotrimoxazole ( 25 g ) , ticarcillin ( 75 mg ) , polymyxin b ( 300 g ) , tobramycin ( 10 g ) , chloramphenicol ( 30 g ) , norfloxacin ( 10 g ) , ofloxacin ( 5 g ) , amikacin ( 30 g ) , gentamicin ( 10 g ) , ampicillin ( 10 g ) , carbenicillin ( 100 g ) , rifampin ( 5 g ) and colistin ( 110 g ) ( mast , merseyside , uk ) . the isolates were cultured on mueller hinton agar plates ( merck , germany ) inoculated with a bacterial suspension equal to 0.5 mcfarland and incubated at 37c for 1824 hours . the diameter of the zone of growth inhibition was measured using the clsi guidelines ( 16 ) . all a. baumannii isolates were grown for 18 hours at 37c in macconkey agar ( difco bd bioscience ) and dna were extracted by sodium dodecyl sulfate ( sds)-proteinase k phenol chloroform method as described previously ( 17 ) . briefly , 4 - 5 fresh colonies were resuspended in 300 l of tris - edta ( te ) buffer containing sds ( 1% ) and proteinase k ( 10 g / ml ) , and incubated at 40c for 3 hours followed by phenol - chloroform extraction and ethanol precipitation . the genes encoding aminoglycoside - modifying enzymes , including phosphotransferases aph ( 3')-via ( apha6 ) , acetyltransferases aac ( 3)-ia ( aacc1 ) , nucleotidyl transferases ant ( 2'')-ia ( aadb ) and ant(3 " ) -ia ( aada1 ) , were detected by pcr . polymerase chain reaction ( pcr ) is a rapid method for in vitro amplification of dna . pcr reactions were performed in a final volume of 25 l ( 24 l pcr master mix plus 1 l of template dna ) . pcr amplification was carried out in a thermal cycler with the following parameters : initial denaturation at 95c for 3 minutes , followed by 35 cycles of 60 seconds , denaturation at 94c , 60 seconds for annealing at primers annealing temperatures ( table 1 ) and 45 seconds for extension at 72c with a final extension at 72c for 5 minutes ( table 2 ) . the presence and sizes of amplicons were analyzed by electrophoresis on 1.2 % agarose gel in a tris - acetate - edta ( tae ) buffer at 90 volts alongside a 1 kb dna ladder ( fermentas , lithuania ) . electrophoresis was performed for one hour and finally the gels were stained with ethidium bromide and visualized using the gel documentation system . the mean age of the population was 51 years , with a range of 14 to 86 years . the isolates were obtained from patients belonging to different age groups : 2039 years ( n = 26 ) , 4059 ( n = 40 ) , 6090 years ( n = 34 ) and three isolates were from patients less than 20 years old . a total of 103 a. baumannii isolates were recovered from clinical specimens of hospitalized patients that had been admitted to the imam reza hospital of tabriz , north - west of iran . the isolates were obtained from invasive and non - invasive sites , including trachea ( 38% ) , urine ( 22% ) , sputum ( 10% ) , blood ( 7% ) , catheter ( 6% ) , bronchial washings ( 6% ) , wound ( 5% ) , abscess drainage ( 3% ) , cerebrospinal fluid ( 2% ) , ascites fluid ( 2% ) and pleural effusion ( 2% ) . the samples were transferred to the laboratory of the microbiology department of tabriz university and were immediately inoculated on to macconkey and blood agar plates . analysis of antimicrobial susceptibility in this study showed that the highest resistance was against cefiexime ( 100% ) , ceftizoxime ( 100% ) and ticaracilin ( 100% ) , whereas the highest susceptibility was observed towards polymyxin b ( 84% ) , colistin ( 77% ) and rifampin ( 73% ) ( table 3 ) . resistance to various aminoglycosides was as follows ; gentamicin 86% , tobramycin 63% and amikacin 81% . screening of aminoglycoside resistant isolates for resistance genes by pcr ( figure 1 - 4 ) revealed that the prevalence of the aacc1 gene , which confers resistance to amikacin and gentamicin , was 65.11% . screening for apha6 , aada1and aadb genes revealed that 52 ( 60.46 % ) isolates were positive for apha6 , 24 ( 27.9 % ) for aada1 and 16 ( 18.6 % ) for aadb , respectively . lane 1 isolate negative for aacc1 gene , lane 2 - 8 isolates with aacc1 gene , lane 9 , no dna and lane 10 related to size marker ( 1 kb dna ladder ) . lane 1 and 7 , isolates negative for apha ( 6 ) gene , lane 2_6 and 8 , isolates with apha ( 6 ) genes , lane 9 , size marker ( 1 kb dna ladder ) . lane 1,4,5 isolates negative for aada1 gene , lane 2 , 3 and 6 , isolates with aada1 gene , lane 7 , no dna and lane 8 related to size marker ( 1 kb dna ladder ) . lane 1 , 2 , 7 and 8 isolates negative for aadb gene , lane 3 , 4 , 5 and 6 isolates with aadb gene , lane 9 , no dna and lane 10 size marker ( 1 kb dna ladder ) . isolates with two ame genes were seen in 32 ( 31.06% ) cases for aacc1 and apha6 , 17 ( 16.5% ) cases for aada1 and apha6 genes , 15 ( 14.56% ) cases for aacc1 and aada1 , 10 ( 9.7% ) cases for apha6 and aadb , 8 ( 7.76% ) cases for aada1 and aadb genes and 6 ( 5.82% ) isolates had aacc1 and aadb genes . nine isolates ( 8.73% ) had three ame genes , aacc1 , aada1 and apha6 . three isolates were positive for aacc1 , apha6 and aadb , and three isolates had a combination of four different ame genes including aacc1 , apha6 , aadb and aada1 . strains with aacc1 or apha6 were found to be resistant to kanamycin and gentamicin or kanamycin and amikacin , respectively , while aadb was associated with resistance to kanamycin , gentamicin and tobramycin ( 18 ) . the mean age of the population was 51 years , with a range of 14 to 86 years . the isolates were obtained from patients belonging to different age groups : 2039 years ( n = 26 ) , 4059 ( n = 40 ) , 6090 years ( n = 34 ) and three isolates were from patients less than 20 years old . a total of 103 a. baumannii isolates were recovered from clinical specimens of hospitalized patients that had been admitted to the imam reza hospital of tabriz , north - west of iran . the isolates were obtained from invasive and non - invasive sites , including trachea ( 38% ) , urine ( 22% ) , sputum ( 10% ) , blood ( 7% ) , catheter ( 6% ) , bronchial washings ( 6% ) , wound ( 5% ) , abscess drainage ( 3% ) , cerebrospinal fluid ( 2% ) , ascites fluid ( 2% ) and pleural effusion ( 2% ) . the samples were transferred to the laboratory of the microbiology department of tabriz university and were immediately inoculated on to macconkey and blood agar plates . analysis of antimicrobial susceptibility in this study showed that the highest resistance was against cefiexime ( 100% ) , ceftizoxime ( 100% ) and ticaracilin ( 100% ) , whereas the highest susceptibility was observed towards polymyxin b ( 84% ) , colistin ( 77% ) and rifampin ( 73% ) ( table 3 ) . resistance to various aminoglycosides was as follows ; gentamicin 86% , tobramycin 63% and amikacin 81% . screening of aminoglycoside resistant isolates for resistance genes by pcr ( figure 1 - 4 ) revealed that the prevalence of the aacc1 gene , which confers resistance to amikacin and gentamicin , was 65.11% . screening for apha6 , aada1and aadb genes revealed that 52 ( 60.46 % ) isolates were positive for apha6 , 24 ( 27.9 % ) for aada1 and 16 ( 18.6 % ) for aadb , respectively . lane 1 isolate negative for aacc1 gene , lane 2 - 8 isolates with aacc1 gene , lane 9 , no dna and lane 10 related to size marker ( 1 kb dna ladder ) . lane 1 and 7 , isolates negative for apha ( 6 ) gene , lane 2_6 and 8 , isolates with apha ( 6 ) genes , lane 9 , size marker ( 1 kb dna ladder ) . lane 1,4,5 isolates negative for aada1 gene , lane 2 , 3 and 6 , isolates with aada1 gene , lane 7 , no dna and lane 8 related to size marker ( 1 kb dna ladder ) . lane 1 , 2 , 7 and 8 isolates negative for aadb gene , lane 3 , 4 , 5 and 6 isolates with aadb gene , lane 9 , no dna and lane 10 size marker ( 1 kb dna ladder ) . isolates with two ame genes were seen in 32 ( 31.06% ) cases for aacc1 and apha6 , 17 ( 16.5% ) cases for aada1 and apha6 genes , 15 ( 14.56% ) cases for aacc1 and aada1 , 10 ( 9.7% ) cases for apha6 and aadb , 8 ( 7.76% ) cases for aada1 and aadb genes and 6 ( 5.82% ) isolates had aacc1 and aadb genes . nine isolates ( 8.73% ) had three ame genes , aacc1 , aada1 and apha6 . three isolates were positive for aacc1 , apha6 and aadb , and three isolates had a combination of four different ame genes including aacc1 , apha6 , aadb and aada1 . strains with aacc1 or apha6 were found to be resistant to kanamycin and gentamicin or kanamycin and amikacin , respectively , while aadb was associated with resistance to kanamycin , gentamicin and tobramycin ( 18 ) . mdr a. baumannii is an important pathogen that is involved in nosocomial infections especially in icu wards . this bacterium is one of the most important problems encountered in hospitals , clinics and public health centers ( 19 ) . this organism is very difficult to eradicate due to its inherent and acquired resistance against multiple classes of antibiotics , so that very few effective therapeutic options remain available . aminoglycosides have been an important group of antibiotics in treatment of serious bacterial infections , especially those with aerobic gram negative bacteria , but recent reports indicated the emergence of resistance to aminoglycosides in acinetobacter isolates in different parts of the world . aminoglycoside resistance in acinetobacter primarily results from inactivation of the antibiotic by specific modifying enzymes such as acetyl transferases , phosphotransferases , and adenylyl transferases . this study focused on resistance to different aminoglycosides in clinically important isolates of a. baumannii , with emphasis on gentamicin , tobramycin and amikacin . the prevalence of ames encoding genes were investigated in a. baumannii isolates recovered from patients hospitalized in tabriz city in the north west of iran . findings of the present study showed that 65.11% of the studied acinetobacter isolates were positive for aacc1 genes . this indicates a high prevalence of resistance due to aacc1 gene in the studied cases . also other aminoglycosidemodifying enzyme genes detected by pcr were apha6 ( 60.46 % ) , aada1 ( 27.9 % ) and aadb ( 18.6 % ) . these results indicate that the rate of resistance by aminoglycoside - modifying enzyme types apha6 , aada1 and aadb have increased significantly over the past years . ( 20 ) evaluated antimicrobial susceptibility and aminoglycoside resistance genes of sixty acinetobacter strains isolated from hospitalized patients in kashan city . they reported the presence of acetyltransferase genes ( aacc1 ) in 63.3% of acinetobacter isolates . other genes including phosphotransferase ( apha6 ) and adenylyl transferase ( aada1 and aadb ) were detected in 65% , 41.7% and 3.3% , of the isolates , respectively . in our study the prevalence of apha6 and aada1 genes was much lower than that reported from kashan city but the prevalence of aadb genes was significantly higher in our study . these findings showed that clinical isolates of acinetobacter in hospitals carry various kinds of aminoglycoside resistance genes . showed the highest resistance rate against amikacin , tobramycin and ceftazidim , respectively ; while isolated bacteria were more sensitive to ampicillic / subactam . another study in iran was done by shahcheraghi and his colleagues in tehran ( 21 ) , which showed the highest resistance ( 100% ) to cefixime that was very similar to our study . the lowest resistance was reported against colistin ( 4.2 % ) whereas in our study , 19% of isolates were resistant to colistin ( 21 ) . in a study done by lee and his colleagues in korea in 2011 ( 12 ) , the majority of aminoglycoside - modifying enzyme genes detected by pcr were aacc1 ( 56% ) , aadb ( 48% ) , and apha6 ( 71% ) ( 12 ) . nemec and his colleagues in czech republic in 2004 investigated the diversity of genes encoding aminoglycosidemodifying enzymes and their association with class 1 integrons in a. baumannii and reported aminoglycoside resistance genes in 95 % of isolates : aacc1 ( n = 68 ) , aada1 ( n = 68 ) , apha6 ( n = 55 ) , and aadb ( n = 31 ) ( 18 ) . the rate of resistance in our study was considerably higher than the rates found for other geographical regions including korea and czech republic . ( 22 ) in 2010 studied the susceptibility of 107 isolates of a. baumannii - calcoaceticus complex to amikacin , gentamicin and tobramycin using the disk diffusion method . the susceptibility to aminoglycoside antibiotics 56.1% of isolates contained two and 3.7% contained three ame genes . in our study resistance to gentamicin and isolates with two ame genes were seen in 32 ( 31.06% ) cases for aacc1 and apha6 ; 17 ( 16.5% ) cases for aada1 and apha6 genes ; 15 ( 14.56% ) cases for aacc1 and aada1 ; 10 ( 9.7% ) cases for apha6 and aadb ; 8 ( 7.76% ) cases for aada1 and aadb genes and 6 ( 5.82% ) isolates had aacc1 and aadb genes . these results are in agreement with some other studies that have found that aacc1 and apha6 genes are the most common ame genes in a. baumannii isolates . ( 12 , 20)detected aacc1 in 56% and 63.3 % of isolates , respectively . in another study that was done by nigro ( 23 ) in australia they investigated the pattern of resistance to aminoglycosides in sixtyone multi - resistant a. baumannii strains isolated between 2000 and 2010 in six australian hospitals . in this work the isolates were screened for ame genes ; aadb , aacc1 , apha1b , apha6 and oxa 23 beta - lactamase gene ( 20 ) . they found that the apha6 gene was present in combination with aacc1 and apha1 in two isolates . our study results showed a remarkable diversity of genes encoding aminoglycosidemodifying enzymes in the study region . the multiple resistance mechanisms in a. baumannii isolates make this bacterium a major clinical and public health concern . our study results indicated that the genes related to ame are prevalent in the a. baumannii strains in the study region which highlighted the necessity of considering preventive measure to control dissemination of resistance genes ( 21 ) .

background : acinetobacter baumannii is one of the major causes of nosocomial infections and is resistant to most available antibiotics . aminoglycosides remain as drugs of choice for treatment of acinetobacter infections yet resistance to aminoglycosides has increased in the recent years.objectives:the present study investigated the prevalence of genes encoding aminoglycoside - modifying enzymes in a. baumannii strains isolated from patients of tabriz city , northwest of iran.materials and methods : a total of 103 acinetobacter isolates were collected from imam reza hospital of tabriz university of medical sciences . antimicrobial susceptibility patterns of the isolates to different antimicrobial agents including cephalosporins , gentamicin , amikacin , tobramycin , colistin and polymyxin , were evaluated by the disc diffusion method . the frequency of aminoglycoside modifying enzymes encoding genes aacc1 , apha6 , aada1 and aadb was analyzed by the pcr method.results:antimicrobial susceptibility analysis showed that the highest resistance was towards betalactam antibiotics including cephalosporins whereas the highest sensitivity was observed towards colistin ( 77% ) and polymyxin ( 84% ) . the resistance rate to aminoglycosides was 81% , 86% and 63% for amikacin , gentamicin and tobramycin , respectively . the pcr results showed that among the 103 a. baumannii isolates , 56 ( 65.11 % ) were positive for aacc1 , 52 ( 60.46 % ) for apha6 , 24 ( 27.9 % ) for aada1 and 16 ( 18.6 % ) for aadb resistant genes.conclusions:the results of this study indicated that the genes encoding aminoglycoside - modifying enzymes are prevalent in a. baumannii isolates in the study region , which highlighted the necessity of considering preventive measures to control dissemination of these resistance genes .

1. Background 2. Objectives 3. Materials and Methods 3.1. Bacterial Isolation and Identification 3.2. Antimicrobial Susceptibility Test 3.3. DNA Extraction and PCR Amplification 3.4. Detection of aminoglycoside-resistance genes 3.5. PCR Analysis 4. Results 4.1. Characteristics of the Study Population 4.2. Isolates and Antimicrobial Susceptibility 4.3. Aminoglycoside-Modifying Enzymes 5. Discussion

acinetobacter baumannii is a gram - negative , aerobic , non - motile , non - fastidious strictly aerobic and glucose - non - fermenting bacterium that has coccobacillary morphology on non - selective agar and is classified as an opportunistic pathogen in hospitals . it was experimentally shown that the majority of a. baumannii strains survive longer than escherichia coli on dry surfaces , and some strains survive for more than four months . a. baumannii has been recognized as the most important cause of nosocomial infections in immunocompromised patients particularly those in the intensive care units ( icus ) ( 1 ) , and is ranked second after pseudomonas aeruginosa among gramnegative nosocomial pathogens ( 2 ) . a. baumannii is resistant to most available antibiotics and there are increasing reports of multidrug - resistant a. baumannii ( mdrab ) outbreaks in clinical settings worldwide ( 5 ) . multidrug - resistant a. baumannii strains are resistant to at least three different classes of antimicrobial agents mainly beta - lactams , aminoglycosides , carbapenems and fluoroquinolones ( 6 ) . however , in the recent years different resistance mechanisms have emerged against these antimicrobial agents . the most prevalent resistance mechanisms to carbapenem antibiotics in a. baumannii is production of oxatype lactamases ( 10 , 11 ) , and resistance to quinolones is related to alterations in the target enzymes , gyra and parc . aminoglycosides have long been used for the treatment of acinetobacter infections and still are an important alternative for therapy of infections caused by mdr strains . however , resistance to aminoglycosides has increased in the recent years in these bacteria ( 12 ) . the major mechanism of aminoglycoside resistance in clinical isolates of gram - negative bacteria is enzymatic modification of amino- or hydroxyl - groups of the aminoglycosides . previous studies indicated that there are several mechanisms of resistance to aminoglycosides in the acinetobacter spp . many of the aminoglycoside modifying enzymes ( ame ) results in clinical resistance , but in general only the aphs and aacs produce high levels of resistance . the aim of this study was to evaluate the prevalence of aminoglycoside resistance and frequency of aacc1 , apha6 , aada1 and aadb aminoglycoside resistance genes among a. baumannii strains isolated from patients referred to imam reza hospital of tabriz city . in this study a total of 103 clinical a. baumannii strains were collected from hospitalized patients at imam reza hospital of tabriz university of medical sciences . the samples were transferred to the laboratory of the department of microbiology in the medicine faculty and all isolates were identified by using standard biochemical tests such as , gram staining , oxidase test , catalase test , motility , citrate utilization , oxidative / fermentative glucose ( o / f ) test and growth ability at 44c ( 15 ) . the antimicrobial resistance of the isolates was determined by using the standard disk diffusion method according to the clinical laboratory standard institute ( clsi ) guidelines ( 2011 ) . the antimicrobial agents used in this study consisted of cefotaxime ( 30 g ) , cefixime ( 5 g ) , ceftizoxime ( 30 g ) , ceftazidime ( 30 g ) , ceftriaxone ( 30 g ) , cephalexin ( 30 g ) , cephalothin ( 30 g ) , tetracycline ( 30 g ) , ciprofloxacin ( 5 g ) , cotrimoxazole ( 25 g ) , ticarcillin ( 75 mg ) , polymyxin b ( 300 g ) , tobramycin ( 10 g ) , chloramphenicol ( 30 g ) , norfloxacin ( 10 g ) , ofloxacin ( 5 g ) , amikacin ( 30 g ) , gentamicin ( 10 g ) , ampicillin ( 10 g ) , carbenicillin ( 100 g ) , rifampin ( 5 g ) and colistin ( 110 g ) ( mast , merseyside , uk ) . all a. baumannii isolates were grown for 18 hours at 37c in macconkey agar ( difco bd bioscience ) and dna were extracted by sodium dodecyl sulfate ( sds)-proteinase k phenol chloroform method as described previously ( 17 ) . the genes encoding aminoglycoside - modifying enzymes , including phosphotransferases aph ( 3')-via ( apha6 ) , acetyltransferases aac ( 3)-ia ( aacc1 ) , nucleotidyl transferases ant ( 2'')-ia ( aadb ) and ant(3 " ) -ia ( aada1 ) , were detected by pcr . the presence and sizes of amplicons were analyzed by electrophoresis on 1.2 % agarose gel in a tris - acetate - edta ( tae ) buffer at 90 volts alongside a 1 kb dna ladder ( fermentas , lithuania ) . in this study a total of 103 clinical a. baumannii strains were collected from hospitalized patients at imam reza hospital of tabriz university of medical sciences . the samples were transferred to the laboratory of the department of microbiology in the medicine faculty and all isolates were identified by using standard biochemical tests such as , gram staining , oxidase test , catalase test , motility , citrate utilization , oxidative / fermentative glucose ( o / f ) test and growth ability at 44c ( 15 ) . the antimicrobial resistance of the isolates was determined by using the standard disk diffusion method according to the clinical laboratory standard institute ( clsi ) guidelines ( 2011 ) . the antimicrobial agents used in this study consisted of cefotaxime ( 30 g ) , cefixime ( 5 g ) , ceftizoxime ( 30 g ) , ceftazidime ( 30 g ) , ceftriaxone ( 30 g ) , cephalexin ( 30 g ) , cephalothin ( 30 g ) , tetracycline ( 30 g ) , ciprofloxacin ( 5 g ) , cotrimoxazole ( 25 g ) , ticarcillin ( 75 mg ) , polymyxin b ( 300 g ) , tobramycin ( 10 g ) , chloramphenicol ( 30 g ) , norfloxacin ( 10 g ) , ofloxacin ( 5 g ) , amikacin ( 30 g ) , gentamicin ( 10 g ) , ampicillin ( 10 g ) , carbenicillin ( 100 g ) , rifampin ( 5 g ) and colistin ( 110 g ) ( mast , merseyside , uk ) . all a. baumannii isolates were grown for 18 hours at 37c in macconkey agar ( difco bd bioscience ) and dna were extracted by sodium dodecyl sulfate ( sds)-proteinase k phenol chloroform method as described previously ( 17 ) . the genes encoding aminoglycoside - modifying enzymes , including phosphotransferases aph ( 3')-via ( apha6 ) , acetyltransferases aac ( 3)-ia ( aacc1 ) , nucleotidyl transferases ant ( 2'')-ia ( aadb ) and ant(3 " ) -ia ( aada1 ) , were detected by pcr . the isolates were obtained from patients belonging to different age groups : 2039 years ( n = 26 ) , 4059 ( n = 40 ) , 6090 years ( n = 34 ) and three isolates were from patients less than 20 years old . a total of 103 a. baumannii isolates were recovered from clinical specimens of hospitalized patients that had been admitted to the imam reza hospital of tabriz , north - west of iran . the isolates were obtained from invasive and non - invasive sites , including trachea ( 38% ) , urine ( 22% ) , sputum ( 10% ) , blood ( 7% ) , catheter ( 6% ) , bronchial washings ( 6% ) , wound ( 5% ) , abscess drainage ( 3% ) , cerebrospinal fluid ( 2% ) , ascites fluid ( 2% ) and pleural effusion ( 2% ) . the samples were transferred to the laboratory of the microbiology department of tabriz university and were immediately inoculated on to macconkey and blood agar plates . analysis of antimicrobial susceptibility in this study showed that the highest resistance was against cefiexime ( 100% ) , ceftizoxime ( 100% ) and ticaracilin ( 100% ) , whereas the highest susceptibility was observed towards polymyxin b ( 84% ) , colistin ( 77% ) and rifampin ( 73% ) ( table 3 ) . resistance to various aminoglycosides was as follows ; gentamicin 86% , tobramycin 63% and amikacin 81% . screening of aminoglycoside resistant isolates for resistance genes by pcr ( figure 1 - 4 ) revealed that the prevalence of the aacc1 gene , which confers resistance to amikacin and gentamicin , was 65.11% . screening for apha6 , aada1and aadb genes revealed that 52 ( 60.46 % ) isolates were positive for apha6 , 24 ( 27.9 % ) for aada1 and 16 ( 18.6 % ) for aadb , respectively . isolates with two ame genes were seen in 32 ( 31.06% ) cases for aacc1 and apha6 , 17 ( 16.5% ) cases for aada1 and apha6 genes , 15 ( 14.56% ) cases for aacc1 and aada1 , 10 ( 9.7% ) cases for apha6 and aadb , 8 ( 7.76% ) cases for aada1 and aadb genes and 6 ( 5.82% ) isolates had aacc1 and aadb genes . nine isolates ( 8.73% ) had three ame genes , aacc1 , aada1 and apha6 . three isolates were positive for aacc1 , apha6 and aadb , and three isolates had a combination of four different ame genes including aacc1 , apha6 , aadb and aada1 . strains with aacc1 or apha6 were found to be resistant to kanamycin and gentamicin or kanamycin and amikacin , respectively , while aadb was associated with resistance to kanamycin , gentamicin and tobramycin ( 18 ) . the isolates were obtained from patients belonging to different age groups : 2039 years ( n = 26 ) , 4059 ( n = 40 ) , 6090 years ( n = 34 ) and three isolates were from patients less than 20 years old . a total of 103 a. baumannii isolates were recovered from clinical specimens of hospitalized patients that had been admitted to the imam reza hospital of tabriz , north - west of iran . the isolates were obtained from invasive and non - invasive sites , including trachea ( 38% ) , urine ( 22% ) , sputum ( 10% ) , blood ( 7% ) , catheter ( 6% ) , bronchial washings ( 6% ) , wound ( 5% ) , abscess drainage ( 3% ) , cerebrospinal fluid ( 2% ) , ascites fluid ( 2% ) and pleural effusion ( 2% ) . the samples were transferred to the laboratory of the microbiology department of tabriz university and were immediately inoculated on to macconkey and blood agar plates . analysis of antimicrobial susceptibility in this study showed that the highest resistance was against cefiexime ( 100% ) , ceftizoxime ( 100% ) and ticaracilin ( 100% ) , whereas the highest susceptibility was observed towards polymyxin b ( 84% ) , colistin ( 77% ) and rifampin ( 73% ) ( table 3 ) . resistance to various aminoglycosides was as follows ; gentamicin 86% , tobramycin 63% and amikacin 81% . screening of aminoglycoside resistant isolates for resistance genes by pcr ( figure 1 - 4 ) revealed that the prevalence of the aacc1 gene , which confers resistance to amikacin and gentamicin , was 65.11% . screening for apha6 , aada1and aadb genes revealed that 52 ( 60.46 % ) isolates were positive for apha6 , 24 ( 27.9 % ) for aada1 and 16 ( 18.6 % ) for aadb , respectively . isolates with two ame genes were seen in 32 ( 31.06% ) cases for aacc1 and apha6 , 17 ( 16.5% ) cases for aada1 and apha6 genes , 15 ( 14.56% ) cases for aacc1 and aada1 , 10 ( 9.7% ) cases for apha6 and aadb , 8 ( 7.76% ) cases for aada1 and aadb genes and 6 ( 5.82% ) isolates had aacc1 and aadb genes . nine isolates ( 8.73% ) had three ame genes , aacc1 , aada1 and apha6 . three isolates were positive for aacc1 , apha6 and aadb , and three isolates had a combination of four different ame genes including aacc1 , apha6 , aadb and aada1 . strains with aacc1 or apha6 were found to be resistant to kanamycin and gentamicin or kanamycin and amikacin , respectively , while aadb was associated with resistance to kanamycin , gentamicin and tobramycin ( 18 ) . mdr a. baumannii is an important pathogen that is involved in nosocomial infections especially in icu wards . this bacterium is one of the most important problems encountered in hospitals , clinics and public health centers ( 19 ) . aminoglycosides have been an important group of antibiotics in treatment of serious bacterial infections , especially those with aerobic gram negative bacteria , but recent reports indicated the emergence of resistance to aminoglycosides in acinetobacter isolates in different parts of the world . this study focused on resistance to different aminoglycosides in clinically important isolates of a. baumannii , with emphasis on gentamicin , tobramycin and amikacin . the prevalence of ames encoding genes were investigated in a. baumannii isolates recovered from patients hospitalized in tabriz city in the north west of iran . findings of the present study showed that 65.11% of the studied acinetobacter isolates were positive for aacc1 genes . also other aminoglycosidemodifying enzyme genes detected by pcr were apha6 ( 60.46 % ) , aada1 ( 27.9 % ) and aadb ( 18.6 % ) . these results indicate that the rate of resistance by aminoglycoside - modifying enzyme types apha6 , aada1 and aadb have increased significantly over the past years . ( 20 ) evaluated antimicrobial susceptibility and aminoglycoside resistance genes of sixty acinetobacter strains isolated from hospitalized patients in kashan city . other genes including phosphotransferase ( apha6 ) and adenylyl transferase ( aada1 and aadb ) were detected in 65% , 41.7% and 3.3% , of the isolates , respectively . these findings showed that clinical isolates of acinetobacter in hospitals carry various kinds of aminoglycoside resistance genes . showed the highest resistance rate against amikacin , tobramycin and ceftazidim , respectively ; while isolated bacteria were more sensitive to ampicillic / subactam . another study in iran was done by shahcheraghi and his colleagues in tehran ( 21 ) , which showed the highest resistance ( 100% ) to cefixime that was very similar to our study . the lowest resistance was reported against colistin ( 4.2 % ) whereas in our study , 19% of isolates were resistant to colistin ( 21 ) . in a study done by lee and his colleagues in korea in 2011 ( 12 ) , the majority of aminoglycoside - modifying enzyme genes detected by pcr were aacc1 ( 56% ) , aadb ( 48% ) , and apha6 ( 71% ) ( 12 ) . nemec and his colleagues in czech republic in 2004 investigated the diversity of genes encoding aminoglycosidemodifying enzymes and their association with class 1 integrons in a. baumannii and reported aminoglycoside resistance genes in 95 % of isolates : aacc1 ( n = 68 ) , aada1 ( n = 68 ) , apha6 ( n = 55 ) , and aadb ( n = 31 ) ( 18 ) . ( 22 ) in 2010 studied the susceptibility of 107 isolates of a. baumannii - calcoaceticus complex to amikacin , gentamicin and tobramycin using the disk diffusion method . in our study resistance to gentamicin and isolates with two ame genes were seen in 32 ( 31.06% ) cases for aacc1 and apha6 ; 17 ( 16.5% ) cases for aada1 and apha6 genes ; 15 ( 14.56% ) cases for aacc1 and aada1 ; 10 ( 9.7% ) cases for apha6 and aadb ; 8 ( 7.76% ) cases for aada1 and aadb genes and 6 ( 5.82% ) isolates had aacc1 and aadb genes . these results are in agreement with some other studies that have found that aacc1 and apha6 genes are the most common ame genes in a. baumannii isolates . in another study that was done by nigro ( 23 ) in australia they investigated the pattern of resistance to aminoglycosides in sixtyone multi - resistant a. baumannii strains isolated between 2000 and 2010 in six australian hospitals . in this work the isolates were screened for ame genes ; aadb , aacc1 , apha1b , apha6 and oxa 23 beta - lactamase gene ( 20 ) . our study results showed a remarkable diversity of genes encoding aminoglycosidemodifying enzymes in the study region . the multiple resistance mechanisms in a. baumannii isolates make this bacterium a major clinical and public health concern . our study results indicated that the genes related to ame are prevalent in the a. baumannii strains in the study region which highlighted the necessity of considering preventive measure to control dissemination of resistance genes ( 21 ) .

healthcare workers are considered to be at particular risk of developing ageist attitudes because they are exposed to a disproportionate percentage of ill or dependent older people . research demonstrates that many factors have influence on healthcare workers ' attitudes towards older people , including age , gender [ 13 ] , education [ 46 ] , exposure to well older people [ 710 ] , area of practice [ 1114 ] , and professional socialization . an attitude is an evaluation of something or someone on a continuum of like to dislike or favourable to unfavourable . ageism has been defined as a systematic stereotyping of discrimination against people because they are old . ageism is perpetuated by the portrayal of older people as frail , ill , suffering mental deterioration , poor and dependent , and the alternative portrayal of living affluent life styles and scrounging off the welfare state . in addition , budget constraints in the uk and ireland which currently pitch health services against social services over costs for supportive care packages negate against timely discharge from hospital and reinforce an overreliance on family careers for support . these consequences are likely to further reinforce the portrayal of older people as a burden on society . stereotypes such as these are in contrast with the reality that the majority of older people lead fit and independent lives . older people consider that inadequate housing , low income , enforced retirement , and deficiencies in nursing home care are evidence of ageism . in addition , the national council of ageing and older people study undertaken in 2005 in the republic of ireland concluded that indirect discrimination , such as barriers to access of services , inadequate transportation , excessive waiting in accident and emergency and outpatient departments , and underfunded community services , is widespread and frequent . internationally , older people care services have difficulty in attracting and retaining staff [ 22 , 23 ] . the status afforded by health care workers to older people in their care has received attention from researchers [ 20 , 24 , 25 ] . provided evidence of older people being neglected or treated as unimportant , and more recently mitchell and mccance indicated that older people perceive that they are commonly denied an active role in making decisions about their health and lives . reports of elder abuse cases in care homes in the republic of ireland , mortality rates and neglect of older patients in a uk nhs hospital , and substandard care of patients with dementia have indicated inadequate training , poor communication and management , and insufficient access to specialist services and rehabilitation therapy as contributing factors . the o neill report of systematic abuse of residents in the leas cross nursing home in the republic of ireland highlighted that managers and clinical leaders did not recognise the care required to meet the needs of their residents . in addition , o neill identified failures at area health board , the health service executive ( hse ) , and government levels for the quality of care the residents received . it was contended that the low priority afforded by these public organisations to the needs of older people and their failure to provide adequate policy and legislation had direct consequences for care standards . the quality of care that older people receive in health services reflects the innate nature of ageism within society . indeed , chronic underfunding and low priority given to the development of services for older people is considered to profoundly influence how healthcare workers think about working with older people [ 12 , 26 ] . the republic of ireland has a population of 4,203,200 , and the proportion of its population aged 65 years and above is 11.0% . initiatives in 2005 to counter ageism and elder abuse in ireland included a rise in the state old age pension to reduce the poverty risk in its older population from 27% to 20% and an annual say no to ageism week . the health service executive in 2008 introduced mandatory training on recognising and responding to elder abuse in residential care setting , which focused on discrimination in general , but particularly ageism . the fair deal legislation introduced in 2009 espouses principles of equality and fairness in access to services but acknowledges that independent financial resources continue to have significant influence on personal choice and timely supportive services . ageing demographics is a global phenomenon , and the republic of ireland is anticipating a 25% increase in its population aged 65 years and over , from year 2004 to 2026 . given the rising percentage of older people using hospital and community services , and almost weekly accounts in the media of substandard care of older people , it is important that the attitudes of healthcare workers towards older people are re - examined . our study was undertaken in a rural county , where common to other rural counties of ireland work outside the home , and the migration of younger family members to urban areas to seek work is challenging care services for older people . in this county , health care is managed through one acute regional hospital , nine community hospitals , two community nursing units , and approximately 58 public health nurses . older people in receipt of nursing care will encounter ward managers , registered staff nurses , healthcare assistants , public health nurses , community - registered nurses , and student nurses , and the decision was taken to include all these groups in a survey of attitudes towards older people . the aim of this study was to explore the attitudes held by these health care workers towards older people in this rural population . to measure , using kogan 's attitudes towards older people scale [ kop ] , the attitudes of health care workers towards older people . to compare scores on kop across groups of health care workers categorised by role title , length of service in current role , and across work place setting . to explore the relationship between highest education attainment and kop scores . to make recommendations to inform education , research , and practice . the design of the study was a survey of attitudes of healthcare workers towards older people . attitudes towards older people were measured using the kogan 's attitudes towards older people scale [ kop ] , developed by kogan in 1961 . in developing the scale , kogan assigned older people the status of a minority group , and the items in the scale originated within ethnic minority stereotype research . the kop is a 34-item likert type scale with 17 matched positive and negative statements ; an example of negative - positive item pair is most older people tend to let their homes become shabby and unattractive and most old people can generally be counted on to maintain a clean , attractive home . it must be acknowledged that the kogan scale was developed in the early 1960s , and it has been subject to criticism . mclafferty has suggested that the scale measures societal attitudes but does not take into account the particular context in which nurses meet older people , and iwasaki and jones highlight the diverse nature of older life and that neither positive nor negative images can reveal the ambiguous nature of people 's attitudes , views , and experiences of ageing . however , the scale has been used extensively to measure attitudes towards older people across a range of professional groups [ 3 , 6 , 8 , 10 , 11 ] . kogan investigated the scale 's reliability and reported spearman - brown reliability coefficients ranging from 0.66 , to 0.83 , and interscale item correlations ranging from 0.46 to 0.52 . in addition , kogan contended concurrent validity by the use of measures of antiauthoritarian and antiminority attitudes . the reliability of kop has since been confirmed by other researchers [ 3 , 6 , 8 , 10 , 11 ] . the kop in our study was scored on a 6-point likert scale : 6 : highly positive , 5 : positive , 4 : slightly positive , 3 : slightly negative , 2 : negative , and 1 : highly negative . the negative statements were reverse scored , so that higher scores were attributed to more positive attitudes . the minimum score possible was 34 , and the maximum score possible was 204 . section 1 was designed to capture biographical and contextual data such as gender , job title , length of time in current role , work setting and highest education qualification , and completion of a healthcare / carers course and care of older people course . section 2 comprised the a participant information letter was provided , and the voluntary nature of completion was stated . consenting participants completed the questionnaire , sealed it in the envelope provided , and placed it in a marked container in the practice setting office for collection . the number of staff on duty during the designated 24hour data collection period in year 2009 was ascertained from service managers and duty rosters to establish the questionnaire response rate . the participants were employed within health service executive sectors as ward managers , registered nurses , public health nurses , healthcare assistants , and student nurses . the sampling strategy adopted was a convenience sample of health care workers in the work settings of an acute regional hospital , community hospitals , nursing units , and in the community . in the regional hospital , the sample comprised of all these healthcare workers from the three general medical wards and two surgical wards and its one rehabilitation ward . in the community , the sample was comprised of all healthcare workers rostered to work in the county 's nine community hospitals and its two nursing units during the 24-hour data collection period . directors of nursing and managers agreed to distribute the questionnaires during the collection period in 2009 . owing to the large geographical nature of the county , a random sample of twenty public health nurses were sent the questionnaire by post , with a stamped addressed envelope for response . the researcher received approval to proceed with the study from the university of ulster 's research ethics committee and the ethics committee in the regional hospital . the regional hospital has responsibility for both hospital and community nursing services in the county . the main ethical implications of this research were informed consent , confidentiality , and anonymity of participants . the service managers for the acute hospital , the community nursing services , the community hospitals , and nursing units were contacted and requested in writing for permission to distribute questionnaires to their health care workers , which was granted . there was a minimal amount of risk associated with this research , as the participants were staff employed by the health service executive . it was anticipated that the research would help to identify those categories of healthcare workers who may express ageist attitudes . whilst there is a link between attitude and predicted behaviour , expressing ageist attitudes does not mean that a group of staff do not give high - quality care . if ageist attitudes were expressed , then recommendations for education interventions for these staff would be proposed . the questionnaires were coded jointly by the first and second author to ensure consistency of analysis , and standard schemes were used to compute the scores for kop . data was analysed using spss for windows v.17 ( spss inc . , chicago , il ) . the kop was computed by using a 6-point likert approach , in which the more positive attitudes towards older people achieved a higher score . the kop was totalled , and cross - referenced to the returned questionnaires to address any errors or missed data coding . missing data on spss was replaced with the midpoint number 3.5 , a strategy supported by palliant . descriptive statistics were used to explore the characteristics of the sample . for statistical tests , the level of significance considered appropriate for this study was a value for p of .05 . one - way analysis of variance with post hoc comparisons were conducted to examine whether attitudes were significantly different between groups of health care workers . a total of 303 questionnaires were distributed to health care workers , and 190 returned the completed questionnaires . the study had an overall response rate of 62.2% , which is comparable to response rates in similar studies of 57% and 69% . three questionnaires with five or more missing item data out of a total of 34 were withdrawn from the study . five returned questionnaires had some missing data in section 1 , which requested information on occupation and qualifications ; these were not excluded from the kop analysis and the missing data remained . the prevalence of health care worker occupations were registered staff nurses 85 ( 45.5% ) , health care assistants 49 ( 26.2% ) , ward managers 21 ( 11.2% ) , student nurses 19 ( 10.2% ) , and public health nurses 11 ( 5.9% ) . the prevalence rate of highest educational attainment was ascertained as outlined in table 1 , and it was observed that 33.7% of nurses were university graduates having attained a nursing degree , higher diploma , or msc . in addition , 83% of the healthcare assistants in this survey had either undertaken a healthcare / carers course , a care of older people course , or both . kolmogorov - smirnov statistic equal to 0.200 was a nonsignificant result , and visual inspection of the distribution of scores indicated normality ( table 2 ) . the measure was found to be reliable on the basis of the data obtained in the questionnaires ( cronback a = 0.751 ) . the variation of intensity of attitudes was illustrated by dividing the potential range of scores into six equal categories , and this showed that health care workers held positive attitudes towards older people ; indeed 97.3% of scores fell into the an independent sample t - test was used to compare kop mean attitude scores between males ( n = 19 ) and females ( n = 167 ) . participants were then categorised in 5 groups according to their job titles : ward manager , registered nurse , public health nurse , student nurse , and healthcare assistant . a one - way between - groups analysis of variance levene statistic was 0.202 , indicating that we had not violated the assumption of homogeneity of variance . although ward managers and public health nurses held more positive attitudes ( figure 1 ) , post - hoc comparisons performed using the tukey hsd test failed to detect a significant difference in kop scores between the groups ( p = .135 ) . a one - way between - groups analysis of variance was conducted to explore the attitudes of participants categorised into 7 groups by length of service in current role : those who had worked 5 years ; 610 years ; 1115 years ; 1620 years ; 2125 years ; 2630 years ; 31 years . levene statistic was 0.953 , indicating that we had not violated the assumption of homogeneity of variance . kop scores between these groups were not significantly different [ f(6,179 ) = 1.8 , p = .106 ] . an independent sample t - test was conducted to explore attitudes between health care workers who worked in acute care settings and those who worked in the community / community hospital settings and mean attitudes were not significantly different between these two groups ( p = .248 ) . an independent sample t - test was conducted to compare kop scores for those who had attained a university degree , higher diploma , or msc and those who had not attained qualifications at university . there was a significant difference in scores for university graduates ( m = 149.34 , sd = 15.4 ) , compared to those who had not attained university qualifications [ m = 144.88 , sd = 12.45 ; t(161 ) = 2.02 , p = .044 ] . this study set out to explore the attitudes held by groups of health care workers towards older people in a rural population . it was reassuring to find that health care workers in this rural county in the republic of ireland generally held positive attitudes towards older people . our study did not detect significant differences in attitude scores measured by kop across gender , job title , length of service in current role , and work place setting . in contrast , our study did detect a significant difference in scores for university graduates , when compared to those who had not attained a university qualification , with university study associated with more positive attitudes . research indicates that health care workers may hold negative attitudes towards the structural context of work and the restrictive practices that can pervade in older people care settings . research which supports this context dimension [ 12 , 13 ] suggests that attitudes can be negatively influenced by the underresourced care environments experienced when working with older people . our study did not detect a significant difference in kop scores between health care workers who worked in acute care services and those in community , community hospital settings , or nursing units . hope and later mclafferty and morrison reported that health care workers in care settings for older people had more positive attitudes towards older people than those working in more acute care settings . however , more recently , and similar to our findings , gallagher et al . 's research did not detect a significant difference in attitudes across work setting . organisational change , health service reform , early supported discharge and ageing demographics in the last two decades , and mediating towards a higher prevalence of older people in acute care wards mean that in almost all care settings , health care workers are increasingly working with older people , and perhaps this explains the similar favourable attitudes . over 83% of the healthcare assistants in our study healthcare / carers course , a care of older people course , or both . healthcare assistants provide much of the face to face care with older people , but their attitudes towards older people have received only scanty attention from researchers [ 4 , 6 ] . in our study , no significant kop score differences were found between staff nurses and healthcare assistants , which contrasts with previous research that reported that healthcare assistants held more negative attitudes towards older people than registered nurses . perhaps the recent irish government initiatives to combat ageism and the high uptake of healthcare / carers courses and care of older people courses by healthcare assistants have led towards the more positive attitudes . it is perhaps significant that 33.7% of participants were university graduates having obtained a degree , higher diploma , or a master 's degree . health care workers are a stable workforce in this rural county , and full - time job vacancies can be rare . individual staff may consider that university academic attainment affords them greater opportunities for career progression than others without university qualifications . education has been identified in previous studies as a predictor for attitudes towards older people [ 5 , 37 ] . our finding that staff who attained a university qualification had significantly higher mean kop scores than those health care workers who had not attained university qualifications is particularly interesting in light of the shift towards nursing becoming an all - graduate profession . the research evidence relating to education and attitudes towards older people is complex , and perhaps this is not surprising , when educational attainment is not a guarantee that individuals have received sufficient education and training in the needs of and care required by those who are older . nurses who work with older people are critical of both staff in acute care sectors and educators of nursing students , for not fully addressing the needs of older people in education programmes [ 13 , 14 ] . in addition , there has been criticism that nurse educators fail to counter negative perceptions of working with older people as a future career option of nursing students . indeed , stevens and crouch considered that nurse educators perpetuated the view that acute care was more demanding of skill and knowledge . criticisms of nurse education has been reinforced by the recent alzheimer 's society survey of 1100 nurses in the uk which reported that 80% of participants felt they had been inadequately trained to work with people with dementia . our findings suggest that public health nurses hold more positive attitudes towards older people than registered nurses , nursing assistants , and students , although it must be noted that differences across role title were not significant . in the republic of ireland , public health nurses commonly hold three professional qualifications : general nursing , midwifery , and public health nursing . public health nurses are community based , and their work is with all levels of that community , working with individuals , families , and groups . they are autonomous practitioners who prioritise their workloads , in their roles as managers , clinicians , and health promoters . it is conceivable that in this rural county , the public health nurses know the older person they are working with , not only as an older person but possibly as a neighbour , distant relative , or friend . in addition , as they are embedded in the community , the person being supported is real and likely to be perceived as living independently . this community embedded knowing the person might be very different from the experience of some of the healthcare workers who primarily work in hospitals , which may partly explain why public health workers hold the most positive attitudes . however , ward managers working in both acute hospital settings and the community nursing units also generally held more positive attitudes than registered nurses and health care assistants . it is likely that as 61.9% of ward managers had studied to degree , higher diploma , or masters , that higher education attainment is also a factor that links ward managers and public health nurses in terms of more positive attitudes . there are a number of limitations in this study that would need to be addressed if this study was to be undertaken again . is one of the significant influencing factors on attitudes , it would have been pertinent to see if that finding was replicated in this study . however , the attitude scores of participants categorised by length of service in current role were scrutinised , and significant differences were not detected . a six - point likert scale was chosen , similar to lookinland and anson and haight et al . . however the authors of this study suggest that missing data could be managed more simply , by using a whole midpoint number , such as in a 5-point likert scale used by ryan et al . . these points indicate the importance of undertaking a pilot study with all questionnaire tools , which would have given the researcher the opportunity to make informed amendments . our study has shown that the vast majority of the participants which included nurses , nursing assistants , and students hold positive attitudes , towards older people . in addition , we found that study to a higher level at university appears to mitigate towards holding more positive attitudes and this is an important finding in light of the shift towards nursing as an all - graduate profession . as previously stated , the study was undertaken in a rural county in the republic of ireland , with a generally stable nursing workforce , and it is possible that healthcare workers are regularly caring for older people they have come to know as individuals . with the global trend towards an increasingly ageing population , it is clear that nurses need to be equipped with the knowledge and skills to fulfil significant roles in responding to future health and support needs . the recent initiatives in the republic of ireland to counter ageism and elder abuse [ 3032 ] are commendable , and in addition the high update of carers and care of older people courses by nursing assistants may be promoting more positive attitudes . budget constraints in the uk and ireland currently pitch health services against social services and stall the development of supportive care packages , with resultant delays in discharge from acute hospital care and overreliance on family carers . these consequences are likely in the future to further reinforce the portrayal of older people as a burden on society . the current reports of elder abuse , neglect , and substandard care receiving media coverage are also in danger of undermining those healthcare workers who are attempting to provide quality care in challenging times . despite this context , it is reassuring that in our study , nurses and nursing assistants hold positive attitudes towards older people .

with the global trend towards an increasingly ageing population , it is clear that nurses need to be equipped with the knowledge and skills to fulfil significant roles in responding to future health and support needs . this paper reports the results of a survey that aimed to identify and evaluate the attitudes of nurses , healthcare assistants , and nursing students towards older people . the survey was undertaken in a rural county in the republic of ireland . it is reassuring that in our study , we found that these healthcare workers hold positive attitudes towards older people . in addition , we found that study to a higher level at university appears to mitigate towards holding more positive attitudes , and this is an important finding in light of the shift towards nursing as an all - graduate profession .

1. Introduction 2. Material and Methods 3. Sampling and Ethical Considerations 4. Data Analysis 5. Results 6. Discussion 7. Conclusions

healthcare workers are considered to be at particular risk of developing ageist attitudes because they are exposed to a disproportionate percentage of ill or dependent older people . research demonstrates that many factors have influence on healthcare workers ' attitudes towards older people , including age , gender [ 13 ] , education [ 46 ] , exposure to well older people [ 710 ] , area of practice [ 1114 ] , and professional socialization . an attitude is an evaluation of something or someone on a continuum of like to dislike or favourable to unfavourable . ageism is perpetuated by the portrayal of older people as frail , ill , suffering mental deterioration , poor and dependent , and the alternative portrayal of living affluent life styles and scrounging off the welfare state . in addition , budget constraints in the uk and ireland which currently pitch health services against social services over costs for supportive care packages negate against timely discharge from hospital and reinforce an overreliance on family careers for support . these consequences are likely to further reinforce the portrayal of older people as a burden on society . stereotypes such as these are in contrast with the reality that the majority of older people lead fit and independent lives . older people consider that inadequate housing , low income , enforced retirement , and deficiencies in nursing home care are evidence of ageism . in addition , the national council of ageing and older people study undertaken in 2005 in the republic of ireland concluded that indirect discrimination , such as barriers to access of services , inadequate transportation , excessive waiting in accident and emergency and outpatient departments , and underfunded community services , is widespread and frequent . internationally , older people care services have difficulty in attracting and retaining staff [ 22 , 23 ] . the status afforded by health care workers to older people in their care has received attention from researchers [ 20 , 24 , 25 ] . provided evidence of older people being neglected or treated as unimportant , and more recently mitchell and mccance indicated that older people perceive that they are commonly denied an active role in making decisions about their health and lives . reports of elder abuse cases in care homes in the republic of ireland , mortality rates and neglect of older patients in a uk nhs hospital , and substandard care of patients with dementia have indicated inadequate training , poor communication and management , and insufficient access to specialist services and rehabilitation therapy as contributing factors . the o neill report of systematic abuse of residents in the leas cross nursing home in the republic of ireland highlighted that managers and clinical leaders did not recognise the care required to meet the needs of their residents . in addition , o neill identified failures at area health board , the health service executive ( hse ) , and government levels for the quality of care the residents received . it was contended that the low priority afforded by these public organisations to the needs of older people and their failure to provide adequate policy and legislation had direct consequences for care standards . the quality of care that older people receive in health services reflects the innate nature of ageism within society . indeed , chronic underfunding and low priority given to the development of services for older people is considered to profoundly influence how healthcare workers think about working with older people [ 12 , 26 ] . the republic of ireland has a population of 4,203,200 , and the proportion of its population aged 65 years and above is 11.0% . initiatives in 2005 to counter ageism and elder abuse in ireland included a rise in the state old age pension to reduce the poverty risk in its older population from 27% to 20% and an annual say no to ageism week . the health service executive in 2008 introduced mandatory training on recognising and responding to elder abuse in residential care setting , which focused on discrimination in general , but particularly ageism . ageing demographics is a global phenomenon , and the republic of ireland is anticipating a 25% increase in its population aged 65 years and over , from year 2004 to 2026 . given the rising percentage of older people using hospital and community services , and almost weekly accounts in the media of substandard care of older people , it is important that the attitudes of healthcare workers towards older people are re - examined . our study was undertaken in a rural county , where common to other rural counties of ireland work outside the home , and the migration of younger family members to urban areas to seek work is challenging care services for older people . in this county , health care is managed through one acute regional hospital , nine community hospitals , two community nursing units , and approximately 58 public health nurses . older people in receipt of nursing care will encounter ward managers , registered staff nurses , healthcare assistants , public health nurses , community - registered nurses , and student nurses , and the decision was taken to include all these groups in a survey of attitudes towards older people . the aim of this study was to explore the attitudes held by these health care workers towards older people in this rural population . to measure , using kogan 's attitudes towards older people scale [ kop ] , the attitudes of health care workers towards older people . to compare scores on kop across groups of health care workers categorised by role title , length of service in current role , and across work place setting . to make recommendations to inform education , research , and practice . the design of the study was a survey of attitudes of healthcare workers towards older people . attitudes towards older people were measured using the kogan 's attitudes towards older people scale [ kop ] , developed by kogan in 1961 . in developing the scale , kogan assigned older people the status of a minority group , and the items in the scale originated within ethnic minority stereotype research . the kop is a 34-item likert type scale with 17 matched positive and negative statements ; an example of negative - positive item pair is most older people tend to let their homes become shabby and unattractive and most old people can generally be counted on to maintain a clean , attractive home . it must be acknowledged that the kogan scale was developed in the early 1960s , and it has been subject to criticism . mclafferty has suggested that the scale measures societal attitudes but does not take into account the particular context in which nurses meet older people , and iwasaki and jones highlight the diverse nature of older life and that neither positive nor negative images can reveal the ambiguous nature of people 's attitudes , views , and experiences of ageing . however , the scale has been used extensively to measure attitudes towards older people across a range of professional groups [ 3 , 6 , 8 , 10 , 11 ] . kogan investigated the scale 's reliability and reported spearman - brown reliability coefficients ranging from 0.66 , to 0.83 , and interscale item correlations ranging from 0.46 to 0.52 . in addition , kogan contended concurrent validity by the use of measures of antiauthoritarian and antiminority attitudes . the kop in our study was scored on a 6-point likert scale : 6 : highly positive , 5 : positive , 4 : slightly positive , 3 : slightly negative , 2 : negative , and 1 : highly negative . the negative statements were reverse scored , so that higher scores were attributed to more positive attitudes . the minimum score possible was 34 , and the maximum score possible was 204 . section 1 was designed to capture biographical and contextual data such as gender , job title , length of time in current role , work setting and highest education qualification , and completion of a healthcare / carers course and care of older people course . section 2 comprised the a participant information letter was provided , and the voluntary nature of completion was stated . consenting participants completed the questionnaire , sealed it in the envelope provided , and placed it in a marked container in the practice setting office for collection . the number of staff on duty during the designated 24hour data collection period in year 2009 was ascertained from service managers and duty rosters to establish the questionnaire response rate . the participants were employed within health service executive sectors as ward managers , registered nurses , public health nurses , healthcare assistants , and student nurses . the sampling strategy adopted was a convenience sample of health care workers in the work settings of an acute regional hospital , community hospitals , nursing units , and in the community . in the regional hospital , the sample comprised of all these healthcare workers from the three general medical wards and two surgical wards and its one rehabilitation ward . in the community , the sample was comprised of all healthcare workers rostered to work in the county 's nine community hospitals and its two nursing units during the 24-hour data collection period . owing to the large geographical nature of the county , a random sample of twenty public health nurses were sent the questionnaire by post , with a stamped addressed envelope for response . the researcher received approval to proceed with the study from the university of ulster 's research ethics committee and the ethics committee in the regional hospital . the regional hospital has responsibility for both hospital and community nursing services in the county . the main ethical implications of this research were informed consent , confidentiality , and anonymity of participants . the service managers for the acute hospital , the community nursing services , the community hospitals , and nursing units were contacted and requested in writing for permission to distribute questionnaires to their health care workers , which was granted . it was anticipated that the research would help to identify those categories of healthcare workers who may express ageist attitudes . the questionnaires were coded jointly by the first and second author to ensure consistency of analysis , and standard schemes were used to compute the scores for kop . , chicago , il ) . the kop was computed by using a 6-point likert approach , in which the more positive attitudes towards older people achieved a higher score . the kop was totalled , and cross - referenced to the returned questionnaires to address any errors or missed data coding . missing data on spss was replaced with the midpoint number 3.5 , a strategy supported by palliant . descriptive statistics were used to explore the characteristics of the sample . a total of 303 questionnaires were distributed to health care workers , and 190 returned the completed questionnaires . three questionnaires with five or more missing item data out of a total of 34 were withdrawn from the study . the prevalence of health care worker occupations were registered staff nurses 85 ( 45.5% ) , health care assistants 49 ( 26.2% ) , ward managers 21 ( 11.2% ) , student nurses 19 ( 10.2% ) , and public health nurses 11 ( 5.9% ) . the prevalence rate of highest educational attainment was ascertained as outlined in table 1 , and it was observed that 33.7% of nurses were university graduates having attained a nursing degree , higher diploma , or msc . in addition , 83% of the healthcare assistants in this survey had either undertaken a healthcare / carers course , a care of older people course , or both . kolmogorov - smirnov statistic equal to 0.200 was a nonsignificant result , and visual inspection of the distribution of scores indicated normality ( table 2 ) . the measure was found to be reliable on the basis of the data obtained in the questionnaires ( cronback a = 0.751 ) . the variation of intensity of attitudes was illustrated by dividing the potential range of scores into six equal categories , and this showed that health care workers held positive attitudes towards older people ; indeed 97.3% of scores fell into the an independent sample t - test was used to compare kop mean attitude scores between males ( n = 19 ) and females ( n = 167 ) . participants were then categorised in 5 groups according to their job titles : ward manager , registered nurse , public health nurse , student nurse , and healthcare assistant . although ward managers and public health nurses held more positive attitudes ( figure 1 ) , post - hoc comparisons performed using the tukey hsd test failed to detect a significant difference in kop scores between the groups ( p = .135 ) . a one - way between - groups analysis of variance was conducted to explore the attitudes of participants categorised into 7 groups by length of service in current role : those who had worked 5 years ; 610 years ; 1115 years ; 1620 years ; 2125 years ; 2630 years ; 31 years . an independent sample t - test was conducted to explore attitudes between health care workers who worked in acute care settings and those who worked in the community / community hospital settings and mean attitudes were not significantly different between these two groups ( p = .248 ) . an independent sample t - test was conducted to compare kop scores for those who had attained a university degree , higher diploma , or msc and those who had not attained qualifications at university . this study set out to explore the attitudes held by groups of health care workers towards older people in a rural population . it was reassuring to find that health care workers in this rural county in the republic of ireland generally held positive attitudes towards older people . our study did not detect significant differences in attitude scores measured by kop across gender , job title , length of service in current role , and work place setting . in contrast , our study did detect a significant difference in scores for university graduates , when compared to those who had not attained a university qualification , with university study associated with more positive attitudes . research indicates that health care workers may hold negative attitudes towards the structural context of work and the restrictive practices that can pervade in older people care settings . research which supports this context dimension [ 12 , 13 ] suggests that attitudes can be negatively influenced by the underresourced care environments experienced when working with older people . our study did not detect a significant difference in kop scores between health care workers who worked in acute care services and those in community , community hospital settings , or nursing units . hope and later mclafferty and morrison reported that health care workers in care settings for older people had more positive attitudes towards older people than those working in more acute care settings . however , more recently , and similar to our findings , gallagher et al . organisational change , health service reform , early supported discharge and ageing demographics in the last two decades , and mediating towards a higher prevalence of older people in acute care wards mean that in almost all care settings , health care workers are increasingly working with older people , and perhaps this explains the similar favourable attitudes . over 83% of the healthcare assistants in our study healthcare / carers course , a care of older people course , or both . healthcare assistants provide much of the face to face care with older people , but their attitudes towards older people have received only scanty attention from researchers [ 4 , 6 ] . in our study , no significant kop score differences were found between staff nurses and healthcare assistants , which contrasts with previous research that reported that healthcare assistants held more negative attitudes towards older people than registered nurses . perhaps the recent irish government initiatives to combat ageism and the high uptake of healthcare / carers courses and care of older people courses by healthcare assistants have led towards the more positive attitudes . it is perhaps significant that 33.7% of participants were university graduates having obtained a degree , higher diploma , or a master 's degree . health care workers are a stable workforce in this rural county , and full - time job vacancies can be rare . education has been identified in previous studies as a predictor for attitudes towards older people [ 5 , 37 ] . our finding that staff who attained a university qualification had significantly higher mean kop scores than those health care workers who had not attained university qualifications is particularly interesting in light of the shift towards nursing becoming an all - graduate profession . the research evidence relating to education and attitudes towards older people is complex , and perhaps this is not surprising , when educational attainment is not a guarantee that individuals have received sufficient education and training in the needs of and care required by those who are older . nurses who work with older people are critical of both staff in acute care sectors and educators of nursing students , for not fully addressing the needs of older people in education programmes [ 13 , 14 ] . in addition , there has been criticism that nurse educators fail to counter negative perceptions of working with older people as a future career option of nursing students . criticisms of nurse education has been reinforced by the recent alzheimer 's society survey of 1100 nurses in the uk which reported that 80% of participants felt they had been inadequately trained to work with people with dementia . our findings suggest that public health nurses hold more positive attitudes towards older people than registered nurses , nursing assistants , and students , although it must be noted that differences across role title were not significant . in the republic of ireland , public health nurses commonly hold three professional qualifications : general nursing , midwifery , and public health nursing . public health nurses are community based , and their work is with all levels of that community , working with individuals , families , and groups . they are autonomous practitioners who prioritise their workloads , in their roles as managers , clinicians , and health promoters . it is conceivable that in this rural county , the public health nurses know the older person they are working with , not only as an older person but possibly as a neighbour , distant relative , or friend . in addition , as they are embedded in the community , the person being supported is real and likely to be perceived as living independently . this community embedded knowing the person might be very different from the experience of some of the healthcare workers who primarily work in hospitals , which may partly explain why public health workers hold the most positive attitudes . however , ward managers working in both acute hospital settings and the community nursing units also generally held more positive attitudes than registered nurses and health care assistants . it is likely that as 61.9% of ward managers had studied to degree , higher diploma , or masters , that higher education attainment is also a factor that links ward managers and public health nurses in terms of more positive attitudes . there are a number of limitations in this study that would need to be addressed if this study was to be undertaken again . is one of the significant influencing factors on attitudes , it would have been pertinent to see if that finding was replicated in this study . however , the attitude scores of participants categorised by length of service in current role were scrutinised , and significant differences were not detected . however the authors of this study suggest that missing data could be managed more simply , by using a whole midpoint number , such as in a 5-point likert scale used by ryan et al . our study has shown that the vast majority of the participants which included nurses , nursing assistants , and students hold positive attitudes , towards older people . in addition , we found that study to a higher level at university appears to mitigate towards holding more positive attitudes and this is an important finding in light of the shift towards nursing as an all - graduate profession . as previously stated , the study was undertaken in a rural county in the republic of ireland , with a generally stable nursing workforce , and it is possible that healthcare workers are regularly caring for older people they have come to know as individuals . with the global trend towards an increasingly ageing population , it is clear that nurses need to be equipped with the knowledge and skills to fulfil significant roles in responding to future health and support needs . the recent initiatives in the republic of ireland to counter ageism and elder abuse [ 3032 ] are commendable , and in addition the high update of carers and care of older people courses by nursing assistants may be promoting more positive attitudes . budget constraints in the uk and ireland currently pitch health services against social services and stall the development of supportive care packages , with resultant delays in discharge from acute hospital care and overreliance on family carers . these consequences are likely in the future to further reinforce the portrayal of older people as a burden on society . the current reports of elder abuse , neglect , and substandard care receiving media coverage are also in danger of undermining those healthcare workers who are attempting to provide quality care in challenging times . despite this context , it is reassuring that in our study , nurses and nursing assistants hold positive attitudes towards older people .

any attempt to answer this question implies the idea that one can prospectively decide about the capabilities of a selected cell without relating it to other cells and without functionally testing its capabilities . it is widely accepted that currently a definite characterization of tissue stem cells is only possible on the basis of their functional capabilities and not on the basis of explicit , directly observable attributes . such a functional perspective is inherently consistent with the biological role of tissue stem cells to maintain tissue homeostasis and to ( re)generate functional tissues . the two key capabilities of tissue stem cells are the ability to self - renew their own population and the ability to produce a large number of fully functional , differentiated cells , implying also the ability to proliferate . however , although these are necessary capabilities , they are not sufficient to guarantee long - term maintenance and reconstitution of a fully functional tissue , which requires a highly coordinated control of cell production and differentiation . this points to another essential property of tissue stem cells : the flexibility in the use of their functional potentials . this flexibility , which had for the first time been incorporated into a definition of tissue stem cells by potten and loeffler , refers to the fact that stem cells might particularly be characterized by their ability to respond to the actual needs of the system . such adaptiveness inevitably requires a communication of stem cells among each other and with their microenvironment . beside feedback regulations on the basis of long - range acting molecules such as cytokines [ 24 ] , this communication also refers to the importance of the so - called stem cell niche [ 59 ] . meanwhile , the existence of stem cell supporting niches has been identified for most ( regenerative ) tissues , including the hematopoietic system [ 10 , 11 ] . moreover , there is increasing evidence that stem cell organization is the result of complex cell - cell and cell - microenvironment interactions rather than the consequence of a predefined stem cell intrinsic program [ 1215 ] . applying the functional definition , the above - stated question whether a particular cell is a stem cell can only be answered retrospectively , having subjected the cell to a functional assay . this , however , will induce a cellular response and will inevitably alter the actual properties of the cell . this means that , in order to answer the question , one unavoidably loses the original cell . this situation is somehow similar to heisenberg 's uncertainty principle in quantum physics which states that the very act of measuring the functional properties of a certain system always changes its characteristics , thus , giving rise to a certain degree of uncertainty in the evaluation of the system properties . although not identical , the uncertainty in the determination of the functional potential of a cell still implies that all prospective statements about stem cell functioning are necessarily probabilistic statements about the cellular behavior under particular conditions . there are a number of experimental observations which challenge the classical conception of a cell intrinsically predefined stem cell program . although these observations are not restricted to one particular tissue , we will discuss them with the focus on the hematopoietic system . hematopoietic stem cells ( hscs ) are heterogeneous with respect to functional properties such as cycling activity , engraftment potential or differentiation status , as well as to the expression of specific markers ( phenotypic heterogeneity ) . although there exist a number of sophisticated purification protocols that are able to select more homogeneous populations of stem cells [ 1620 ] , there is always a certain functional overlap of the obtained subpopulations . furthermore , there is accumulating evidence that the phenotypic properties of hsc are reversibly changing ( phenotypic reversibility ) [ 2128 ] and that tissue stem cells specified for one type of tissue can be manipulated such that they can act as stem cells of another tissue ( stem cell plasticity ) [ 2932 ] . even though there are most likely a number of constraints in the developmental options , these observations point to the fact that the functional potential of a stem cell can not be uniquely determined by its actual phenotypic appearance . therefore , although a specific purification protocol might select a population of cells with a homogeneous phenotype , showing a certain behavior within a particular functional assay , this behavior might change over time or if the cells are exposed to different assay conditions . because classical stem cell concepts are not able to explain all these experimental findings however , to be validated , such concepts need a rigorous examination by quantitative and predictive modeling approaches . particularly with respect to the uncertainty in the prospective characterization of stem cell function , a well - defined theoretical framework will help to cope with the complexity of experimental systems and will , therefore , considerably contribute to a deeper understanding of functional principles of stem cell organization . in conjunction with predictive quantitative models , such a theory will assist biologists to select , design , and optimize experimental strategies , and can help to systematically anticipate the impact of manipulations to a system . theoretical approaches and simulation techniques support the identification of latent mechanisms and crucial parameters of biological processes , and may predict new phenomena . furthermore , the application of a common model structure to different systems ( i.e. , tissues or cell types ) may help to understand generic construction and regulation principles . to serve as the basis for a theoretical framework of tissue stem cell organization and to allow for a stringent experimental validation of the theory , quantitative models have to fulfill a number of general requirements . they have to provide experimentally testable predictions . because functional assays are the only way to definitely characterize tissue stem cells , the models must be able to account for the readouts of these assays . this requires that system - measurement interactions have to be considered in the model . furthermore , stem cell models must be based on populations of individual cells to follow clonal development , to enable considerations of population fluctuations , and to conform to the uncertainty principle . because of the increasing evidence that stem cell behavior is not the result of a cell - autonomous program , but instead the consequence of complex cell - cell and cell - growth environment interactions , these interactions have to be represented in such models . to be able to correctly describe regulatory processes , the model systems have to be dynamic in time , and possibly also in space . particularly , they must be comprehensive in the sense of being applicable to normal homeostasis as well as to perturbed situations . the functional definition of tissue stem cells implies that stemness should be regarded as a functional endpoint rather than as an explicit attribute of individual cells . therefore , any concept of tissue stem cells has to specify assumptions about the mechanisms that potentially control the regenerative and proliferative potential of these cells . thus , a dynamic model should adequately represent processes that drive and control cellular attributes . apparently , these processes are determined by the genetic and epigenetic statuses of the cells as well as by the activity of various signaling and metabolic pathways . since it is presently impossible to describe the entirety of these processes in any reasonable detail , one major goal is the derivation of a simplified basic scheme accounting for the generic principles underlying the cellular dynamics . because many experimental results show the necessity to consider flexibility and reversibility of cellular properties as important constituents of stem cell organization , we propose to give up the view of tissue stem cells as being entities with a preprogrammed development this view should be replaced by a concept that makes cellular capabilities for flexible and regulated tissue self - organizing the new paradigm . such a concept incorporates context - dependent phenotypic reversibility and generation of stem cell heterogeneity as the result of a dynamically regulated process . it consequentially avoids assumptions that lead to a direct or indirect a priori labeling of particular cells as stem cells ; cells are purely characterized on the basis of functional potentials . these cellular potentials as well as their actual use are able to change in response to cell - cell and cell - microenvironment interactions , such that the cell population fulfils the functional criteria of the stem cell definition . in this sense , a cell with high potential for long - term repopulation will not necessarily act as a long - term repopulating cell . in contrast , a cell with only a low long - term repopulating potential might , under certain circumstances , be selected to act as a stem cell . it should be stressed that although this concept includes a considerable degree of flexibility in the cellular development , it does not exclude the existence of restrictions in the developmental potential of individual cells . therefore , also the complete loss of repopulating potential at a certain stage of development ( e.g. , due to terminal differentiation ) is compatible with the proposed concept . to put such a theoretical framework to a quantitative test , comparing it with various types of experimental observations , the general concept has been translated into a stochastic , single - cell - based model for hsc which is summarized in the next section . as already described in the context of the general concept , we assume that cellular properties of hsc can reversibly change within a range of potential options . herein , the direction of cellular development and the decision whether a certain property is actually expressed depend on the internal state of the cell and on signals from its growth environment . particularly , individual cells are considered to reside in one of two growth environments ( denoted as ge - a and ge- ) . the state of each cell is characterized by its actual growth environment , by its position in the cell cycle ( g1 , s , g2 , m , or g0 ) , and by a property a , which describes its affinity to reside in ge - a . whereas cells in ge- are assumed to gradually loose affinity a , cells in ge - a are able to gradually regain a ( up to a maximal value amax ) . furthermore , whereas cells in ge - a are assumed to be nonproliferating , cells in ge- are able to proliferate with an average generation time c . the transition of cells between the two growth environments is modeled as a stochastic process . the transition intensities ( i.e. , the probabilities of growth environment change per time step , denoted as and ) depend on the actual value of the affinity a and on the number of stem cells residing in ge - a and ge- , respectively . if affinity a of an individual cell has fallen below a prespecified threshold ( amin ) , the ability to home to ge - a and , therefore , the potential to regain affinity a is lost . these cells start the formation of differentiated clones with a fixed life span , that is , they continue to proliferate for a fixed period of time and are finally removed from the system . we demonstrated that this model of hsc organization consistently describes a broad variety of observed phenomena such as heterogeneity of clonogenic and repopulation potentials , changing cell cycle activity of primitive progenitors , or different types of clonal competition including the development and treatment of specific human leukemias [ 3336 ] . particularly , the proposed single - cell - based model structure allows to analyze cellular dynamics not only on the population , but also on the individual clone level . this is of particular interest in applications where the dynamic properties of individual ( potentially manipulated ) stem cells or stem cell clones are essential targets . examples of such applications are gene - therapeutic approaches , and also the ex vivo expansion of stem or progenitor cells . in both cases , the competitive repopulation potential and the in vivo persistence of ( clonally derived ) stem cell transplants should be controlled and possibly optimized . to illustrate the theoretical investigation of individual cell fates and of clonal dynamics and to highlight important benefits of a model analysis , we will consider two particular phenomena classes : fluctuating contribution of individually marked stem cell clones and cell fate asymmetry of paired progenitors . to simulate the dynamics of individual stem cell clones , all model cells are individually labeled with an inheritable marker at one point in time . using this procedure we would like to unmistakably point out that here and throughout the paper , a clone is defined as the entire progeny of one particular cell . this implies that a clone is always characterized relative to a particular marking event , specifying the founder cell of the clone . it is also possible that different marking events define nested clones , implying that identical cells can be considered as members of different clones . consider the case that the individual cell marking procedure is completely neutral ( i.e. , not inducing any competitive growth advantage ) and has been applied to a homeostatic hematopoietic system . this means that the number of traceable clones equals the total number of cells contributing to the system at this particular time point . starting from such a configuration , our model predicts that the system will inevitably convert from this polyclonal state to an oligo- and finally to a monoclonal situation . in other words , asymptotically all cells will belong to only one clone ( i.e. , all having one common ancestor ) even in the case of completely neutral marking . however , the time scale of such a monoclonality conversion might be very large . for the murine homeostatic reference situation ( see for detailed model parameters ) with about 300 model stem cells , the time to monoclonality has been estimated to be approximately 65 years . during a normal mouse life span of about 2 years , the number of stem cell clones is predicted to reduce to about 30 . the cause of this clonality conversion is the stochastic fluctuation of cells between the two growth environments , with a certain positive probability of final differentiation ( here , in the sense of reaching a < amin ) for cells in ge-. of course , the kinetics of the conversion depends on the model parameters which determine the differentiation probability , such as the average generation time of stem cells c ( figure 2(a ) ) . furthermore , it is predicted that the process of clone exhaustion can be accelerated by system perturbations , for example , due to repeated cell kill events ( figure 2(b ) ) . there is another point that might considerably affect the interpretation of experimental observations on clonal contribution . this is the fact that clone sizes ( i.e. , cell numbers per clone ) are predicted to fluctuate over time . therefore , also clones that actually contribute to hematopoiesis might be overlooked , for example , due to a threshold - dependent detection procedure . to illustrate this effect , figure 3(a ) illustrates the fluctuating size of 50 individual clones within a homeostatic system . in contrast , figures 3(b ) and 3(c ) are depicting different projections of this data . whereas figure 3(b ) shows all existing clones ( i.e. , clone sizes larger than or equal to one cell ) , figure 3(c ) indicates measurable clones , assuming a detection threshold of 10 stem cells per clone . the emerging pattern looks very different although the underlying system is identical . applying these simulation results to different observations can help to identify misleading aspects in the interpretation of experimental findings and to disentangle seemingly contradictory results . one example is the ongoing debate , whether hematopoiesis is mono- , oligo- or polyclonal in nature . opposing results , reaching from oligoclonality with large long - lived clones to polyclonal situations with many short - lived clones , have been reported [ 3743 ] . to discuss the model analysis of these phenomena , let us consider two particular results on the clonal composition of the hematopoietic system . whereas jordan and lemischka observed an oligoclonal hematopoiesis with a few dominant persistent clones , drize et al . reported a polyclonal composition with many small short - lived clones . although a similar general experimental setup for the tracing of retrovirally marked clones had been applied in both studies , the sampling strategies as well as the measurement protocols differed . in contrast to jordan & lemischka who analyzed repeated blood / spleen samples with a high cell number but with relatively low detection sensitivity for individual marker signals , drize et al . analyzed single - cell - induced spleen colonies obtained by injecting repeated bone marrow samples into irradiated recipient mice . because only a small proportion of bone marrow cells seed in the spleen , the sample size of analyzed cells is small . however , this procedure ensures a high detection sensitivity due to the amplification of the marker signal in the clonally derived colonies . to simulate these two experimental strategies , model systems are initiated with individually labeled stem cells sampled from a homeostatic reference system . according to the two described experimental protocols , different numbers of marked cells , with n = 10 cells for the jordan - like simulation and n = 100 cells for the simulation of the drize experiment , were used . experimentally detectable clones have been simulated by the sampling of individual model stem cells ( representing spleen - colony forming cells ) with a probability of 0.01 for the drize - like setting , and by counting all differentiated clones ( representing the entirety of bone marrow / spleen cells ) which exceed a size threshold of 10 000 cells per clone for the jordan - like setting . this procedure is applied at sequential time points ( 3-month intervals ) . as demonstrated by our simulations ( figure 4 ) , the different experimental observations can be consistently explained by differences in the sampling techniques and detection thresholds applied to an identical underlying biological system . although our model of a self - organized stem cell population does explicitly preclude asymmetric cell divisions , it still accounts for asymmetric cell fates . this asymmetry , however , is not caused by a predefined cell intrinsic program , but emerges as the result of cell - cell and cell - microenvironment interactions . for illustration ( cf . figure 5 ) , consider a model cell with initial affinity a1 . however , during completion of a cell division , also the affinity a changes from a1 to a new value a2 < a1 . now , one daughter cell might change to ge - a , subsequently regaining the affinity to its initial value a1 , while the other daughter cell continues to decrease a. beside such an asymmetric development , also two scenarios of symmetric cell fates can be obtained : whenever both daughter cells regenerate their affinity , the number of cells with the original functional potential is amplified . in contrast , a symmetric differentiation is generated if both daughter cells remain under the influence of ge-. it is also possible to quantitatively describe experimental data on asymmetric stem cell behavior within the context of our model . as an example , consider the cycling activity of stem cells , as described by punzel et al . . these authors analyzed the in vitro cell cycle activity of purified human cord blood cells . in short , individual cd34/cd38 cells were seeded into 96-well plates , previously coated with either bovine serum albumin ( bsa ) , fibronectin ( fn ) , or a specific stromal cell line feeder layer ( aft024 ) . using time - lapse fluorescence microscopy , a division is denoted as asymmetric if one first - generation daughter cell did not divide during the culture period while the other first - generation daughter divided at least once . occurrence of asymmetric cell division was quantified by the percentage of cells showing asymmetric division with respect to all cells deposited ( ad index ) . the determined ad values for the stroma - free cultures ( bsa , fn ) were 22.9% and 22.8% , respectively . in contrast , an ad value of 31.1% has been observed for the aft024 cultures , suggesting that stromal coculture is able to increase the asymmetric behavior . to test whether these results can quantitatively be reproduced without the assumption of asymmetric cell division events , these systems have been traced for 10 days according to the experimental protocol . to compare the ad index of simulations and experiments , a model division is denoted as asymmetric whenever only one of the two first - generation daughter cells is performing further cell divisions . the simulations revealed that the proportion of asymmetric cell fate is particularly sensitive to the initial affinity a of in silico culture - initiating cells . the higher the initial ge - a affinity a of the cells is , the higher the proportion of asymmetric cell divisions is ( figure 6 ) . because affinity a directly correlates to the probability of a cell to long - term repopulate a model system , these results predict that cells with high long - term repopulating potential more frequently exhibit an asymmetric cell fate in vitro . furthermore , the experimental observation that stromal coculture of stem cells enhances the proportion of asymmetric divisions can be explained by the assumption of different regeneration coefficients r. whereas r = 1 ( i.e. , no regeneration of affinity a in ge - a ) produces lower proportions of asymmetric cell fates ( figure 6(a ) ) , r > 1 leads to an increase in the amount of asymmetric cell fates ( figure 6(b ) ) . therefore , the heterogeneity of the in vitro stem cell supporting potential of different stromal cell types can consistently be represented in the model by growth environments ( ge - a ) allowing for variable degrees of a - regeneration . based on these results , we are able to quantitatively reproduce the published experimental results . again starting from the previously derived reference parameter set that consistently describes different in vivo assays using c57bl/6 mice , a variation of the initial affinity range and of the regeneration coefficient lead to a good quantitative fit of simulation results and experimental data ( figure 7 ) . whereas the stroma - free situation is described by a regeneration coefficient of r = 1 , r = 1.05 is assumed for the situation of a stroma - supported culture . note that the latter r - value is still smaller than 1.1 , which is the regeneration coefficient assumed for the in vivo situation . both simulation scenarios use initial affinities a , uniformly distributed on the interval [ 0.5 ; 1 ] . particularly with regard to stem cell fate and individual clonal dynamics , there are a number of predictions arising from the proposed mathematical model . one basic conclusion is that the developmental fate of a stem cell can not be predicted with certainty , even if the actual state of the cell could be determined exactly . however , probabilistic statements about the future development of individual clones as well as about the potential of a population of well - characterized cells are certainly possible . in terms of the model , the likelihood for a certain developmental fate of a stem cell is assumed to depend on the general potential of the cell , on its actual state , and on the microenvironmental signals the cell receives . as demonstrated for a chimeric mouse model , genetic differences in the potential of cells ( e.g. , reactivity on microenvironmental signals ) it has be shown that even very small differences in cellular properties , which would not affect the general repopulation ability of the cells in a nonchimeric situation , are sufficient to sensitively affect the cellular development in the competition scenario . also epigenetically determined ( as , e.g. , suggested by the group of mller - sieburg [ 45 , 46 ] ) or induced ( e.g. , by insertional mutagenesis [ 47 , 48 ] ) differences between stem cell clones within one genetic background could influence the probabilities for certain developmental fates . another related prediction is the clonality conversion as a consequence of system immanent fluctuations . even in the oversimplified case of an identical potential of all stem cell clones , still , it is not possible to unequivocally specify the successful clones in advance . however , as stated above , even small differences in the cellular potential of stem cell clones are able to bias the competitive potential considerably . our model is able to estimate the effect of differences in cellular parameters on competitive growth characteristics , and therefore , to provide statistical predictions about future clonal contributions . this might particularly be important to understand the effects of insertional mutagenesis as well as to quantitatively characterize the outcome of gene - therapeutic interventions . even though a developmental asymmetry of stem cells is inevitably required to provide a continuous production of differentiated cells without exhausting the stem cell population , this asymmetry is not necessarily linked to cell division events . alternatively a flexible functional asymmetry can be achieved by a self - organizing population of interacting cells , including a certain degree of reversibility in cellular properties and functionalities . summarizing our results , we demonstrated that it is possible to understand tissue stem cell systems without assumptions on unidirectional developmental hierarchies , preprogrammed asymmetric division events , or other assumptions implying the existence of a predetermined stem cell entity . as illustrated for the hematopoietic system , a self - organizing perspective would change the paradigm of thinking about stem cells . within such a concept , cellular properties are considered to permanently fluctuate with some cells meeting a situation of clonal expansion . this means that stem cells are selected and modified in response to cell - cell and cell - microenvironment interactions , rather than being specialized a priori . thus , it is their potential and the flexibility to use it , but not a particular actually expressed property , that distinguishes them from other cells .

many experimental findings on heterogeneity , flexibility , and plasticity of tissue stem cells are currently challenging stem cell concepts that assume a cell intrinsically predefined , unidirectional differentiation program . in contrast to these classical concepts , nonhierarchical self - organizing systems provide an elegant and comprehensive alternative to explain the experimental data . here we present the application of such a self - organizing concept to quantitatively describe the hematopoietic stem cell system . focusing on the analysis of individual - stem - cell fates and clonal dynamics , we particularly discuss implications of the theoretical results on the interpretation of experimental findings . we demonstrate that it is possible to understand hematopoietic stem cell organization without assumptions on unidirectional developmental hierarchies , preprogrammed asymmetric division events or other assumptions implying the existence of a predetermined stem cell entity . the proposed perspective , therefore , changes the general paradigm of thinking about stem cells .

1. INTRODUCTION 2. CHALLENGES IN STEM CELL BIOLOGY 3. THEORETICAL CONCEPTS AND QUANTITATIVE MODELS IN STEM CELL BIOLOGY 4. A NEW PERSPECTIVE ON STEM CELL SYSTEMS 5. A NEW MODEL OF HEMATOPOIETIC STEM CELL ORGANIZATION 6. CLONALITY ANALYSIS ON THE SINGLE-CELL LEVEL 7. ASYMMETRY OF CELLULAR FATE 8. CONCLUSIONS

any attempt to answer this question implies the idea that one can prospectively decide about the capabilities of a selected cell without relating it to other cells and without functionally testing its capabilities . it is widely accepted that currently a definite characterization of tissue stem cells is only possible on the basis of their functional capabilities and not on the basis of explicit , directly observable attributes . such a functional perspective is inherently consistent with the biological role of tissue stem cells to maintain tissue homeostasis and to ( re)generate functional tissues . the two key capabilities of tissue stem cells are the ability to self - renew their own population and the ability to produce a large number of fully functional , differentiated cells , implying also the ability to proliferate . however , although these are necessary capabilities , they are not sufficient to guarantee long - term maintenance and reconstitution of a fully functional tissue , which requires a highly coordinated control of cell production and differentiation . this points to another essential property of tissue stem cells : the flexibility in the use of their functional potentials . this flexibility , which had for the first time been incorporated into a definition of tissue stem cells by potten and loeffler , refers to the fact that stem cells might particularly be characterized by their ability to respond to the actual needs of the system . beside feedback regulations on the basis of long - range acting molecules such as cytokines [ 24 ] , this communication also refers to the importance of the so - called stem cell niche [ 59 ] . meanwhile , the existence of stem cell supporting niches has been identified for most ( regenerative ) tissues , including the hematopoietic system [ 10 , 11 ] . moreover , there is increasing evidence that stem cell organization is the result of complex cell - cell and cell - microenvironment interactions rather than the consequence of a predefined stem cell intrinsic program [ 1215 ] . applying the functional definition , the above - stated question whether a particular cell is a stem cell can only be answered retrospectively , having subjected the cell to a functional assay . this situation is somehow similar to heisenberg 's uncertainty principle in quantum physics which states that the very act of measuring the functional properties of a certain system always changes its characteristics , thus , giving rise to a certain degree of uncertainty in the evaluation of the system properties . although not identical , the uncertainty in the determination of the functional potential of a cell still implies that all prospective statements about stem cell functioning are necessarily probabilistic statements about the cellular behavior under particular conditions . there are a number of experimental observations which challenge the classical conception of a cell intrinsically predefined stem cell program . although these observations are not restricted to one particular tissue , we will discuss them with the focus on the hematopoietic system . hematopoietic stem cells ( hscs ) are heterogeneous with respect to functional properties such as cycling activity , engraftment potential or differentiation status , as well as to the expression of specific markers ( phenotypic heterogeneity ) . although there exist a number of sophisticated purification protocols that are able to select more homogeneous populations of stem cells [ 1620 ] , there is always a certain functional overlap of the obtained subpopulations . furthermore , there is accumulating evidence that the phenotypic properties of hsc are reversibly changing ( phenotypic reversibility ) [ 2128 ] and that tissue stem cells specified for one type of tissue can be manipulated such that they can act as stem cells of another tissue ( stem cell plasticity ) [ 2932 ] . even though there are most likely a number of constraints in the developmental options , these observations point to the fact that the functional potential of a stem cell can not be uniquely determined by its actual phenotypic appearance . therefore , although a specific purification protocol might select a population of cells with a homogeneous phenotype , showing a certain behavior within a particular functional assay , this behavior might change over time or if the cells are exposed to different assay conditions . because classical stem cell concepts are not able to explain all these experimental findings however , to be validated , such concepts need a rigorous examination by quantitative and predictive modeling approaches . particularly with respect to the uncertainty in the prospective characterization of stem cell function , a well - defined theoretical framework will help to cope with the complexity of experimental systems and will , therefore , considerably contribute to a deeper understanding of functional principles of stem cell organization . in conjunction with predictive quantitative models , such a theory will assist biologists to select , design , and optimize experimental strategies , and can help to systematically anticipate the impact of manipulations to a system . theoretical approaches and simulation techniques support the identification of latent mechanisms and crucial parameters of biological processes , and may predict new phenomena . furthermore , the application of a common model structure to different systems ( i.e. , tissues or cell types ) may help to understand generic construction and regulation principles . to serve as the basis for a theoretical framework of tissue stem cell organization and to allow for a stringent experimental validation of the theory , quantitative models have to fulfill a number of general requirements . because functional assays are the only way to definitely characterize tissue stem cells , the models must be able to account for the readouts of these assays . furthermore , stem cell models must be based on populations of individual cells to follow clonal development , to enable considerations of population fluctuations , and to conform to the uncertainty principle . because of the increasing evidence that stem cell behavior is not the result of a cell - autonomous program , but instead the consequence of complex cell - cell and cell - growth environment interactions , these interactions have to be represented in such models . to be able to correctly describe regulatory processes , the model systems have to be dynamic in time , and possibly also in space . the functional definition of tissue stem cells implies that stemness should be regarded as a functional endpoint rather than as an explicit attribute of individual cells . therefore , any concept of tissue stem cells has to specify assumptions about the mechanisms that potentially control the regenerative and proliferative potential of these cells . apparently , these processes are determined by the genetic and epigenetic statuses of the cells as well as by the activity of various signaling and metabolic pathways . since it is presently impossible to describe the entirety of these processes in any reasonable detail , one major goal is the derivation of a simplified basic scheme accounting for the generic principles underlying the cellular dynamics . because many experimental results show the necessity to consider flexibility and reversibility of cellular properties as important constituents of stem cell organization , we propose to give up the view of tissue stem cells as being entities with a preprogrammed development this view should be replaced by a concept that makes cellular capabilities for flexible and regulated tissue self - organizing the new paradigm . such a concept incorporates context - dependent phenotypic reversibility and generation of stem cell heterogeneity as the result of a dynamically regulated process . it consequentially avoids assumptions that lead to a direct or indirect a priori labeling of particular cells as stem cells ; cells are purely characterized on the basis of functional potentials . these cellular potentials as well as their actual use are able to change in response to cell - cell and cell - microenvironment interactions , such that the cell population fulfils the functional criteria of the stem cell definition . in contrast , a cell with only a low long - term repopulating potential might , under certain circumstances , be selected to act as a stem cell . it should be stressed that although this concept includes a considerable degree of flexibility in the cellular development , it does not exclude the existence of restrictions in the developmental potential of individual cells . therefore , also the complete loss of repopulating potential at a certain stage of development ( e.g. , due to terminal differentiation ) is compatible with the proposed concept . to put such a theoretical framework to a quantitative test , comparing it with various types of experimental observations , the general concept has been translated into a stochastic , single - cell - based model for hsc which is summarized in the next section . as already described in the context of the general concept , we assume that cellular properties of hsc can reversibly change within a range of potential options . herein , the direction of cellular development and the decision whether a certain property is actually expressed depend on the internal state of the cell and on signals from its growth environment . , the probabilities of growth environment change per time step , denoted as and ) depend on the actual value of the affinity a and on the number of stem cells residing in ge - a and ge- , respectively . if affinity a of an individual cell has fallen below a prespecified threshold ( amin ) , the ability to home to ge - a and , therefore , the potential to regain affinity a is lost . particularly , the proposed single - cell - based model structure allows to analyze cellular dynamics not only on the population , but also on the individual clone level . this is of particular interest in applications where the dynamic properties of individual ( potentially manipulated ) stem cells or stem cell clones are essential targets . examples of such applications are gene - therapeutic approaches , and also the ex vivo expansion of stem or progenitor cells . to illustrate the theoretical investigation of individual cell fates and of clonal dynamics and to highlight important benefits of a model analysis , we will consider two particular phenomena classes : fluctuating contribution of individually marked stem cell clones and cell fate asymmetry of paired progenitors . to simulate the dynamics of individual stem cell clones , all model cells are individually labeled with an inheritable marker at one point in time . it is also possible that different marking events define nested clones , implying that identical cells can be considered as members of different clones . starting from such a configuration , our model predicts that the system will inevitably convert from this polyclonal state to an oligo- and finally to a monoclonal situation . however , the time scale of such a monoclonality conversion might be very large . for the murine homeostatic reference situation ( see for detailed model parameters ) with about 300 model stem cells , the time to monoclonality has been estimated to be approximately 65 years . during a normal mouse life span of about 2 years , the number of stem cell clones is predicted to reduce to about 30 . of course , the kinetics of the conversion depends on the model parameters which determine the differentiation probability , such as the average generation time of stem cells c ( figure 2(a ) ) . furthermore , it is predicted that the process of clone exhaustion can be accelerated by system perturbations , for example , due to repeated cell kill events ( figure 2(b ) ) . there is another point that might considerably affect the interpretation of experimental observations on clonal contribution . therefore , also clones that actually contribute to hematopoiesis might be overlooked , for example , due to a threshold - dependent detection procedure . in contrast , figures 3(b ) and 3(c ) are depicting different projections of this data . , clone sizes larger than or equal to one cell ) , figure 3(c ) indicates measurable clones , assuming a detection threshold of 10 stem cells per clone . applying these simulation results to different observations can help to identify misleading aspects in the interpretation of experimental findings and to disentangle seemingly contradictory results . to discuss the model analysis of these phenomena , let us consider two particular results on the clonal composition of the hematopoietic system . in contrast to jordan & lemischka who analyzed repeated blood / spleen samples with a high cell number but with relatively low detection sensitivity for individual marker signals , drize et al . analyzed single - cell - induced spleen colonies obtained by injecting repeated bone marrow samples into irradiated recipient mice . to simulate these two experimental strategies , model systems are initiated with individually labeled stem cells sampled from a homeostatic reference system . according to the two described experimental protocols , different numbers of marked cells , with n = 10 cells for the jordan - like simulation and n = 100 cells for the simulation of the drize experiment , were used . experimentally detectable clones have been simulated by the sampling of individual model stem cells ( representing spleen - colony forming cells ) with a probability of 0.01 for the drize - like setting , and by counting all differentiated clones ( representing the entirety of bone marrow / spleen cells ) which exceed a size threshold of 10 000 cells per clone for the jordan - like setting . although our model of a self - organized stem cell population does explicitly preclude asymmetric cell divisions , it still accounts for asymmetric cell fates . however , during completion of a cell division , also the affinity a changes from a1 to a new value a2 < a1 . now , one daughter cell might change to ge - a , subsequently regaining the affinity to its initial value a1 , while the other daughter cell continues to decrease a. beside such an asymmetric development , also two scenarios of symmetric cell fates can be obtained : whenever both daughter cells regenerate their affinity , the number of cells with the original functional potential is amplified . it is also possible to quantitatively describe experimental data on asymmetric stem cell behavior within the context of our model . to test whether these results can quantitatively be reproduced without the assumption of asymmetric cell division events , these systems have been traced for 10 days according to the experimental protocol . to compare the ad index of simulations and experiments , a model division is denoted as asymmetric whenever only one of the two first - generation daughter cells is performing further cell divisions . because affinity a directly correlates to the probability of a cell to long - term repopulate a model system , these results predict that cells with high long - term repopulating potential more frequently exhibit an asymmetric cell fate in vitro . furthermore , the experimental observation that stromal coculture of stem cells enhances the proportion of asymmetric divisions can be explained by the assumption of different regeneration coefficients r. whereas r = 1 ( i.e. , no regeneration of affinity a in ge - a ) produces lower proportions of asymmetric cell fates ( figure 6(a ) ) , r > 1 leads to an increase in the amount of asymmetric cell fates ( figure 6(b ) ) . therefore , the heterogeneity of the in vitro stem cell supporting potential of different stromal cell types can consistently be represented in the model by growth environments ( ge - a ) allowing for variable degrees of a - regeneration . based on these results , we are able to quantitatively reproduce the published experimental results . again starting from the previously derived reference parameter set that consistently describes different in vivo assays using c57bl/6 mice , a variation of the initial affinity range and of the regeneration coefficient lead to a good quantitative fit of simulation results and experimental data ( figure 7 ) . both simulation scenarios use initial affinities a , uniformly distributed on the interval [ 0.5 ; 1 ] . particularly with regard to stem cell fate and individual clonal dynamics , there are a number of predictions arising from the proposed mathematical model . one basic conclusion is that the developmental fate of a stem cell can not be predicted with certainty , even if the actual state of the cell could be determined exactly . however , probabilistic statements about the future development of individual clones as well as about the potential of a population of well - characterized cells are certainly possible . in terms of the model , the likelihood for a certain developmental fate of a stem cell is assumed to depend on the general potential of the cell , on its actual state , and on the microenvironmental signals the cell receives . , reactivity on microenvironmental signals ) it has be shown that even very small differences in cellular properties , which would not affect the general repopulation ability of the cells in a nonchimeric situation , are sufficient to sensitively affect the cellular development in the competition scenario . , by insertional mutagenesis [ 47 , 48 ] ) differences between stem cell clones within one genetic background could influence the probabilities for certain developmental fates . even in the oversimplified case of an identical potential of all stem cell clones , still , it is not possible to unequivocally specify the successful clones in advance . however , as stated above , even small differences in the cellular potential of stem cell clones are able to bias the competitive potential considerably . our model is able to estimate the effect of differences in cellular parameters on competitive growth characteristics , and therefore , to provide statistical predictions about future clonal contributions . this might particularly be important to understand the effects of insertional mutagenesis as well as to quantitatively characterize the outcome of gene - therapeutic interventions . even though a developmental asymmetry of stem cells is inevitably required to provide a continuous production of differentiated cells without exhausting the stem cell population , this asymmetry is not necessarily linked to cell division events . alternatively a flexible functional asymmetry can be achieved by a self - organizing population of interacting cells , including a certain degree of reversibility in cellular properties and functionalities . summarizing our results , we demonstrated that it is possible to understand tissue stem cell systems without assumptions on unidirectional developmental hierarchies , preprogrammed asymmetric division events , or other assumptions implying the existence of a predetermined stem cell entity . as illustrated for the hematopoietic system , a self - organizing perspective would change the paradigm of thinking about stem cells . this means that stem cells are selected and modified in response to cell - cell and cell - microenvironment interactions , rather than being specialized a priori .

the advent of rotary nickel titanium ( niti ) instrumentation has brought significant progress to root canal preparation in terms of preserving native root canal morphology and preventing apical transportation . however , separation of instruments during continuous rotary motion is the most common accident in clinical practice . niti instruments may exhibit premature fatigue failure , thus reducing the life of the instrument or torsional stress failure , which is the result of file binding to the canal walls . therefore , rotary instrumentation requires manual or mechanical cervical preflaring to mitigate fracture risk . in 2008 , yared used a single rotating instrument ( protaper ) in reciprocating motion for canal preparation and shaping and demonstrated that the technique provides greater speed , good cost - benefit ratio , and reduced fracture rates . a new approach to root canal preparation was introduced in 2010 with the reciproc ( rc ) system ( vdw , munich , germany ) and later by the waveone ( wo ) system ( dentsply maillefer , ballaigues , switzerland ) . both use a single niti m - wire alloy instrument driven by reciprocating motion , whereby the instrument cuts the dentin wall of the root canal during the counterclockwise rotation , when taper lock is imminent . the following clockwise motion then disengages the instrument from dentin , reducing the incidence of separation . according to manufacturer recommendations , a glidepath must be prepared using small ( # 10 and # 15 ) rotary or hand files , but cervical preflaring is unnecessary . within the context of widespread use of reciprocating systems , the present study assessed the influence of multiple reuse ( after resterilization ) and cervical preflaring on fracture strength , plastic deformation , and/or surface cracking in rc and wo reciprocating instruments after root canal instrumentation in extracted human teeth . the null hypothesis was that both instrument systems would be equally fracture resistant when driven by reciprocating motion , with and without cervical preflaring . the present study was approved by the local research ethics committee ( caae : 13831213.4.0000.5052 ) . the sample comprised extracted human upper molars ( length 1822 mm ) with fully formed roots , constricted root canals , and root curvature 35. for selection purposes , teeth were identified and imaged by conventional radiography and cone beam computed tomography . only teeth in which the entire root canal system could be visualized were included in the sample for experimental group allocation . teeth with root curvature > 35 , partially or completely calcified canals , or excessively wide canals were excluded from the study . after application of the selection criteria , 550 teeth were selected from a set of 1153 extracted human molars . figure 1 provides a schematic illustration of the study methods . in all selected teeth , canal orifices were located with an endodontic explorer and special files under 32 magnification ( mc - m1233 operating microscope , dfv com . and ind . ltda , valena , rj , brazil ) , and teeth were then randomly allocated across the different study groups . the presence of a fourth canal was identified in 41.2% of the teeth used in the sample ( n = 200 ) . forty 25-mm reciprocating instruments were used in this study : 20 reciproc r25 , tip size 25 , 0.08 variable taper ( rc ) ( vdw gmbh , munich , germany ) and 20 waveone primary , tip size 25 , 0.08 variable taper ( wo ) ( dentsply - maillefer , ballaigues , switzerland ) , all powered by a silver reciproc motor and sirona endo 6:1 contra - angle handpiece ( vdw gmbh , munich , germany ) . rc files were driven by the rc all program and wo files by the wo all program . four study groups ( n = 10 each ) were defined , according to root canal instrumentation procedure : g1 : rc;g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc;g3 : wo;g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc ; g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . procedures were performed by a single operator , who followed the manufacturer - issued instructions for each instrument . after coronal access , teeth were irrigated with 5 ml sodium hypochlorite ( naocl ) 2.5% ( biodinmica qumica e farmacutica ltda , ibipor , pr , brazil ) and explored with hand files as described elsewhere . the root canal systems of all teeth were evacuated with a # 10 k - file , which was used to identify patency . working length determination was performed under operating microscope magnification ( mc - m1233 , dfv com . & ind . ltda , valena , rj , brazil ) , 1 mm short of the apex . in all teeth , glidepaths were prepared with a # 13/0.02 mm / mm pathfile ( dentsply - maillefer , ballaigues , switzerland ) , which enabled standardized canal preparation . in groups g1 and g3 , instrumentation was performed with a single file ( rc and wo , respectively ) in reciprocating motion , advancing into each root third in 3-mm increments until the desired length was reached , as recommended by the manufacturers . in groups g2 and g4 , instrumentation was preceded by cervical preflaring , performed using # 2 and # 3 gates - glidden drills ( dentsply - maillefer , ballaigues , switzerland ) , in descending order , and driven by a reciproc silver motor programmed appropriately for these instruments . after this step , rc and wo instruments were used as in groups g1 and g3 . after each use , any dentin chip adhered to the instruments was removed with moist gauze dressings , and the root canals were irrigated again with 5 ml of naocl and recapitulated with a # 10 k - file to full length . once instrumentation of each tooth was complete , the instruments were decontaminated and cleaned in an ultrasonic unit , examined under the operating microscope for potential plastic deformation , and autoclaved for later reuse . the identification ring of each instrument was removed with a scalpel blade so as to allow reinsertion into the sirona contra - angle handpiece . each instrument was used repeatedly until fracture occurred , at which time the instrument was identified , and the number of uses until separation was recorded . the surfaces of all instruments were analyzed by scanning electron microscopy ( sem ) , performed with a dsm-940a system ( carl zeiss , jena , germany ) , and checked for surface changes potentially caused by irrigant exposure and for cracks or plastic deformation caused by continued instrumentation . sem images ( magnification 100 , 250 , 500 , and 1000 ) were obtained at three time points for each instrument : before use [ figure 2 ] ; after first use [ figure 3 ] ; and after fracture [ figure 4 ] . scanning electron microscope images of reciproc ( a ) and waveone ( b ) instruments before use surface images of waveone instruments after the first use . note the presence of surface cracks on the waveone instrument ( arrows ) ( scanning electron microscope images magnification : a 250 ; b 500 ; c 1000 ) reciproc ( used in 12 teeth gi ) and waveone ( used in 6 teeth gii ) instruments after separation ( scanning electron microscope images magnification : 250 , 500 , 1000 ) . note cracks inside the metal mass of reciproc ( arrow ) statistical procedures were performed with the statistical package for the social sciences ( spss 17.0 for windows , spss inc . , chicago , usa ) . because values were normally distributed across all groups , one - way analysis of variance was used for comparative purposes . the present study was approved by the local research ethics committee ( caae : 13831213.4.0000.5052 ) . the sample comprised extracted human upper molars ( length 1822 mm ) with fully formed roots , constricted root canals , and root curvature 35. for selection purposes , teeth were identified and imaged by conventional radiography and cone beam computed tomography . only teeth in which the entire root canal system could be visualized were included in the sample for experimental group allocation . teeth with root curvature > 35 , partially or completely calcified canals , or excessively wide canals were excluded from the study . after application of the selection criteria , 550 teeth were selected from a set of 1153 extracted human molars . in all selected teeth , endodontic access was established using high - speed diamond burs . canal orifices were located with an endodontic explorer and special files under 32 magnification ( mc - m1233 operating microscope , dfv com . and ind . ltda , valena , rj , brazil ) , and teeth were then randomly allocated across the different study groups . the presence of a fourth canal was identified in 41.2% of the teeth used in the sample ( n = 200 ) . forty 25-mm reciprocating instruments were used in this study : 20 reciproc r25 , tip size 25 , 0.08 variable taper ( rc ) ( vdw gmbh , munich , germany ) and 20 waveone primary , tip size 25 , 0.08 variable taper ( wo ) ( dentsply - maillefer , ballaigues , switzerland ) , all powered by a silver reciproc motor and sirona endo 6:1 contra - angle handpiece ( vdw gmbh , munich , germany ) . rc files were driven by the rc all program and wo files by the wo all program . four study groups ( n = 10 each ) were defined , according to root canal instrumentation procedure : g1 : rc;g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc;g3 : wo;g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc ; g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . procedures were performed by a single operator , who followed the manufacturer - issued instructions for each instrument . after coronal access , teeth were irrigated with 5 ml sodium hypochlorite ( naocl ) 2.5% ( biodinmica qumica e farmacutica ltda , ibipor , pr , brazil ) and explored with hand files as described elsewhere . the root canal systems of all teeth were evacuated with a # 10 k - file , which was used to identify patency . working length determination was performed under operating microscope magnification ( mc - m1233 , dfv com . ltda , valena , rj , brazil ) , 1 mm short of the apex . in all teeth , glidepaths were prepared with a # 13/0.02 mm / mm pathfile ( dentsply - maillefer , ballaigues , switzerland ) , which enabled standardized canal preparation . in groups g1 and g3 , instrumentation was performed with a single file ( rc and wo , respectively ) in reciprocating motion , advancing into each root third in 3-mm increments until the desired length was reached , as recommended by the manufacturers . in groups g2 and g4 , instrumentation was preceded by cervical preflaring , performed using # 2 and # 3 gates - glidden drills ( dentsply - maillefer , ballaigues , switzerland ) , in descending order , and driven by a reciproc silver motor programmed appropriately for these instruments . after this step , rc and wo instruments were used as in groups g1 and g3 . after each use , any dentin chip adhered to the instruments was removed with moist gauze dressings , and the root canals were irrigated again with 5 ml of naocl and recapitulated with a # 10 k - file to full length . once instrumentation of each tooth was complete , the instruments were decontaminated and cleaned in an ultrasonic unit , examined under the operating microscope for potential plastic deformation , and autoclaved for later reuse . the identification ring of each instrument was removed with a scalpel blade so as to allow reinsertion into the sirona contra - angle handpiece . each instrument was used repeatedly until fracture occurred , at which time the instrument was identified , and the number of uses until separation was recorded . the surfaces of all instruments were analyzed by scanning electron microscopy ( sem ) , performed with a dsm-940a system ( carl zeiss , jena , germany ) , and checked for surface changes potentially caused by irrigant exposure and for cracks or plastic deformation caused by continued instrumentation . sem images ( magnification 100 , 250 , 500 , and 1000 ) were obtained at three time points for each instrument : before use [ figure 2 ] ; after first use [ figure 3 ] ; and after fracture [ figure 4 ] . scanning electron microscope images of reciproc ( a ) and waveone ( b ) instruments before use surface images of waveone instruments after the first use . note the presence of surface cracks on the waveone instrument ( arrows ) ( scanning electron microscope images magnification : a 500 ; c 1000 ) reciproc ( used in 12 teeth gi ) and waveone ( used in 6 teeth gii ) instruments after separation ( scanning electron microscope images magnification : 250 , 500 , 1000 ) . statistical procedures were performed with the statistical package for the social sciences ( spss 17.0 for windows , spss inc . , chicago , usa ) . because values were normally distributed across all groups , one - way analysis of variance was used for comparative purposes . rc files exhibited significantly greater fracture strength than wo files ( p < 0.001 ) , under equal conditions . comparison of groups in which the same instruments were used revealed that cervical preflaring significantly increased the fracture strength ( p < 0.01 ) of both instrument systems . on comparison between different instrument systems with and without preflaring , rc files exhibited the same fracture strength as wo files did after preflaring , providing further evidence of the superiority of the rc system over the wo system . mean and standard deviation number of teeth and root canals instrumented per instrument per study group on sem , surface cracking was only seen in wo instruments [ figure 3 ] . it should be stressed that although these cracks were already apparent after the first instrument use , the files remained fracture resistant and could be reused at least five times before separation . major changes in the techniques and principles of endodontics have occurred in the last 20 years . this is demonstrated clearly by recent advances in instrumentation of root canal systems , such as the development of safer techniques , files made from more elastic and stable alloys , and a lower rate of errors during endodontic instrumentation . the present study assessed the fracture strength and development of plastic deformations and/or surface cracks on rc r25 and wo primary reciprocating instruments , with and without cervical preflaring , by means of operating microscope and sem images . the null hypothesis of the study was rejected as the results demonstrated that cervical preflaring had an impact on the fracture strength of the tested instruments . the choice of instruments for this study was based on the fact that both were made from the same material ( niti m - wire alloy ) , are driven by reciprocating motion , and share similar manufacturing processes . the method of reusing sterilized instruments on extracted human teeth simulates a real - world clinical setting , particularly in terms of the risk of instrument separation , although not all anatomical features of the pulp cavity can be standardized . however , the large sample size , random allocation of teeth across study groups , and glidepath standardization with a # 13 pathfile prevented this variable from having any significant influence on results . negotiating the apical foramen and determining apical patency with # 15 or smaller files are essential clinical steps and are firmly enshrined in current practice as they prevent plugging and/or deviation of the root canal . hence , the inclusion of these steps in our methods brings our experiment even closer to the reality of daily clinical practice as clearance of potential interferents from the root canal system and determination of apical patency play a critical role in ensuring safety and improving the prognosis of endodontic treatment . likewise , the use of cervical preflaring a clinical procedure widely employed in endodontic practice in groups g2 and g4 of this study further demonstrates our concern with making our experimental condition reproduce clinical aspects as closely as possible . preflaring ensures there is a sufficient space for penetration of the mechanical instrument , prevents coronal binding , and reduces the risk of instrument separation . in addition , it enhances the range of action of irrigating solution by enabling advancement of the irrigation needle and penetration of the ultrasonic tip during passive ultrasonic irrigation ( pui ) . although the rc and wo instruments were conceived for single use only , these devices have a high level of embedded technology , which led us to analyze their fracture strength after repeated sterilization before each reuse . this analysis showed that both instruments remained safe for reuse . according to the current literature , the advent of reciprocating motion systems has made root canal shaping and cleaning safer . in addition , the past studies suggest the possibility of using only one instrument to prepare the entire root canal with satisfactory results , particularly because a single instrument has increased fracture strength as compared to full - sequence rotary instrumentation . the influence of cross - section , alloy type , manufacturing process , and type of driving motion on the cyclic fatigue resistance of niti instruments has been the object of several recent studies . nevertheless , how and why the design of the instrument might influence its behavior under cyclic fatigue stress remains unclear . cheung and darvell showed , in various instruments , that fatigue resistance does not appear to be affected by instrument design , suggesting that neither the cross - section area of an instrument nor its shape is the primary determinant of fatigue life . however , other studies have suggested that difference in cross - section appears to be an important determinant of cyclic fatigue resistance across different files . in a study conducted by plotino et al . , rc instruments exhibited significantly greater fatigue strength than wo instruments , which suggests that this difference may be related to the differences in cross - sectional area and reciprocating motion between the two systems . earlier studies also demonstrated the superiority of rc instrumentation over the wo system in fatigue resistance testing and have shown that the angles employed in instrument motion have a direct influence on cyclic fatigue . believe another factor that enhances resistance in rc and wo instruments is the type of alloy used in their manufacture . the authors showed that a synergistic effect between the m - wire alloy and reciprocating motion provides a significant increase in cyclic fatigue resistance . recently , a new continuous rotary instrument was developed by coltene / whaledent ag ( hyflex edm ) . manufactured using the electrical discharge machining technique , this instrument has greater resistance to cyclic fatigue when compared with rc and wo . in our sample , the rc system was superior to the wo system regardless of cervical preflaring , which suggests that clearance of the root canal system through glidepath preparation before instrumentation is of the utmost clinical importance . this step is recommended by the manufacturer of wo but deemed unnecessary when using the rc system . perhaps , choosing not to prepare a glidepath could bias results significantly in favor of the rc system as demonstrated elsewhere in the literature . in a study of discarded niti files , alapati et al . found cracks along the metal body of the instruments . in our sample , sem analysis of the instruments after the first use revealed cracks only on the wo files . we did not observe any plastic deformation , which may be explained by the greater resistance of the m - wire alloy as compared with other niti alloys . it bears stressing that the back - and - forth motion recommended by manufacturers and followed during specimen preparation in this study is largely intended to reduce torsional strain and , consequently , torsion fatigue , whereas its effect on flexural stresses is probably less evident . to date , rc and wo have been marketed for single - file root canal instrumentation and are approved for single use only so as to prevent structural weakening as a result of cyclic fatigue . means the same instrument may still be used to instrument three or four root canals , each of which may have complex , tortuous anatomy . therefore , single use reduces but does not altogether eliminate the risk of cumulative fatigue damage and potential instrument separation . hence , we conclude that cyclic fatigue testing is as valuable for reciprocating instrumentation as for rotary instruments . it should be noted that the high level of fatigue resistance of reciprocating instruments may play an important role in improving root canal cleaning and shaping procedures as shaping can be done with remarkable speed . within this context , the principles of endodontics are shifting away from simultaneous cleaning and shaping toward a paradigm , whereby the root canal system is first shaped to enable effective cleaning by means of enhanced irrigation procedures . evidence suggests that enhanced apical preparation provides a greater reduction of bacterial load and dentin debris , primarily by enabling more effective irrigation , which is made easier by cervical preparation . in the present study , cervical preflaring before instrumentation played a relevant role in increasing the fatigue life of the tested instruments . another relevant clinical factor is localization , negotiation , and determination of apical patency , especially as endodontic treatment of older adults who often present with constricted pulp chambers filled with dystrophic calcifications therefore , canal clearance and apical debridement are considered important procedures to reduce instrument stress against the walls of the canal system and thus improve the safety of endodontic treatment . nevertheless , in a recent study by de - deus et al . , the rc system demonstrated satisfactory fracture strength even in the absence of patent canals . the present study sought to assess the fracture strength of rc and wo instruments after repeated sterilization and reuse cycles . our results suggest that both the rc and wo systems can be safely reused several times , preceded by some preliminary manipulation to prevent the instrument from binding and thus reduce the risk of separation . based on our findings , we suggest that files can be reused safely up to 3 times . this number also facilitates control of the number of reuses of a single file . on the first use , the instrument is taken directly from its original packaging and its identifier ring is intact . after the first reprocessing , the identifier ring is enlarged and must be removed before the instrument can fit into the handpiece ; this serves as a milestone for the second use of the instrument . finally , at the time of the third use , the instrument will already be missing its identifier ring , which demonstrates it should be discarded after use . despite these conclusions , we believe that further research is needed to assess the ability of these reciprocating systems to ensure adequate canal antisepsis , as the duration of exposure to naocl irrigating solution tends to be reduced in single - file preparations , and to evaluate the influence of this clinical practice on treatment success over time . notably , one of the instrument specimens produced dark discoloration of the irrigation solution shortly after the start of its first use . the canal was irrigated with fresh solution and dark discoloration occurred again . upon removal of the instrument from the root canal , it was found to have been markedly corroded by the 2.5% naocl solution , with loss of material . on sem examination , energy dispersive x - ray analysis did not show any difference in alloy properties in relation to the other instruments used in the study [ figure 6 ] . it bears stressing that no instances of such damage , i.e. , structural corrosion of niti instruments by naocl solution , have ever been reported in the literature . this event was probably due to a flaw in the manufacturing process as other instruments from the same lot exhibited stable behavior . scanning electron microscope images ( 100 ) of a reciproc r25 instrument showing corrosion and loss of metal mass energy dispersive x - ray analyses of the surface ( a ) and eroded area ( b ) of the instrument damaged by 2.5% sodium hypochlorite solution considering the proposed objective of this study , we conclude that both rc and wo instruments exhibited good fracture strength after autoclave sterilization and reuse . cervical preflaring significantly increased the number of times rc and wo files could be reused safely .

objective : this study sought to assess the influence of cervical preflaring and reuse after resterilization , on fracture strength and plastic deformation and/or surface cracking in reciprocating nickel titanium [ niti ] endodontic instruments after root canal instrumentation of extracted human teeth.materials and methods : forty engine - driven reciprocating niti instruments ( 20 reciproc [ rc ] , vdw ; 20 waveone [ wo ] , dentsply ) were used in root canal instrumentation of extracted human molars . a total of 485 human upper molars with similar anatomical features were selected and randomly distributed across four groups according to the instrumentation procedures performed ( g1 and g3 : rc ; g2 and g4 : wo ) . reciprocating instruments were used as per manufacturer instructions . in groups g3 and g4 , cervical preflaring was performed prior to instrumentation . after each use of each instrument , sterilization was repeated and scanning electron microscope ( sem ) images were obtained to check for plastic deformations and/or cracks on instrument surfaces . each instrument was used repeatedly until fracture occurred.results:regardless of flaring , rc files exhibited greater fracture strength than wo instruments ( p < 0.01 ) with and without preflaring . cervical preflaring significantly reduced the risk of instrument fracture ( p < 0.01 ) . no plastic deformations were observed before fracturing . however , cracks were found on wo instruments shortly after their first use . rc and wo instruments resisted fracturing after autoclave sterilization.conclusions:cervical preflaring allowed a significant increase in the number of times rc and wo files could be reused safely .

INTRODUCTION MATERIALS AND METHODS Ethical aspects and sample selection Preparation of specimens Division of experimental groups Root canal preparation Scanning electron microscopy Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

the advent of rotary nickel titanium ( niti ) instrumentation has brought significant progress to root canal preparation in terms of preserving native root canal morphology and preventing apical transportation . however , separation of instruments during continuous rotary motion is the most common accident in clinical practice . niti instruments may exhibit premature fatigue failure , thus reducing the life of the instrument or torsional stress failure , which is the result of file binding to the canal walls . therefore , rotary instrumentation requires manual or mechanical cervical preflaring to mitigate fracture risk . in 2008 , yared used a single rotating instrument ( protaper ) in reciprocating motion for canal preparation and shaping and demonstrated that the technique provides greater speed , good cost - benefit ratio , and reduced fracture rates . a new approach to root canal preparation was introduced in 2010 with the reciproc ( rc ) system ( vdw , munich , germany ) and later by the waveone ( wo ) system ( dentsply maillefer , ballaigues , switzerland ) . both use a single niti m - wire alloy instrument driven by reciprocating motion , whereby the instrument cuts the dentin wall of the root canal during the counterclockwise rotation , when taper lock is imminent . according to manufacturer recommendations , a glidepath must be prepared using small ( # 10 and # 15 ) rotary or hand files , but cervical preflaring is unnecessary . within the context of widespread use of reciprocating systems , the present study assessed the influence of multiple reuse ( after resterilization ) and cervical preflaring on fracture strength , plastic deformation , and/or surface cracking in rc and wo reciprocating instruments after root canal instrumentation in extracted human teeth . the null hypothesis was that both instrument systems would be equally fracture resistant when driven by reciprocating motion , with and without cervical preflaring . the sample comprised extracted human upper molars ( length 1822 mm ) with fully formed roots , constricted root canals , and root curvature 35. for selection purposes , teeth were identified and imaged by conventional radiography and cone beam computed tomography . only teeth in which the entire root canal system could be visualized were included in the sample for experimental group allocation . after application of the selection criteria , 550 teeth were selected from a set of 1153 extracted human molars . the presence of a fourth canal was identified in 41.2% of the teeth used in the sample ( n = 200 ) . forty 25-mm reciprocating instruments were used in this study : 20 reciproc r25 , tip size 25 , 0.08 variable taper ( rc ) ( vdw gmbh , munich , germany ) and 20 waveone primary , tip size 25 , 0.08 variable taper ( wo ) ( dentsply - maillefer , ballaigues , switzerland ) , all powered by a silver reciproc motor and sirona endo 6:1 contra - angle handpiece ( vdw gmbh , munich , germany ) . rc files were driven by the rc all program and wo files by the wo all program . four study groups ( n = 10 each ) were defined , according to root canal instrumentation procedure : g1 : rc;g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc;g3 : wo;g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc ; g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . procedures were performed by a single operator , who followed the manufacturer - issued instructions for each instrument . the root canal systems of all teeth were evacuated with a # 10 k - file , which was used to identify patency . working length determination was performed under operating microscope magnification ( mc - m1233 , dfv com . in groups g1 and g3 , instrumentation was performed with a single file ( rc and wo , respectively ) in reciprocating motion , advancing into each root third in 3-mm increments until the desired length was reached , as recommended by the manufacturers . in groups g2 and g4 , instrumentation was preceded by cervical preflaring , performed using # 2 and # 3 gates - glidden drills ( dentsply - maillefer , ballaigues , switzerland ) , in descending order , and driven by a reciproc silver motor programmed appropriately for these instruments . after this step , rc and wo instruments were used as in groups g1 and g3 . after each use , any dentin chip adhered to the instruments was removed with moist gauze dressings , and the root canals were irrigated again with 5 ml of naocl and recapitulated with a # 10 k - file to full length . once instrumentation of each tooth was complete , the instruments were decontaminated and cleaned in an ultrasonic unit , examined under the operating microscope for potential plastic deformation , and autoclaved for later reuse . the identification ring of each instrument was removed with a scalpel blade so as to allow reinsertion into the sirona contra - angle handpiece . each instrument was used repeatedly until fracture occurred , at which time the instrument was identified , and the number of uses until separation was recorded . the surfaces of all instruments were analyzed by scanning electron microscopy ( sem ) , performed with a dsm-940a system ( carl zeiss , jena , germany ) , and checked for surface changes potentially caused by irrigant exposure and for cracks or plastic deformation caused by continued instrumentation . sem images ( magnification 100 , 250 , 500 , and 1000 ) were obtained at three time points for each instrument : before use [ figure 2 ] ; after first use [ figure 3 ] ; and after fracture [ figure 4 ] . scanning electron microscope images of reciproc ( a ) and waveone ( b ) instruments before use surface images of waveone instruments after the first use . note the presence of surface cracks on the waveone instrument ( arrows ) ( scanning electron microscope images magnification : a 250 ; b 500 ; c 1000 ) reciproc ( used in 12 teeth gi ) and waveone ( used in 6 teeth gii ) instruments after separation ( scanning electron microscope images magnification : 250 , 500 , 1000 ) . because values were normally distributed across all groups , one - way analysis of variance was used for comparative purposes . the sample comprised extracted human upper molars ( length 1822 mm ) with fully formed roots , constricted root canals , and root curvature 35. for selection purposes , teeth were identified and imaged by conventional radiography and cone beam computed tomography . only teeth in which the entire root canal system could be visualized were included in the sample for experimental group allocation . after application of the selection criteria , 550 teeth were selected from a set of 1153 extracted human molars . the presence of a fourth canal was identified in 41.2% of the teeth used in the sample ( n = 200 ) . forty 25-mm reciprocating instruments were used in this study : 20 reciproc r25 , tip size 25 , 0.08 variable taper ( rc ) ( vdw gmbh , munich , germany ) and 20 waveone primary , tip size 25 , 0.08 variable taper ( wo ) ( dentsply - maillefer , ballaigues , switzerland ) , all powered by a silver reciproc motor and sirona endo 6:1 contra - angle handpiece ( vdw gmbh , munich , germany ) . rc files were driven by the rc all program and wo files by the wo all program . four study groups ( n = 10 each ) were defined , according to root canal instrumentation procedure : g1 : rc;g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc;g3 : wo;g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . g2 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) ( dentsply - maillefer , ballaigues , switzerland ) + rc ; g4 : cervical preflaring with gates - glidden drills ( # 3 and # 2 ) + wo . procedures were performed by a single operator , who followed the manufacturer - issued instructions for each instrument . the root canal systems of all teeth were evacuated with a # 10 k - file , which was used to identify patency . working length determination was performed under operating microscope magnification ( mc - m1233 , dfv com . in groups g1 and g3 , instrumentation was performed with a single file ( rc and wo , respectively ) in reciprocating motion , advancing into each root third in 3-mm increments until the desired length was reached , as recommended by the manufacturers . in groups g2 and g4 , instrumentation was preceded by cervical preflaring , performed using # 2 and # 3 gates - glidden drills ( dentsply - maillefer , ballaigues , switzerland ) , in descending order , and driven by a reciproc silver motor programmed appropriately for these instruments . after this step , rc and wo instruments were used as in groups g1 and g3 . after each use , any dentin chip adhered to the instruments was removed with moist gauze dressings , and the root canals were irrigated again with 5 ml of naocl and recapitulated with a # 10 k - file to full length . once instrumentation of each tooth was complete , the instruments were decontaminated and cleaned in an ultrasonic unit , examined under the operating microscope for potential plastic deformation , and autoclaved for later reuse . the identification ring of each instrument was removed with a scalpel blade so as to allow reinsertion into the sirona contra - angle handpiece . each instrument was used repeatedly until fracture occurred , at which time the instrument was identified , and the number of uses until separation was recorded . the surfaces of all instruments were analyzed by scanning electron microscopy ( sem ) , performed with a dsm-940a system ( carl zeiss , jena , germany ) , and checked for surface changes potentially caused by irrigant exposure and for cracks or plastic deformation caused by continued instrumentation . sem images ( magnification 100 , 250 , 500 , and 1000 ) were obtained at three time points for each instrument : before use [ figure 2 ] ; after first use [ figure 3 ] ; and after fracture [ figure 4 ] . scanning electron microscope images of reciproc ( a ) and waveone ( b ) instruments before use surface images of waveone instruments after the first use . note the presence of surface cracks on the waveone instrument ( arrows ) ( scanning electron microscope images magnification : a 500 ; c 1000 ) reciproc ( used in 12 teeth gi ) and waveone ( used in 6 teeth gii ) instruments after separation ( scanning electron microscope images magnification : 250 , 500 , 1000 ) . because values were normally distributed across all groups , one - way analysis of variance was used for comparative purposes . rc files exhibited significantly greater fracture strength than wo files ( p < 0.001 ) , under equal conditions . comparison of groups in which the same instruments were used revealed that cervical preflaring significantly increased the fracture strength ( p < 0.01 ) of both instrument systems . on comparison between different instrument systems with and without preflaring , rc files exhibited the same fracture strength as wo files did after preflaring , providing further evidence of the superiority of the rc system over the wo system . mean and standard deviation number of teeth and root canals instrumented per instrument per study group on sem , surface cracking was only seen in wo instruments [ figure 3 ] . it should be stressed that although these cracks were already apparent after the first instrument use , the files remained fracture resistant and could be reused at least five times before separation . major changes in the techniques and principles of endodontics have occurred in the last 20 years . this is demonstrated clearly by recent advances in instrumentation of root canal systems , such as the development of safer techniques , files made from more elastic and stable alloys , and a lower rate of errors during endodontic instrumentation . the present study assessed the fracture strength and development of plastic deformations and/or surface cracks on rc r25 and wo primary reciprocating instruments , with and without cervical preflaring , by means of operating microscope and sem images . the null hypothesis of the study was rejected as the results demonstrated that cervical preflaring had an impact on the fracture strength of the tested instruments . the choice of instruments for this study was based on the fact that both were made from the same material ( niti m - wire alloy ) , are driven by reciprocating motion , and share similar manufacturing processes . the method of reusing sterilized instruments on extracted human teeth simulates a real - world clinical setting , particularly in terms of the risk of instrument separation , although not all anatomical features of the pulp cavity can be standardized . however , the large sample size , random allocation of teeth across study groups , and glidepath standardization with a # 13 pathfile prevented this variable from having any significant influence on results . negotiating the apical foramen and determining apical patency with # 15 or smaller files are essential clinical steps and are firmly enshrined in current practice as they prevent plugging and/or deviation of the root canal . hence , the inclusion of these steps in our methods brings our experiment even closer to the reality of daily clinical practice as clearance of potential interferents from the root canal system and determination of apical patency play a critical role in ensuring safety and improving the prognosis of endodontic treatment . likewise , the use of cervical preflaring a clinical procedure widely employed in endodontic practice in groups g2 and g4 of this study further demonstrates our concern with making our experimental condition reproduce clinical aspects as closely as possible . preflaring ensures there is a sufficient space for penetration of the mechanical instrument , prevents coronal binding , and reduces the risk of instrument separation . although the rc and wo instruments were conceived for single use only , these devices have a high level of embedded technology , which led us to analyze their fracture strength after repeated sterilization before each reuse . according to the current literature , the advent of reciprocating motion systems has made root canal shaping and cleaning safer . in addition , the past studies suggest the possibility of using only one instrument to prepare the entire root canal with satisfactory results , particularly because a single instrument has increased fracture strength as compared to full - sequence rotary instrumentation . the influence of cross - section , alloy type , manufacturing process , and type of driving motion on the cyclic fatigue resistance of niti instruments has been the object of several recent studies . however , other studies have suggested that difference in cross - section appears to be an important determinant of cyclic fatigue resistance across different files . , rc instruments exhibited significantly greater fatigue strength than wo instruments , which suggests that this difference may be related to the differences in cross - sectional area and reciprocating motion between the two systems . believe another factor that enhances resistance in rc and wo instruments is the type of alloy used in their manufacture . the authors showed that a synergistic effect between the m - wire alloy and reciprocating motion provides a significant increase in cyclic fatigue resistance . recently , a new continuous rotary instrument was developed by coltene / whaledent ag ( hyflex edm ) . manufactured using the electrical discharge machining technique , this instrument has greater resistance to cyclic fatigue when compared with rc and wo . in our sample , the rc system was superior to the wo system regardless of cervical preflaring , which suggests that clearance of the root canal system through glidepath preparation before instrumentation is of the utmost clinical importance . in our sample , sem analysis of the instruments after the first use revealed cracks only on the wo files . we did not observe any plastic deformation , which may be explained by the greater resistance of the m - wire alloy as compared with other niti alloys . it bears stressing that the back - and - forth motion recommended by manufacturers and followed during specimen preparation in this study is largely intended to reduce torsional strain and , consequently , torsion fatigue , whereas its effect on flexural stresses is probably less evident . to date , rc and wo have been marketed for single - file root canal instrumentation and are approved for single use only so as to prevent structural weakening as a result of cyclic fatigue . therefore , single use reduces but does not altogether eliminate the risk of cumulative fatigue damage and potential instrument separation . it should be noted that the high level of fatigue resistance of reciprocating instruments may play an important role in improving root canal cleaning and shaping procedures as shaping can be done with remarkable speed . within this context , the principles of endodontics are shifting away from simultaneous cleaning and shaping toward a paradigm , whereby the root canal system is first shaped to enable effective cleaning by means of enhanced irrigation procedures . in the present study , cervical preflaring before instrumentation played a relevant role in increasing the fatigue life of the tested instruments . , the rc system demonstrated satisfactory fracture strength even in the absence of patent canals . the present study sought to assess the fracture strength of rc and wo instruments after repeated sterilization and reuse cycles . our results suggest that both the rc and wo systems can be safely reused several times , preceded by some preliminary manipulation to prevent the instrument from binding and thus reduce the risk of separation . based on our findings , we suggest that files can be reused safely up to 3 times . this number also facilitates control of the number of reuses of a single file . on the first use , the instrument is taken directly from its original packaging and its identifier ring is intact . after the first reprocessing , the identifier ring is enlarged and must be removed before the instrument can fit into the handpiece ; this serves as a milestone for the second use of the instrument . despite these conclusions , we believe that further research is needed to assess the ability of these reciprocating systems to ensure adequate canal antisepsis , as the duration of exposure to naocl irrigating solution tends to be reduced in single - file preparations , and to evaluate the influence of this clinical practice on treatment success over time . notably , one of the instrument specimens produced dark discoloration of the irrigation solution shortly after the start of its first use . upon removal of the instrument from the root canal , it was found to have been markedly corroded by the 2.5% naocl solution , with loss of material . on sem examination , energy dispersive x - ray analysis did not show any difference in alloy properties in relation to the other instruments used in the study [ figure 6 ] . , structural corrosion of niti instruments by naocl solution , have ever been reported in the literature . this event was probably due to a flaw in the manufacturing process as other instruments from the same lot exhibited stable behavior . scanning electron microscope images ( 100 ) of a reciproc r25 instrument showing corrosion and loss of metal mass energy dispersive x - ray analyses of the surface ( a ) and eroded area ( b ) of the instrument damaged by 2.5% sodium hypochlorite solution considering the proposed objective of this study , we conclude that both rc and wo instruments exhibited good fracture strength after autoclave sterilization and reuse . cervical preflaring significantly increased the number of times rc and wo files could be reused safely .

several recent studies have highlighted the tremendous importance of telomeres in clinical medicine ( yamaguchi et al , 2005 ; armanios et al , 2007 ; calado et al , 2009 ) . these advances provide an opportunity to revisit some of the concepts and data that provide a link between telomeres , the turnover of various ( stem ) cells and the diverse pathology that is now linked to telomere dysfunction . this aim of this essay is not to provide a comprehensive review of the many papers that have implicated telomeres in human disease . instead , i hope to provide some insight into this broad and complex topic by reviewing ideas and data from selected publications in very diverse areas . the russian evolutionary biologist theodosius dobzhansky , wrote nothing in biology makes sense except in the light of evolution ' ( dobzhansky , 1973 ) . the essential truth in this statement is in my view exemplified by a remarkable difference in telomere function between human beings and most other eukaryotes . strikingly , complete loss of telomerase is tolerated for several generations in yeast , worms , plants and mice ( table i ) . in each of these species , the loss of telomerase results in a measurable and predictable decline in average telomere length with each subsequent generation . after four or more generations without major changes in growth or development , chromosome ends without sufficient telomere repeats typically are prone to fusion and the resulting dicentric chromosomes compromise the ability of cells to continue cell division . chromosome fusions and other molecular changes in cells of late generation telomerase - null mutant animals or plants result in a plethora of phenotypes . most typical are a failure to reproduce , variable growth defects , accelerated ageing ' in some tissues and failed or delayed responses to tissue injury . in stark contrast to these consequences in late generation telomerase - null animals or plants are the often severe and life - threatening clinical phenotypes observed in patients with a modest , two - fold reduction in telomerase levels ( e.g. resulting from haplo - insufficiency for one of the two essential telomerase genes ) ( table i ) in evolutionary terms , the cells of the germline are the most important cells : only the gametes produced by the cells of the germline have the potential to cross generations , whereas somatic cells are inevitably lost with each generation ( figure 1 ) . the disposable soma theory provides a useful paradigm to think about reproduction , somatic cells and ageing ( kirkwood and holliday , 1979 ) . all somatic cells and tissues , including our brain , reflect an evolutionary strategy to propagate germline dna . most people do not ( like to ) see themselves as the mortal carriers of precious , highly selected dna contained in cells of the germline . however , the notion that our soma serves to propagate germline dna and is disposable ' in the bigger scheme of things is useful in considering possible differences in telomere biology and dna repair between cells , tissues and species . at the single cell level , many signalling and cell cycle checkpoint pathways are integrated to allow cells to make life or death decisions ( cook et al , 2009 ) . however , constant selective pressure has also resulted in very specific differences between cells and between species . no doubt , many aspects of the pathways involved in cell signalling and cell cycle checkpoints and their connectivity remain to be uncovered and better understood within specific cells . differences in such pathways within cells of a given species need to be understood better before the added complexity of species - specific differences can be fruitfully dissected . however , in the context of growth control and malignant transformation , some differences between species have already been described . for example , dna damage foci originating from short , dysfunctional telomeres ( d'adda di fagagna et al , 2003 ) increase in number with accumulated cell divisions and with age in human and primate cells , but not in murine somatic cells and tissues ( sedivy , 2007 ) . these and other observations ( hahn et al , 1999 ; smogorzewska and de lange , 2002 ) support the idea that loss of telomeric dna prevents tumour growth and contributes to ageing in human beings and primates , but probably does not function in this way in laboratory mice ( with long telomeres ) , plants , worms , flies and yeast . interestingly , telomere loss can be made to also limit cell proliferation and tissue renewal in mice ( hao et al , 2005 ; deng et al , 2008 ) . in these studies , cell proliferation appears to be limited primarily by telomere loss and only indirectly by telomerase levels . the comparable somatic ( stem ) cells from such species are expected to have different requirements in terms of the type and efficiency of various dna repair pathways to ensure that germline dna is successfully propagated across generations . according to the disposable soma ' hypothesis , ageing results primarily from failure to maintain genome integrity in somatic cells after reproduction ( kirkwood and holliday , 1979 ) . the key concept here is that organisms with cells that do spend more energy on dna repair and other cellular maintenance ' functions than is strictly required for production of viable offspring are likely to be at a disadvantage relative to those that do not . thus , differences in lifespan and reproductive strategy between species will be reflected in differences between the type and efficiency of various dna repair pathways . the notion that the efficiency of dna repair is subject to natural selection is not as popular as the more commonly emphasized ideas that evolution proceeds by selection of variants within a species that carry favourable dna mutations or the simpler notion that ageing is a consequence of dna damage ( schumacher et al , 2008 ) . the emphasis on dna repair rather than dna damage in the context of ageing has practical implications : a focus on increasing the efficiency of dna repair is not the same as a focus on reducing dna damage . indeed , limited exposure to dna damage could stimulate repair and be more effective at increasing lifespan ( kaiser , 2003 ) than attempts to limit dna damage altogether ( e.g. with anti - oxidants ' ) . this line of reasoning is supported by the known benefit of ( moderate ) exercise and low - dose irradiation on the lifespan in various organisms ( calabrese and baldwin , 2003 ; radak et al , 2008 ) . no doubt , many differences in the type and efficiency of dna repair pathways in specific cells within tissues and comparable ( stem ) cells between species remain to be uncovered . for example , dna repair changes dramatically during neuronal differentiation ( nouspikel and hanawalt , 2000 ) and the repair of double strand ( ds ) dna breaks through molecules such as ku70/80 and dna - pk in the non - homologous end joining ( nhej ) pathway is much more efficient in human than in murine cells ( banuelos et al , 2008 and references therein ) . differences in nhej between mice and man could reflect the fact that human ( stem ) cells reside for much longer periods in a dormant g0 or g1 state of the cell cycle compared with their murine counterparts . repair of ds dna breaks through homologous recombination is less efficient in g0 or g1 ( aylon et al , 2004 ) and better nejh repair of dna ( ds ) breaks in human cells may reflect the slower turnover of human cells ( wilson et al , 2008 ) . the main point here is that the efficiency and the type of dna repair in somatic as well as germline cells have been and are subject to natural selection . as a result , the efficiency of dna repair in stem cells from species that reproduce rapidly is expected to be less than that in stem cells from species that take years to reproduce . highly effective dna repair required in somatic stem cells from long - lived species creates a novel risk : abnormal somatic stem cells , though perhaps less likely to arise , are more likely to propagate their abnormal genomes and survive . such risks are superimposed on the inherent risk of living longer : more time for mutations to accumulate . it seems plausible that the additional risks to stem cells in long - lived species have favoured the evolution of telomere attrition as tumour suppression mechanism ( reviewed in aubert and lansdorp , 2008 ) . the lack of a similar telomere - related counting mechanism in short - lived species and model organisms provides an explanation for the different consequences of telomerase deficiency in human beings and model organisms ( table i ) . unfortunately , the tumour suppressor function of telomeres in human cells poses a major hurdle in studies of human telomere biology . for example , suitable murine models for human carcinomas , the most common type of human malignancies , are only obtained after telomere function is severely compromised ( artandi et al , 2000 ) . the difference in telomere biology between mice and man has also hampered the development of suitable murine models for other human diseases including werner 's syndrome ( chang et al , 2004 ) . if one accepts that telomere loss indeed evolved to limit the growth of pre - malignant cells in human beings , it should be noted that this mechanism has two serious flaws ( figure 2 ) . first , the mechanism is subject to failure when two chromosome ends with insufficient telomere repeats fuse with each other . such fusion events ( e.g. between sister chromatids or between different chromosome ends ) will extinguish the dna damage signals that originated from the short telomeres . dicentric chromosomes formed by end - to - end fusion are likely to break on mitosis and initiate cycles of chromosome bridge / breakage / fusion ( de lange , 1995 ) . this type of genome instability greatly facilitates deletion and amplification of genes and , as a result , the malignant evolution of ( pre- ) malignant cells . second , by limiting the growth of ( pre- ) malignant cells , the dna damage response triggered by short telomeres ( d'adda di fagagna et al , 2003 ) will become subject to strong negative selective pressure . such selection favours cells with defective dna damage responses . indeed , this seems a plausible mechanism , whereby most human tumours acquire defective dna damage responses ( e.g. over half of all human cancers have mutations in the p53 gene ( vousden and lane , 2007 ) ) . as dna damage responses are upstream in many different dna repair pathways , cells selected on the basis of their inability to respond appropriately to short telomeres will display defects in several different dna repair pathways . indeed , it seems plausible that genome instability triggered by unstable chromosome ends in cells with defective dna damage response result in the mutator cell ' phenotype that is characteristic of many tumour cells ( loeb , 2001 ) . that chromosomal instability can be initiated by disruption of telomere function and that microsatellite instability and other genomic alterations often seen in cancer cells could arise from defective dna damage responses indirectly selected by telomere shortening does not seem to be widely accepted ( michor et al , 2005 ) . these ideas nevertheless seem plausible and worthy of further investigation . apart from the difficulty of finding suitable model organisms , such studies are complicated by the transient period of rampant genome instability driven by telomere dysfunction . sooner or later , most abnormal cells typically stabilize their chromosome ends ( and at that stage typically highly abnormal genomes ) by upregulation of telomerase activity ( kim et al , 1994 ) . the frequent amplification of the telomerase reverse transcriptase gene in human lung cancer suggests that break fusion bridge cycles involving chromosome 5p is frequently involved in the upregulation of telomerase activity in those cells ( weir et al , 2007 ; kang et al , 2008 ) . if telomere attrition evolved to limit the number of somatic cell divisions in long - lived species , one might ask how many times stem cells in tissues actually divide . unfortunately , and perhaps surprisingly , haematopoietic stem cells ( hscs ) are probably the most extensively studied stem cell population . from studies of both human and murine hscs , it has become clear that hscs represent a very heterogeneous population of cells that do not self - renew in the absolute sense of the word ( lansdorp , 1997 ) . the functional properties of purified candidate ' stem cell populations change markedly during development in both man ( lansdorp et al , 1993 ) and mouse ( bowie et al , 2006 ; kim et al , 2007 ) . such functional changes correspond to a rather abrupt change in hsc turnover early in life , which is reflected in a rapid decline in the rate of telomere attrition after 34 years in human beings ( rufer et al , 1999 ) and 12 years in baboons ( baerlocher et al , 2007 ) . the turnover of murine hscs also drops quite abruptly , but at 46 weeks after birth ( bowie et al , 2007 ; kim et al , 2007 ) . the majority of transplantable hscs in adult mice are derived from a very small pool of quiescent cells that divide less than 10 times over a lifetime ( wilson et al , 2008 ) . the picture that is emerging is that hscs are dividing very infrequently in adults and only switch from dormancy to self - renewal following transplantation or other stress conditions . very limited turnover of adult stem cells is also compatible with the minimal loss of telomere length observed in granulocytes from normal human individuals between 20 and 60 years of age ( lansdorp , 2008 ) and the sequential recruitment of quiescent progenitors that was observed to sustain cell production in human long - term bone marrow cultures ( lansdorp and dragowska , 1993 ) . on the basis of telomere length data from different mammalian species , it has been proposed that hscs divide less than 200 times over a lifetime and that this number is evolutionarily conserved ( shepherd et al , 2007 ) . if this number is correct , it suggests that very few cell divisions in the haematopoietic system are wasted ' . the total number of blood cells required for life - long blood cell production in man can be calculated to be in the order of 4 10 cells ( 10 cells / day 365 days 100 years ) . in theory , only 55 divisions of a single cell could satisfy this need ( 2=4 10 ) . with inclusion of additional cell divisions required to compensate for inevitable cell death and loss through differentiation , estimates of less than 200 cell divisions over a lifetime seem reasonable . the estimate of the number of divisions in hscs is at odds with the thousands of divisions proposed , for example stem cells in the gut ( potten et al , 2002 ) . the notion that stem cells in the intestine of mice divide much more than those in the bone marrow is also at odds with data from mice that lack telomerase ( blasco et al , 1997 ; liu et al , 2000 ) and turnover estimates of tissues , including blood and intestine , derived from c dating ( spalding et al , 2005 ) . mice that lack telomerase show a loss of around 5 kb per generation ( blasco et al , 1997 ) . assuming a loss of 100 bp of telomeric dna per cell division , subsequent generations of telomerase - null mice are calculated to be separated by 50 divisions . this estimate is in close agreement with independent estimates of 34133 cell divisions in the male and 25 in the female germline ( figure 3 , adopted from drost and lee , 1995 ) . if intestinal stem cells were indeed to divide thousands of times , one would expect a severe gut phenotype in the first generation of terc ko mice unless intestinal stem cells could somehow avoid telomere loss or maintain telomeres in the absence of telomerase . more likely , intestinal cells , such as lgr5 cells , that divide every 24 h ( barker et al , 2007 ) include a small subset of lgr5 stem cells and a large proportion of lgr5 progenitor cells similar to cd34 cells in human bone marrow . only a very small fraction ( < 1% ) of bm cd34 cells are probably true hscs ( terstappen et al , 1991 ; novelli et al , 1998 ) , and only a minor fraction of sorted single lgr5 cells is capable of initiating crypt formation in vitro ( sato et al , 2009 ) . uncertainties about the number of stem cells in various tissues and their turnover are important in the discussion of human telomerase deficiency disorders . it seems likely that most , if not all , phenotypic consequences of human telomerase deficiency result from compromised telomere function that limits the proliferation of ( stem ) cells . if this is correct , the phenotypic manifestations of telomerase disorders could in fact instruct us about the turnover of stem cells in various tissues . this reasoning explains in part why telomeres and telomerase disorders ' are of interest to a growing number of stem cell ' researchers . the first and most well - studied telomere disorder ' is dyskeratosis congenita ( dc ) . patients with dc typically present with three distinctive clinical characteristics : skin pigmentation abnormalities , nail dystrophy and abnormalities of the oral mucosa known as leukoplakia ( walne and dokal , 2008 ; savage and alter , 2009 ) . the most common fatal complications of dc are related to bone marrow failure , pulmonary fibrosis and cancer ( walne and dokal , 2008 ; savage and alter , 2009 ) . the most frequent cancers are head and neck cancer ( squamous cell carcinoma ) , cancer of the tongue and acute myeloid leukaemia ( alter et al , 2009 ) . in addition , a variety of other clinical abnormalities have been described in dc patients ( reviewed in kirwan and dokal , 2008 ) . hepatic cirrhosis has been described in dc and severe liver disease is not uncommon after hsc transplantation for marrow failure in patients with dc ( calado and young , 2008 ) . several genes have now been implicated in dc ; most notably , all three genes encoding components of the minimal ( cohen et al , 2007 ) telomerase enzyme complex ( walne and dokal , 2008 ; savage and alter , 2009 ) . mutations were initially discovered in dkc1 , located on the x chromosome ( heiss et al , 1998 ) . a link with telomerase was first suggested when the nucleolar protein encoded by dkc1 , dyskerin , was found to stabilize telomerase rna and maintain telomere length through telomerase ( mitchell et al , 1999 ) . mutations in hterc and htert themselves were later discovered in autosomal dominant dc ( vulliamy et al , 2001 ; fogarty et al , 2003 ; armanios et al , 2005 ) . in total , 50% of patients with dc have a mutation in one of the three genes of the telomerase complex ( aubert and lansdorp , 2008 ) . this notion is supported by the recent discoveries of mutations in genes of dc patients such as tinf2 , nhp2 and nop10 ( walne et al , 2007 ; savage et al , 2008 ; vulliamy et al , 2008 ) . mutations in htert and hterc have also been found in diseases other than dc , including other bone marrow failure syndromes such as aplastic anaemia ( aa ) ( yamaguchi et al , 2003 , 2005 ; marrone et al , 2004 ; ly et al , 2005 ; vulliamy et al , 2005 ) and myelodysplastic syndrome ( mds ) ( yamaguchi et al , 2003 ) , and in diseases not typically associated with blood disorders such as idiopathic pulmonary fibrosis ( ipf ) ( armanios et al , 2007 ) . in aa , 3 of 200 patients were found to have mutations in hterc ( yamaguchi et al , 2003 ) and an additional 3.5% ( 7/200 ) had mutations in htert ( yamaguchi et al , 2005 ) . in ipf , 1.4% ( 1/73 ) of patients were identified with hterc mutations , whereas 6.8% ( 5/73 ) of patients with familial ipf had mutations in htert ( armanios et al , 2007 ) . anticipation ' , meaning that the disease typically presents at an earlier age in each subsequent generation ( vulliamy et al , 2004 ; armanios et al , 2005 ) . the most likely explanation for disease anticipation in telomerase disorders is that limiting amounts of telomerase activity in the germline result in gametes with shorter telomeres and , consequently , offspring with shorter telomeres . shorter telomeres in stem cells at birth reduce the number of stem cell divisions before short telomeres trigger a dna damage response and , eventually , senescence or cell death . it is implied here that all disease manifestations of telomerase disorders are likely to result from compromised cell and tissue renewal . from the spectrum of phenotypes of patients with known telomerase deficiencies , one can deduce that the stem cells of the oral mucosa , the skin ( especially in the head and neck area ) , the nails , the lung , the bone marrow and the liver rely on telomerase to sustain proliferation the most or have the highest turnover of all the stem cells in the human body . why specific tissues seem to be affected more than others in individual patients is incompletely understood . it seems possible that environmental factors contribute to disease manifestations , for example smoking in patients with pulmonary fibrosis or alcohol consumption in patients presenting with hepatic cirrhosis . in such patients , a reduced stem cell reserve could lead to symptoms by increasing the demand on the turnover of remaining tissue - specific stem cells by environmental toxins . among environmental toxins ' , factors induced by viral or other infections , such as interferon alpha ( essers et al , 2009 ) , or factors stimulated by , for example , sustained psychological stress ( epel et al , 2004 ) should probably be included . indeed , such factors are likely to accelerate tissue turnover and thereby ageing even in individuals without specific telomere maintenance ' disorders . other known or unknown extrinsic ' factors , including specific diseases , could also affect tissue - specific stem cell numbers . telomere length was found to be much shorter in endothelial cells from the aorta compared with those from pulmonary arteries of the same organ donor , and it was suggested that this could reflect differences in hemodynamic stress to the vessel wall ( chang and harley , 1995 ) . striking cytogenetic abnormalities have also been described to accumulate in aorta endothelial cells with age ( aviv et al , 2001 ) . despite uncertainties about the replicative or regenerative potential of endothelial cells , it seems plausible that this capacity will not exceed that of other somatic ( stem ) cells . extrinsic factors , such as smoking , diabetes , high blood pressure , diet , etc . , could result in loss of endothelial cells or function and trigger cardiovascular disease in the absence of additional genetic factors that compromise the replicative potential of cells such as heritable telomerase deficiencies ( farhat et al , 2008 ; wong et al , 2009 ) . reduced telomerase levels as well as short telomeres are expected to compromise the number as well as the replicative potential of stem cells that remain in specific tissues after extrinsic or intrinsic damage from the environment . for example , cell - specific turnover could be increased by a specific genetic defect or a polymorphic trait that increases the likelihood of cell death in response to injury . presumably , such genetic co - factors can be distinguished from environmental co - factors by careful studies of both environmental exposure as well as the genotype of a given patient relative to family members that are not affected or that display other disease manifestations . finally , it seems possible that disease manifestations of human telomerase disorders are the result of stochastic telomere loss events at individual telomeres during development . telomere repeats are lost not only as a result of the end - replication problem ' , but also as a result of sporadic recently , it was found that within the haematopoietic system the most primitive haematopoietic cells have fewer abruptly shortened telomeres compared with more differentiated cells ( hills et al , 2009 ) . the level of telomerase could be important when more than a few sporadic telomere loss events occur or accumulate in a cell at any given time ( aubert and lansdorp , 2008 ) . in cells with overall shorter telomeres and lower telomerase levels to start with , cell death events could occur more frequently during normal development . both the number and the replicative potential of remaining tissue - specific stem cells could be compromised stochastically depending on when cell death occurred during development and whether remaining cells had to go through extra divisions to make up for the loss of cells . the distinction between environmental , genetic and stochastic reasons for the variable clinical presentation of patients with telomerase disorders is a major research challenge . this challenge is augmented further by the distinct possibility that the specific disease manifestation in an individual patient results from a combination of these and other factors . telomerase is upregulated in a majority of human cancers ( kim et al , 1994 ; cao et al , 2008 ) . this upregulation is believed to be required to allow malignant cells to divide after genetic rearrangements enabled by telomere dysfunction . in view of the telomerase requirement in ( late stage ) cancer growth , the recent finding that heritable defects appear to predispose for the development of acute myeloid leukaemia seems perhaps counterintuitive ( calado et al , 2009 ) . however , patients with dc or bone marrow failure resulting from mutations in telomerase genes often develop leukaemia or other types of cancer ( alter et al , 2009 ) . leukaemia and other malignancies are also well - known complications of high - dose chemotherapy regimens and mds . most likely , the loss of stem cells by telomere dysfunction or treatment creates an environment in which abnormal cells can flourish . this could reflect persistent feedback signals stimulating stem cell proliferation from the microenvironment . with many stem cells present , such stimulation is expected to occur only transiently , whereas with few or no normal stem cells responding , such stimulatory signals could persist and stimulate the growth of abnormal cells ( e.g. cells with defective dna damage responses ) . the malignant progression of such abnormal cells could be further facilitated by mutations induced by earlier treatment . in other words , normal stem cell numbers could indirectly suppress malignant transformation of cells , whereas loss of stem cells could predispose for malignant transformation . the difficulties related to the identification of stem cells in vivo that were discussed above greatly complicate studies on the role of the number of stem cells in tumour development . however , it seems possible that a decline in the number of tissue - specific stem cells could contribute to the exponential increase in tumour development with age . for example , calado et al , reported in a recent paper that heritable hypomorphic mutations in the telomerase reverse transcriptase gene predispose for acute myeloid leukaemia ( calado et al , 2009 ) . in this study of aml patients ( n=594 ) , a common variant of the telomerase reverse transcriptase gene htert , a1062 t , was present three times more frequently in patients than in controls ( n=1110 , p=0.0009 ) . the mutant telomerase allele was found to decrease the enzymatic activity of telomerase by 50% in the trap telomerase assay ( calado et al , 2009 ) . however , the a1062 t htert variant did not appear to compromise telomerase activity in another study ( alder et al , 2008 ) , and the precise functional consequences of a1062 t expression in human ( stem ) cells remain to be clarified . as in most patients only one tert allele was affected , telomerase activity in the stem cells of these patients is expected to be around 75% of normal levels . how reduced telomerase levels in these patients relate to the development of aml is not clear . one possibility is that telomeres were on average shorter at birth and that shorter telomeres limited normal stem cell proliferation with selection of abnormal cells as discussed above . second , it is possible that full telomerase levels are critical in hscs to maintain telomere function of critically short telomeres . even a slight reduction in telomerase levels could increase the probability of cell death after , for example , oxidative damage to telomeric dna in stem cells . telomeres in the most primitive haematopoietic cells were found to have the lowest frequency of abruptly shortened telomeres in the haematopoietic system ( hills et al , 2009 ) and oxidative damage is a major cause of telomere attrition ( von zglinicki , 2002 ) . two recent studies have suggested that risk of developing lung cancer is associated with genetic markers ( snp 's ) that map to the tert locus ( mckay et al , 2008 ; wang et al , 2008 ) . these results were confirmed in a large snp study , which also looked at a variety of other tumours ( rafnar et al , 2009 ) . in this large study of over 30 000 cancer cases and 45 000 controls , a significant association of snp rs401681[c ] within the tert locus on chromosome 5p15.33 was found with basal cell carcinoma , lung cancer and cancer of the urinary bladder , prostate and cervix . interestingly , the same snp seems to confer protection against cutaneous melanoma , and the cancers that are associated with this snp all have a strong environmental component to their risk . although the precise role of genetic variation within the tert locus remains to be elucidated , it is tempting to speculate that this snp is linked to hypomorphic tert alleles similar to the a1062 t variant that was found to be more frequently in patients with aml than normal controls . recent studies have put telomerase and telomere research square at the centre of cancer research . not only is amplification of the htert gene one of the most common genetic abnormalities in lung adenocarcinoma , one of the most common human cancers ( weir et al , 2007 ; kang et al , 2008 ) , it now is becoming clear that snp 's within the tert locus are among the most reproducible risk factors for the development of different types of cancer ( mckay et al , 2008 ; wang et al , 2008 ; rafnar et al , 2009 ) . the finding that hypomorphic tert mutations increase the risk for the development of acute myeloid leukaemia ( calado et al , 2009 ) , chronic lymphocytic leukaemia ( hills and lansdorp , nyas in press ) and probably other cancers , together with the increased risk for leukaemia development associated with bone marrow failure in aa , mds and dc , strongly implicates stem cell failure and reduced stem cell numbers as risk factors for tumour development . more direct measures of stem cell numbers in vivo are needed to examine the relationship between stem cell numbers and tumour development in patients with defective telomere maintenance as well as in normal individuals as a function of age . measurements of the average telomere length as well as the length at individual chromosome ends in specific cells and tissues are expected to provide valuable information about the involvement of telomeres in normal ageing and tumour biology .

the telomeres of most eukaryotes are characterized by guanine - rich repeats synthesized by the reverse transcriptase telomerase . complete loss of telomerase is tolerated for several generations in most species , but modestly reduced telomerase levels in human beings are implicated in bone marrow failure , pulmonary fibrosis and a spectrum of other diseases including cancer . differences in telomerase deficiency phenotypes between species most likely reflect a tumour suppressor function of telomeres in long - lived mammals that does not exist as such in short - lived organisms . another puzzle provided by current observations is that family members with the same genetic defect , haplo - insufficiency for one of the telomerase genes , can present with widely different diseases . here , the crucial role of telomeres and telomerase in human ( stem cell ) biology is discussed from a darwinian perspective . it is proposed that the variable phenotype and penetrance of heritable human telomerase deficiencies result from additional environmental , genetic and stochastic factors or combinations thereof .

Introduction The chicken or the egg? Differences in DNA repair between cells and species? How many stem cell divisions? Telomerase deficiencies and other telomere disorders Telomere defects and cancer predisposition Concluding remarks

several recent studies have highlighted the tremendous importance of telomeres in clinical medicine ( yamaguchi et al , 2005 ; armanios et al , 2007 ; calado et al , 2009 ) . these advances provide an opportunity to revisit some of the concepts and data that provide a link between telomeres , the turnover of various ( stem ) cells and the diverse pathology that is now linked to telomere dysfunction . this aim of this essay is not to provide a comprehensive review of the many papers that have implicated telomeres in human disease . strikingly , complete loss of telomerase is tolerated for several generations in yeast , worms , plants and mice ( table i ) . in each of these species , the loss of telomerase results in a measurable and predictable decline in average telomere length with each subsequent generation . in stark contrast to these consequences in late generation telomerase - null animals or plants are the often severe and life - threatening clinical phenotypes observed in patients with a modest , two - fold reduction in telomerase levels ( e.g. resulting from haplo - insufficiency for one of the two essential telomerase genes ) ( table i ) in evolutionary terms , the cells of the germline are the most important cells : only the gametes produced by the cells of the germline have the potential to cross generations , whereas somatic cells are inevitably lost with each generation ( figure 1 ) . however , the notion that our soma serves to propagate germline dna and is disposable ' in the bigger scheme of things is useful in considering possible differences in telomere biology and dna repair between cells , tissues and species . differences in such pathways within cells of a given species need to be understood better before the added complexity of species - specific differences can be fruitfully dissected . for example , dna damage foci originating from short , dysfunctional telomeres ( d'adda di fagagna et al , 2003 ) increase in number with accumulated cell divisions and with age in human and primate cells , but not in murine somatic cells and tissues ( sedivy , 2007 ) . these and other observations ( hahn et al , 1999 ; smogorzewska and de lange , 2002 ) support the idea that loss of telomeric dna prevents tumour growth and contributes to ageing in human beings and primates , but probably does not function in this way in laboratory mice ( with long telomeres ) , plants , worms , flies and yeast . the comparable somatic ( stem ) cells from such species are expected to have different requirements in terms of the type and efficiency of various dna repair pathways to ensure that germline dna is successfully propagated across generations . thus , differences in lifespan and reproductive strategy between species will be reflected in differences between the type and efficiency of various dna repair pathways . this line of reasoning is supported by the known benefit of ( moderate ) exercise and low - dose irradiation on the lifespan in various organisms ( calabrese and baldwin , 2003 ; radak et al , 2008 ) . no doubt , many differences in the type and efficiency of dna repair pathways in specific cells within tissues and comparable ( stem ) cells between species remain to be uncovered . differences in nhej between mice and man could reflect the fact that human ( stem ) cells reside for much longer periods in a dormant g0 or g1 state of the cell cycle compared with their murine counterparts . the main point here is that the efficiency and the type of dna repair in somatic as well as germline cells have been and are subject to natural selection . as a result , the efficiency of dna repair in stem cells from species that reproduce rapidly is expected to be less than that in stem cells from species that take years to reproduce . highly effective dna repair required in somatic stem cells from long - lived species creates a novel risk : abnormal somatic stem cells , though perhaps less likely to arise , are more likely to propagate their abnormal genomes and survive . it seems plausible that the additional risks to stem cells in long - lived species have favoured the evolution of telomere attrition as tumour suppression mechanism ( reviewed in aubert and lansdorp , 2008 ) . the lack of a similar telomere - related counting mechanism in short - lived species and model organisms provides an explanation for the different consequences of telomerase deficiency in human beings and model organisms ( table i ) . unfortunately , the tumour suppressor function of telomeres in human cells poses a major hurdle in studies of human telomere biology . for example , suitable murine models for human carcinomas , the most common type of human malignancies , are only obtained after telomere function is severely compromised ( artandi et al , 2000 ) . if one accepts that telomere loss indeed evolved to limit the growth of pre - malignant cells in human beings , it should be noted that this mechanism has two serious flaws ( figure 2 ) . first , the mechanism is subject to failure when two chromosome ends with insufficient telomere repeats fuse with each other . second , by limiting the growth of ( pre- ) malignant cells , the dna damage response triggered by short telomeres ( d'adda di fagagna et al , 2003 ) will become subject to strong negative selective pressure . that chromosomal instability can be initiated by disruption of telomere function and that microsatellite instability and other genomic alterations often seen in cancer cells could arise from defective dna damage responses indirectly selected by telomere shortening does not seem to be widely accepted ( michor et al , 2005 ) . sooner or later , most abnormal cells typically stabilize their chromosome ends ( and at that stage typically highly abnormal genomes ) by upregulation of telomerase activity ( kim et al , 1994 ) . the frequent amplification of the telomerase reverse transcriptase gene in human lung cancer suggests that break fusion bridge cycles involving chromosome 5p is frequently involved in the upregulation of telomerase activity in those cells ( weir et al , 2007 ; kang et al , 2008 ) . if telomere attrition evolved to limit the number of somatic cell divisions in long - lived species , one might ask how many times stem cells in tissues actually divide . unfortunately , and perhaps surprisingly , haematopoietic stem cells ( hscs ) are probably the most extensively studied stem cell population . from studies of both human and murine hscs , it has become clear that hscs represent a very heterogeneous population of cells that do not self - renew in the absolute sense of the word ( lansdorp , 1997 ) . the functional properties of purified candidate ' stem cell populations change markedly during development in both man ( lansdorp et al , 1993 ) and mouse ( bowie et al , 2006 ; kim et al , 2007 ) . such functional changes correspond to a rather abrupt change in hsc turnover early in life , which is reflected in a rapid decline in the rate of telomere attrition after 34 years in human beings ( rufer et al , 1999 ) and 12 years in baboons ( baerlocher et al , 2007 ) . the majority of transplantable hscs in adult mice are derived from a very small pool of quiescent cells that divide less than 10 times over a lifetime ( wilson et al , 2008 ) . very limited turnover of adult stem cells is also compatible with the minimal loss of telomere length observed in granulocytes from normal human individuals between 20 and 60 years of age ( lansdorp , 2008 ) and the sequential recruitment of quiescent progenitors that was observed to sustain cell production in human long - term bone marrow cultures ( lansdorp and dragowska , 1993 ) . on the basis of telomere length data from different mammalian species , it has been proposed that hscs divide less than 200 times over a lifetime and that this number is evolutionarily conserved ( shepherd et al , 2007 ) . the estimate of the number of divisions in hscs is at odds with the thousands of divisions proposed , for example stem cells in the gut ( potten et al , 2002 ) . the notion that stem cells in the intestine of mice divide much more than those in the bone marrow is also at odds with data from mice that lack telomerase ( blasco et al , 1997 ; liu et al , 2000 ) and turnover estimates of tissues , including blood and intestine , derived from c dating ( spalding et al , 2005 ) . assuming a loss of 100 bp of telomeric dna per cell division , subsequent generations of telomerase - null mice are calculated to be separated by 50 divisions . if intestinal stem cells were indeed to divide thousands of times , one would expect a severe gut phenotype in the first generation of terc ko mice unless intestinal stem cells could somehow avoid telomere loss or maintain telomeres in the absence of telomerase . more likely , intestinal cells , such as lgr5 cells , that divide every 24 h ( barker et al , 2007 ) include a small subset of lgr5 stem cells and a large proportion of lgr5 progenitor cells similar to cd34 cells in human bone marrow . uncertainties about the number of stem cells in various tissues and their turnover are important in the discussion of human telomerase deficiency disorders . it seems likely that most , if not all , phenotypic consequences of human telomerase deficiency result from compromised telomere function that limits the proliferation of ( stem ) cells . if this is correct , the phenotypic manifestations of telomerase disorders could in fact instruct us about the turnover of stem cells in various tissues . this reasoning explains in part why telomeres and telomerase disorders ' are of interest to a growing number of stem cell ' researchers . patients with dc typically present with three distinctive clinical characteristics : skin pigmentation abnormalities , nail dystrophy and abnormalities of the oral mucosa known as leukoplakia ( walne and dokal , 2008 ; savage and alter , 2009 ) . the most common fatal complications of dc are related to bone marrow failure , pulmonary fibrosis and cancer ( walne and dokal , 2008 ; savage and alter , 2009 ) . several genes have now been implicated in dc ; most notably , all three genes encoding components of the minimal ( cohen et al , 2007 ) telomerase enzyme complex ( walne and dokal , 2008 ; savage and alter , 2009 ) . in total , 50% of patients with dc have a mutation in one of the three genes of the telomerase complex ( aubert and lansdorp , 2008 ) . mutations in htert and hterc have also been found in diseases other than dc , including other bone marrow failure syndromes such as aplastic anaemia ( aa ) ( yamaguchi et al , 2003 , 2005 ; marrone et al , 2004 ; ly et al , 2005 ; vulliamy et al , 2005 ) and myelodysplastic syndrome ( mds ) ( yamaguchi et al , 2003 ) , and in diseases not typically associated with blood disorders such as idiopathic pulmonary fibrosis ( ipf ) ( armanios et al , 2007 ) . anticipation ' , meaning that the disease typically presents at an earlier age in each subsequent generation ( vulliamy et al , 2004 ; armanios et al , 2005 ) . the most likely explanation for disease anticipation in telomerase disorders is that limiting amounts of telomerase activity in the germline result in gametes with shorter telomeres and , consequently , offspring with shorter telomeres . shorter telomeres in stem cells at birth reduce the number of stem cell divisions before short telomeres trigger a dna damage response and , eventually , senescence or cell death . it is implied here that all disease manifestations of telomerase disorders are likely to result from compromised cell and tissue renewal . from the spectrum of phenotypes of patients with known telomerase deficiencies , one can deduce that the stem cells of the oral mucosa , the skin ( especially in the head and neck area ) , the nails , the lung , the bone marrow and the liver rely on telomerase to sustain proliferation the most or have the highest turnover of all the stem cells in the human body . other known or unknown extrinsic ' factors , including specific diseases , could also affect tissue - specific stem cell numbers . telomere length was found to be much shorter in endothelial cells from the aorta compared with those from pulmonary arteries of the same organ donor , and it was suggested that this could reflect differences in hemodynamic stress to the vessel wall ( chang and harley , 1995 ) . despite uncertainties about the replicative or regenerative potential of endothelial cells , it seems plausible that this capacity will not exceed that of other somatic ( stem ) cells . , could result in loss of endothelial cells or function and trigger cardiovascular disease in the absence of additional genetic factors that compromise the replicative potential of cells such as heritable telomerase deficiencies ( farhat et al , 2008 ; wong et al , 2009 ) . reduced telomerase levels as well as short telomeres are expected to compromise the number as well as the replicative potential of stem cells that remain in specific tissues after extrinsic or intrinsic damage from the environment . presumably , such genetic co - factors can be distinguished from environmental co - factors by careful studies of both environmental exposure as well as the genotype of a given patient relative to family members that are not affected or that display other disease manifestations . telomere repeats are lost not only as a result of the end - replication problem ' , but also as a result of sporadic recently , it was found that within the haematopoietic system the most primitive haematopoietic cells have fewer abruptly shortened telomeres compared with more differentiated cells ( hills et al , 2009 ) . in cells with overall shorter telomeres and lower telomerase levels to start with , cell death events could occur more frequently during normal development . both the number and the replicative potential of remaining tissue - specific stem cells could be compromised stochastically depending on when cell death occurred during development and whether remaining cells had to go through extra divisions to make up for the loss of cells . the distinction between environmental , genetic and stochastic reasons for the variable clinical presentation of patients with telomerase disorders is a major research challenge . this challenge is augmented further by the distinct possibility that the specific disease manifestation in an individual patient results from a combination of these and other factors . in view of the telomerase requirement in ( late stage ) cancer growth , the recent finding that heritable defects appear to predispose for the development of acute myeloid leukaemia seems perhaps counterintuitive ( calado et al , 2009 ) . however , patients with dc or bone marrow failure resulting from mutations in telomerase genes often develop leukaemia or other types of cancer ( alter et al , 2009 ) . most likely , the loss of stem cells by telomere dysfunction or treatment creates an environment in which abnormal cells can flourish . this could reflect persistent feedback signals stimulating stem cell proliferation from the microenvironment . in other words , normal stem cell numbers could indirectly suppress malignant transformation of cells , whereas loss of stem cells could predispose for malignant transformation . the difficulties related to the identification of stem cells in vivo that were discussed above greatly complicate studies on the role of the number of stem cells in tumour development . for example , calado et al , reported in a recent paper that heritable hypomorphic mutations in the telomerase reverse transcriptase gene predispose for acute myeloid leukaemia ( calado et al , 2009 ) . in this study of aml patients ( n=594 ) , a common variant of the telomerase reverse transcriptase gene htert , a1062 t , was present three times more frequently in patients than in controls ( n=1110 , p=0.0009 ) . the mutant telomerase allele was found to decrease the enzymatic activity of telomerase by 50% in the trap telomerase assay ( calado et al , 2009 ) . however , the a1062 t htert variant did not appear to compromise telomerase activity in another study ( alder et al , 2008 ) , and the precise functional consequences of a1062 t expression in human ( stem ) cells remain to be clarified . as in most patients only one tert allele was affected , telomerase activity in the stem cells of these patients is expected to be around 75% of normal levels . how reduced telomerase levels in these patients relate to the development of aml is not clear . one possibility is that telomeres were on average shorter at birth and that shorter telomeres limited normal stem cell proliferation with selection of abnormal cells as discussed above . second , it is possible that full telomerase levels are critical in hscs to maintain telomere function of critically short telomeres . even a slight reduction in telomerase levels could increase the probability of cell death after , for example , oxidative damage to telomeric dna in stem cells . telomeres in the most primitive haematopoietic cells were found to have the lowest frequency of abruptly shortened telomeres in the haematopoietic system ( hills et al , 2009 ) and oxidative damage is a major cause of telomere attrition ( von zglinicki , 2002 ) . in this large study of over 30 000 cancer cases and 45 000 controls , a significant association of snp rs401681[c ] within the tert locus on chromosome 5p15.33 was found with basal cell carcinoma , lung cancer and cancer of the urinary bladder , prostate and cervix . interestingly , the same snp seems to confer protection against cutaneous melanoma , and the cancers that are associated with this snp all have a strong environmental component to their risk . although the precise role of genetic variation within the tert locus remains to be elucidated , it is tempting to speculate that this snp is linked to hypomorphic tert alleles similar to the a1062 t variant that was found to be more frequently in patients with aml than normal controls . not only is amplification of the htert gene one of the most common genetic abnormalities in lung adenocarcinoma , one of the most common human cancers ( weir et al , 2007 ; kang et al , 2008 ) , it now is becoming clear that snp 's within the tert locus are among the most reproducible risk factors for the development of different types of cancer ( mckay et al , 2008 ; wang et al , 2008 ; rafnar et al , 2009 ) . the finding that hypomorphic tert mutations increase the risk for the development of acute myeloid leukaemia ( calado et al , 2009 ) , chronic lymphocytic leukaemia ( hills and lansdorp , nyas in press ) and probably other cancers , together with the increased risk for leukaemia development associated with bone marrow failure in aa , mds and dc , strongly implicates stem cell failure and reduced stem cell numbers as risk factors for tumour development . more direct measures of stem cell numbers in vivo are needed to examine the relationship between stem cell numbers and tumour development in patients with defective telomere maintenance as well as in normal individuals as a function of age . measurements of the average telomere length as well as the length at individual chromosome ends in specific cells and tissues are expected to provide valuable information about the involvement of telomeres in normal ageing and tumour biology .

based on international surveys during the past forty years , the prevalence of overweight or obesity in children has accelerated in most global regions . latest available estimates from 34 member countries within the organization for economic cooperation and development indicate that among school age children , 21% of girls and 23% of boys are overweight . worldwide , an estimated twenty - two million children 5 years of age were overweight in 2007 and at present , approximately 1020% of infants and toddlers in the united states ( us ) are overweight , ( 85th body mass index ( bmi ) percentile for age ) , values similar to those reported for young children in multiple countries [ 59 ] . equally concerning is the finding that nearly 10% of infants and toddlers , from birth2 years are also obese , at or above the 95th percentile of the weight for recumbent length growth charts . more than half the overweight children from one longitudinal study became overweight before age 2 , and 25% were overweight by 5 months of age . many overweight infants remain overweight into their childhood years , and childhood obesity has long been known as a strong predictor of adult obesity . recent results from a retrospective medical - chart review of 257 children demonstrated that an increased bmi , as early as 2 weeks of age , was associated with a significant increased risk of overweight at 6 , 12 , 36 , and 60 months . similarly , reports of overweight at 618 months of age being strongly predictive of weight in preschool years are becoming more often documented [ 1315 ] . longitudinal data from 762 infants and children ( age 018 years ) indicated that body weight as early as 2 years of age begins a positive tracking period for adult overweight ; weight status from 26 years was the most critical growth period for prediction and realization of adult overweight . an overweight child at age 24 years has a 5-fold increased risk of being overweight at age 12 , compared to children not overweight during their preschool years . although limited longitudinal data are available to categorize the proportion of overweight infants that remain overweight throughout life , infant weight status is predictive of bmi or measures of obesity during the adolescent and early adult years [ 15 , 1721 ] . the earlier a child becomes overweight , and the longer excess weight is maintained , the greater the risk that the child 's overweight will follow into adulthood . interventions to successfully reduce rates of overweight in very young populations are emerging areas of research , but have yet to be given adequate attention . critical periods for establishing dietary intake patterns , eating habits , and food preferences begin in infancy , and although inconsistent , may be set as early as 2 years of age [ 22 , 23 ] . as the infant progresses to table foods , the family diet exerts a strong influence on food consumption consistent with a general adoption of the eating practices of the family . parental feeding practices , if not causative for weight status in young children , are strongly associated with body weight and healthy food choices throughout childhood . research suggests that interventions to adjust food composition or caloric intake and increase physical activity , especially after infancy , have a small impact on children 's weight or measures of adiposity [ 26 , 27 ] and have been inadequate in curtailing the increased prevalence of overweight affecting young children . obesity prevention efforts that begin during the school age years offer an approach that is insufficient in addressing the epidemic . preschool age children are already overweight , a finding that may be mirrored among other young children throughout the world [ 1 , 9 ] . the primary objective of this narrative review is to discuss risk factors associated with early childhood overweight , with emphasis on current evidence that supports specific modifiable risk factors for obesity prevention within very young children . a review of the available , but few , interventions designed to prevent overweight and obesity within the infant age population is included . it is hoped that this review will contribute to the development of future strategies , policy , and practices consistent with the public health need for an early life obesity prevention plan . antecedents of early childhood obesity are clearly multifactorial , and associations of varying strength have been documented for genetic , biologic , dietary , environmental , and behavioral factors . to comprehensively identify factors associated with early overweight , the referencing databases of medline , pubmed , cochrane central register of controlled trials , and web of science were searched , through september 15 , 2011 , for studies indicating a relationship between early weight gain , overweight , or obesity and the aforementioned multifactorial categories among infants and toddlers with an average study age up to age 2 years . potentially modifiable factors associated with weight were defined as feeding and related dietary , environmental , or behavioral practices that could be potentially modified with interventions beginning at birth . genetic and biological factors were identified as genetic predisposition , epigenetic effects , and those within the prenatal environment . an a priori exclusion was established for studies not published in the english language , without test statistics or probability levels , with less than 10 subjects completing the study , or the primary study objective was not to specifically evaluate or describe factors associated with weight or adiposity status among full term the search strategy further applied individual and combinations of pertinent key words to the title and abstracts of referencing databases including : infant , toddler , overweight , obesity , prevention , weight , weight change , nutrition , diet , education , behavior change , and parenting practices to assess the pool of studies addressing potentially modifiable factors that may not have been secured with the more broad terms identified above . of the 6255 citations generated , those describing interventions or observational studies with focus on associations with weight , or reducing overweight or obesity in children younger than 2 years were selected and the introduction , discussion , and reference sections of each publication were read . the process of obtaining additional potential references was extended until no new studies were generated from these sources . a total of 143 publications were identified as meeting selection criteria of factors significantly associated to early weight gain , overweight or obesity in children through 2 years of age . due to the limited number of publications for some of the modifiable risk factor associations that surfaced , the search was subsequently extended to children with an average study age of 5 years . a summary of the genetic and biologic ( e.g. , prenatal ) , and potentially modifiable ( e.g. , feeding and related dietary , environmental , and behavioral ) factors associated with overweight within infants and young children is discussed within sections of the narrative review below . although strong evidence supports the role of yet non - modifiable genetic factors in early - onset obesity [ 28 , 29 ] , in and by themselves , these appear as an insufficient argument to support the increased prevalence in childhood obesity over the last three decades ; genetic factors alone can not account for the rapid secular increase . genetic polymorphism that increases the risk for obesity [ 30 , 31 ] may explain a small fraction of cases of childhood - onset obesity . however , in the majority children , obesity is attributed to the interaction between multiple genetic factors and an accommodating environment . continued integration of data from multiple sources of environment , genotype , and expression will help clarify obesity - related contributions from these areas [ 3335 ] . increasingly , it appears that epigenetic factors , heritable shifts in gene function , that do not involve changes in dna sequence , are gaining more attention as important factors associated with childhood obesity . genetic predispositions related to children 's weight , food intake , and dietary patterns are modulated by experience and significantly influenced by the environment , including the family environment . skidmore and colleagues suggested that an obesogenic postnatal environment is more important than the fetal environment for the development of obesity in female twins . even racial and ethnic differences in the prevalence of pediatric obesity may be partly explained by differences in potentially modifiable risk factors during early infancy . not surprisingly , parental weight status is a strong predictor of childhood obesity , as parents provide genes , environment , and a diet , within a context of their particular social and behavioral settings . children of overweight parents are at increased risk for development of obesity and those , up to approximately 5 years of age , with both parents being obese have been associated with a much greater risk ( ~10 fold ) for later obesity than those with one obese parent . although findings of an independent association with paternal weight and childhood weight status have been demonstrated , maternal weight status is consistently reported as one of the strongest correlations with their children 's weight . children of overweight mother 's are nearly 3 times as likely to be overweight as children with mothers of a healthy weight . obese women tend to have large babies , and large for their gestational age babies are at a higher risk of becoming obese children , though not all studies have confirmed this association [ 15 , 43 ] . in sum , genetic relationships are clearly associated with childhood obesity , but can not explain all cases of childhood overweight or obesity . it has long been recognized that the intrauterine environment can exert effects on the developing fetus that may have long - lasting influence on health and disease into adulthood . recent epidemiological studies , from populations worldwide , have confirmed that indicators of nutritional deficit in pregnancy , such as infant low birth weight , are associated with greater risk of the infant developing adult cardiovascular disease , hypertension , or type 2 diabetes mellitus , all of which have independent and positive association to weight . potential mechanisms for these developmental origins of disease are lacking , but abnormalities in the hypothalamic pituitary axis influencing metabolism , impaired fat oxidation , increased central fat distribution , and abnormal control of energy intake have been suggested [ 46 , 47 ] . prenatal exposure to maternal smoking during pregnancy increases risk for later obesity , and meta - analyses results confirmed that children whose mothers smoked during pregnancy were at elevated risk for overweight ( pooled adjusted odds ratio ( or ) 1.50 , 95% ci : 1.36 , 1.65 ) at ages 333 years , compared with children whose mothers did not smoke during pregnancy . prenatal exposure to other environmental toxins that may increase the risk of childhood obesity , such as endocrine disrupting chemicals are gaining attention as potential prenatal obesogenic factors . the intrauterine environment may also be a viable source of extra macronutrients that influence birth weight . infants that experience excess maternal gestational weight gain in utero , or that are born to mothers with diabetes , have an increased risk of being born large for their gestational age . these infants will also have a greater risk of becoming overweight , or of developing increased adiposity during their preschool [ 5053 ] , or school age years [ 5456 ] . although associations from observational studies described above are generally consistent with regard to infant or childhood risk of excess weight , causality has not unequivocally been established . nevertheless , these strong associations underscore the fact that maternal obesity , or excess maternal weight gain during pregnancy , along with its attendant endocrine and other biologic disruptions , may contribute to a generational perpetuation of the problem and argues for the potential of prenatal interventions to modify the fetal environment . while meritorious , it also appears clear that if modification of maternal dietary and environmental interventions were to be attempted , some of these potential preventive measures would need to begin prior to the earliest days of gestation . recent research has brought attention to specific parental feeding practices , commencing at birth , that may interact with genetic predispositions or prenatal covariates to inadvertently promote an obesogenic environment during infancy . these risk factors associated with early overweight or obesity have been identified in observational studies from both prospective and retrospective data review , with various population groups and sample sizes . studies of mother - infant dyads from birth , with measured length and weight during infancy or toddler years , reveal important associations . however , such reports are few . conclusions from observational studies of potentially modifiable factors associated with pediatric overweight or obesity in children up to age 5 years are presented on table 1 . results from several systematic reviews are consistent in demonstrating strong evidence of a positive association between rapid infancy weight gain and later risk of obesity [ 15 , 129 , 130 ] . of several body composition methods employed , or surrogate markers used for adiposity estimation , the association among rapid weight gain in infancy and later risk of overweight has been consistently noted from studies , worldwide [ 20 , 5759 , 131134 ] . for example , weight gain during the first 2 months of life , and from 2 to 9 months , measured by dual - energy x - ray absorptiometry , was recently demonstrated in the united states to be associated with fat mass , percentage of fat mass , and fat - free mass ratio in 10-year - old children . similarly , weight gain during the first 3 months of life , and between 312 months , was also positively correlated with who bmi z - scores at age 7 in china , and german infants that gained weight rapidly during the first 2 years of life had greater adiposity during preschool years than those with a normal rate of growth . additionally , increases in weight - for - length or bmi during the first 18 months of life were positively associated with higher bmi , subcutaneous adiposity and obesity at 3 - 4 years of age [ 13 , 14 , 57 ] . studies with skinfold thickness measurements , bioimpedance [ 17 , 19 ] , or a combination of methods [ 18 , 136 ] reveal similar findings that early growth patterns are critical periods for development of overweight . the more rapid and earlier an infant gains excess weight , the greater the likelihood for undesirable weight in subsequent months , and years . effect , rather than a specific obesogenic factor in early life , and the underlying mechanisms of rapid weight gain are unclear , this risk for latter obesity clearly signals the need for intervening in the early period of life . the potential to prevent early excess weight gain , through parental feeding and related practices , beginning at birth , is strong . breastfeeding is recognized as the ideal feeding for infants , due to its potential for health maintenance and disease prevention . among the multiple health benefits associated with breastfeeding , worldwide initiation rates of breastfeeding are currently near the who standard , yet globally only 34.6% of infants up to 6 months of age are exclusively breastfed , with values ranging from 43.2% within the who south - east asia region , to 17.7% among babies in the european region . the u.s . , healthy people 2020 objectives for exclusive breastfeeding through 3 and 6 months of age are 46% and 25% , respectively , yet nearly 25% to 42% of breastfed infants in some cohorts receive formula while still in the hospital ( before two days of age ) , decreasing the likelihood of development of a full milk supply by the mother . moreover , recent survey data indicate that 8% of mothers offered infants a combination of breastmilk and formula , from birth , during the first week of life . among infants provided both breast milk and formula , overall breastfeeding duration was lower than those offered no formula , and when compared to infants provided 4 months of exclusive breastfeeding , the infants provided the combination of breastmilk and formula , or only formula , were at increased risk of overweight or obesity between ages 2 and 6 years . numerous studies , and at least 5 meta - analyses and systematic reviews , have examined the role of breastfeeding in relation to childhood or later obesity [ 141145 ] . not all conclusions are in agreement with regard to the strength of the obesity protective effect of breastfeeding ; or , more correctly expressed , the increased risk of overweight with partial or full infant formula feeding in the first 46 months of life . however , the vast majority of studies show some degree of an inverse association between breastfeeding and risk of obesity . conclusions vary , primarily due to inconsistency with a definition of breastfeeding , particularly that of exclusive breastfeeding , as well as length of followup , definition of weight status , duration of breastfeeding , and confounding factors , which some analyses considered , and others did not . the statistical analyses procedures utilized to aggregate individual clinical trials may also explain differences in meta - analyses results . for example , a protective effect of breastfeeding on overweight ( binary data analysis ) has been reported by meta - analyses using logistic regression , whereas studies using linear regression and bmi ( continuous data analysis ) failed to detect meaningful significant associations . three of the five meta - analyses of observational studies found that obesity risk at school age was reduced by 1525% with when at least three confounding factors ( such as birth weight , parental weight , parental smoking , dietary factors , physical activity , or socioeconomic status ) were considered , results indicated that the likelihood of obesity was 22% lower among breastfed children , compared to those not breastfed . one review demonstrated a reduction in risk of obesity from 24% , before statistical adjustment for confounding variables , to a 7% reduced risk of later overweight after considering paternal weight status , smoking , and socioeconomic status . a separate meta - analysis determined that the risk of becoming overweight was reduced by 4% for each month of breastfeeding , yet the effect realized a plateau after nine months of breastfeeding . taken together , results from the five meta - analyses identified above , published in 20042006 , provide evidence that breastfeeding may have a relatively small , but consistent protective effect against obesity for children . newer observational studies and reviews provide additional support [ 59 , 6172 ] , and as exclusive breastfeeding becomes consistently defined among studies , and tracked among study participants with future reports , results may likely be stronger for a protective breastfeeding effect on overweight prevention . given available data , one recently released national guideline unequivocally concluded that babies who are breastfed are less likely to become overweight or obese compared to those not provided breastmilk . the importance of breastfeeding , and specifically , exclusive breastfeeding , that is , avoidance of infant formula for the first 6 months of life , is a public health message that can not be over emphasized . breastfed infants tend to be leaner and gain weight more slowly throughout infancy than formula - fed infants , particularly after 3 months of age . differences in weight gain during the first 12 months among breast fed and formula fed infants differed by 0.65 kg in one well matched study of 46 breastfed and 41 formula - fed infants ; length and head circumference values were similar among groups . increased intake of energy , protein , or both energy and protein in formula fed infants , compared to breast fed infants , have been proposed as a factor [ 148 , 149 ] . in addition , a stronger infant self - regulation of intake has been suggested as a plausible explanation for differences among caloric intake of breastfed and formula fed infants . breastfeeding is associated with other advantages for decreasing the risk overweight development , such as parent feeding practices of a lower frequency of introducing complementary foods at ages less than 4 months , and less frequently offering high fat or high sucrose foods to infants at 1 year , compared to mothers that formula fed their infants [ 140 , 151 ] . while causality remains to be demonstrated , interactions among many obesity preventative feeding strategies may coexist with breastfeeding . moreover , the finely regulated supply - and - demand relationship of milk production between the mother infant dyad may be disrupted with bottle feeding . consequently , reading and interpretation of hunger and satiety cues may be significantly affected . in summary although the exact subpopulation of infants most likely to benefit from obesity prevention effects has yet to be determined , numerous other health benefits of breastfeeding are indisputable . any intervention in an infant population aimed at potentially reducing the risk of obesity would by necessity include effective encouragement , establishment , and continuation of exclusive breastfeeding for at least the first 6 months of life . the who recommendation for introduction of complementary feeding at 6 months of age has been adopted by many countries . the american academy of pediatrics section on breastfeeding , and the american academy of pediatrics committee on nutrition recommend delaying the introduction of complementary foods to at least the age of 4 months , and preferably 6 months . age - appropriate complementary foods are recommended to be introduced as indicated by the individual child 's nutritional and developmental needs . although age and size often correspond with developmental readiness , these should not be used as sole considerations for deciding what and how to feed babies . provision of complementary food that is not synchronized to developmental milestones or to physiologic and immune readiness may be linked to allergies and digestive problems [ 140 , 155 ] , and early introduction of complementary foods ( e.g. , < 4 months ) has been positively associated with rate of weight gain during infancy , increased weight , or measures of adiposity in infants , toddlers , and preschool age children [ 69 , 7581 ] . estimates for the prevalence of parents providing complementary foods prior to guidelines for developmental readiness vary . one survey of approximately 3,000 parents , completed in 2002 , documented that 26% of infants were introduced to solid foods before 4 months of age , and a second survey , using a similar methodology and sample size in 2008 , indicated that approximately 1015% of parents introduced infants to complementary food prior to 4 months of age . although it appears from these studies that an overall improvement in the practice of providing early complementary feeding has occurred in recent years , some racial / ethnic groups of parents may still engage in this practice at high frequencies . teaching parents to identify the appropriate developmental readiness milestones , through an anticipatory guidance approach , may be useful in delaying the inappropriate introduction of complementary foods at an early age which has been associated with early or excessive weight gain . a classic study by stunkard and colleagues was one of the first to identify that energy intake contributes significantly to body weight and measures of adiposity at 1 year of age . the contribution of energy intake ( assessed via weighted food records ) , but not energy expenditure ( measure via indirect calorimetry for sleeping and total energy expenditure ) , was significant in the prediction of body size in the 78 infants at one year , after considering breastfeeding status , as well as maternal bmi . although not all studies have employed such sophisticated evaluation techniques , and some have reported neutral results for the association between bmi and energy intake within preschool age children [ 158 , 159 ] , several have reported that total energy intake is positively associated with risk or prevalence of overweight in infants , toddlers , and preschool age children [ 81 , 8385 ] . a higher dietary protein intake during infancy has also been positively associated with early weight gain during the first 2 years of life , although with correlations smaller than that for energy intake . randomized trials that compared growth of children fed formula of different protein concentrations showed either no association [ 129 , 130 , 160 ] , or a significant effect of increased protein on early infant weight gain [ 148 , 161 ] . given that formula contains more protein , and most have a slightly higher energy density than breastmilk , these findings could explain , in part , associations among formula fed infants being at higher risk for overweight than breastfed infants . when considering the total dietary protein intake , no association with later increased bmi ( at 6 - 7 years ) was reported at 6-month - old protein intake , but a higher protein intake at 12 months was independently related to increased measures of overweight in one cohort , and only in boys in another . it appears that protein intake during the transition to the family diet is a critical point for overweight risk . by replacing carbohydrate - rich foods ( fruit , vegetables ) of which are low in protein and also relatively low in calories , for other energy dense foods , diet quality will naturally change . such an alteration in macronutrient contribution and energy intake may be an effective approach to decreasing early childhood risk of obesity . some of the most recent comprehensive assessments of diet quality and quantity among infants and toddlers are the feeding infants and toddlers studies ( fitss ) . these 24-hour dietary intake surveys , conducted by phone interview in 2002 and 2008 with representative , cross - sectional samples ( n > 3000 ) of us parents and caregivers , in infants from birth to 48 months of age , provide detailed information on energy , nutrient intakes , and eating patterns of infants and young children [ 164 , 165 ] . results from these studies confirm that obesity associated dietary factors are highly prevalent in the u.s . infant population . in 2008 , infants participating in the fits survey , age birth to 5 months , consumed nearly 14% more calories ( 83 kcal / d ) than estimated needs , and average energy intakes remained above estimated requirements throughout the toddler years . assuming the excess energy from the recent fits study for the youngest infant category , and applying the first law of thermodynamics in its simplest form , after 6 weeks of 83 additional kcal / day , an estimated 0.45 kg weight gain would be possible , and after 6 months , an additional 1.9 kg of weight could be predicted . utilizing an average weight of 7.9 kg for a 6-month - old infant boy , the weight differential of 1.9 kg corresponds to the difference between a weight for age between the 50th percentile and the 98th percentile . high - caloric dessert foods such as cookies , cakes , candies or sweetened beverages were consumed by nearly 20% of the 69-month - old infants and by nearly 45% of 912 month old infants . at 1 year of age , approximately 55% of infants were consuming desserts , sweets , or sweetened beverages and by 15 months of age , two thirds of toddlers consumed this category of foods on the survey day at a level of intake that tended to remain constant throughout the toddler years . given the energy density of these foods , and frequency of consumption , they are likely contributing to the caloric intake to a substantial degree . consumption of sugar sweetened beverages has been positively related to measure of adiposity or overweight in toddler and preschool age children [ 84 , 8694 ] . specific data relating an independent association of sweetened beverage intake and weight measures are not available for infants , yet fits data confirm that even young infants frequently consume calories from the dessert , sweets , or sweetened beverage category . meta - analyses have established the degree to which sweetened beverage intakes in children contributes to obesity ; effect sizes range from a 0.03 to 0.08 unit change in bmi per 12 fl oz of soda per day , depending upon the length of followup , of which varied among studies . considering the contribution of approximately 90 calories from a 6 ounce serving of a regular cola - type beverage , reducing sweetened beverage consumption among children may have a measurable impact on weight status . children with high consumption or availability of fruit and/or vegetables consume less total energy and are associated with a more desirable body composition or body weight during preschool years [ 9599 ] . however , according to the 2008 fits data , approximately 37% of infants age 69 months and 28% of 912-month olds did not consume a single serving of vegetables on a given day . less than 15% of infants and toddlers consumed foods from the micronutrient rich , lower - calorie , dark green vegetable category on the study survey day ; instead , white potato , particularly fried potatoes , were the most frequently consumed vegetable of children aged 1215 months of age ( 18.5% ) and remained a daily dietary habit of many toddlers . of children aged 1 - 2 years , 33% were eating meals or snacks at a fast - food restaurant , on a given study day , which may explain the frequency of fried potato intake . the daily consumption of lower - calorie vegetables , within the infant and toddler population , could help provided a more balanced energy intake . the american academy of pediatrics recommends that infants less than six months of age should not be served juice . pureed , mashed , or whole fruit is appropriate for infants once complementary feeding begins , up to one year of age , and beyond . children aged 16 years should be limited to a total of four to six ounces of juice per day . in summary , the studies mentioned above , all point to specific examples of modifiable practices regarding complementary food introduction which may have greater effectiveness and efficacy impact in infancy than attempting modifications in diet patterns once these are established . for example , simple messages that educate parents and caregivers about healthy feeding and dietary habits for infants and toddlers include those that encourage a wide variety of nutritious foods , especially fruits and vegetables , in forms and times that are developmentally appropriate for the child [ 169 , 170 ] , and with frequency and persistence of offerings to generate acceptance . planning toddler 's snacks , of which contribute about 25% of a toddler 's daily energy intakes to complement meals by including fruits , vegetables and whole grains , rather than sugar - sweetened drinks and desert - type foods , as well as limiting exposure to fried fast food restaurant foods , all provide additional practical examples of modifiable healthy food practices that parents can foster . if provided the opportunity , infants and young toddlers will exert a relatively robust innate ability to regulate energy intake [ 172175 ] . however , innate self - regulation of caloric intake can be easily overridden by environmental factors , including well - meaning , yet misguided parent feeding practices . although a complex relationship has been proposed , correlational evidence is increasingly being documented linking parent feeding practices to infant or childhood weight status [ 104 , 105 , 176178 ] , even when considering several confounding variables . however , as described below , depending on the particular feeding practice , and age of the child , the directionality of the relationship varies . parental inattention to an infant 's hunger and satiety cues has been associated with weight gain at 4 - 5 months , predictive of weight gain at 612 months , and related to infant and toddler weight - for - length z scores . inadequate feeding practices associated with bottle feeding of infant formula , where hunger and satiety cues may require more attention to be recognized compared to breastfeeding , or the use of bottle feeding as a method for soothing infants , may also contribute to greater than expected energy intake in early life . lack of responsive parent feeding practices , such as inattention to a child 's hunger or satiety cues , has been positively associated with infant overfeeding or overweight [ 100103 ] . in contrast , responsive feeding , where the parents or caregivers recognize and respond to infant cues for hunger and satiety helps foster trust and appears to reduce potential overfeeding . feeding infants on cue , rather than on a schedule , they are in control of frequency and amount of feedings . as a consequence , very early attention and appropriate responsiveness by parents to hunger and satiety cues it is unlikely that parent feeding practices of low attention to infant cues for hunger and satiety are transient , or that children do not adapt to such by learning to overeat . at 2 years of age , children participating in laboratory tasks designed to assess their self - regulation skills that ranked lower on inhibitory control and higher on reward sensitivity skills were more likely to be overweight at 5 years of age than their counterparts . in addition , children ( 35 years ) in higher weight categories had lower satiety response and higher response to food cues , even after controlling for parental education and bmi . parental education about reading appropriate hunger cues presented by the infant and responding appropriately with timing , amount , and pacing of early feeding of nutritious food , as well as a parental acceptance of alternative soothing options , rather than immediate feeding , may be helpful to prevent overfeeding . maternal control during feeding at the weaning period or childhood years , and parental use of food as a solace or reward for their children , have been rather consistently implicated as factors associated with unhealthy eating or weight gain in children . offering food as a reward or punishment places undue importance on food and may have negative effects leading to obesity or poor eating behavior . when parental control is applied in a general atmosphere of involvement and parental warmth ( e.g. , authoritative parenting style from the child development literature ) , it has led to positive food choices by young children , healthy eating [ 184186 ] , and obesity prevention [ 176 , 187 ] . children are responsible for participating in choices about food selection ( within the healthy food options provided by the parent ) and should be allowed to take responsibility for determining how much is consumed at each eating occasion . using this approach , along with providing small portions of new foods and praising the child for eating healthy foods in contrast , parental restriction of children 's eating or restricting their access to food [ 25 , 106110 , 189193 ] , particularly if fueled by parental concern about overeating or child 's weight , has been frequently associated with childhood weight gain , particularly in older children . however , parental restriction did not predict change in a sample of preschool age children 's eating behavior , and a favorable influence of parental food restriction on the intake of energy - dense foods and snacks has been reported among toddlers . research to address the directionality of relationships among some parent feeding behaviors with infants and toddlers is needed . although associations between parental feeding practices and subsequent early childhood risk of overweight are well documented , due the nature of the observational research , a causal relation can not be concluded . it is difficult to discern if certain child factors evoke parenting feeding practices , or whether parent feeding practices influence these child factors . moreover , as reviewed by ventura and birch , and anzman and colleagues , the majority of research in the arena of parent feeding practices and childhood weight is cross - sectional , or performed in a feeding - laboratory setting . few studies have systematically examined whether an intervention program designed to educate parents on the potential role their infant or child feeding practices could play on the weight status of their young children are available . given the above findings , educational interventions on feeding practices with high responsiveness to hunger and satiety cues , and allowing for early self - regulation of food intake by the infant , are needed to curb the potential adverse effects that parent feeding practices can have on children 's innate ability to regulate energy intake . interventions for obesity prevention that do not address parenting approaches to feeding are unlikely to be successful . the american academy of pediatrics consensus statement on prevention and treatment of childhood obesity recommends that children 2 years old and younger should not be exposed to television , and children over age 2 should limit daily media exposure to only 1 - 2 hours of quality programming for tv viewing and screen use . in contrast to these recommendations , survey data show that by 3 months of age , approximately 40% of infants regularly watch videos , dvd 's , or television and 90% of children under age 2 watch television daily . moreover , these early screen - viewing patterns appear to persist into childhood . there is solid evidence that increased tv viewing and screen time has been associated with overweight , obesity , or adiposity in toddler and pre - school children within multiple cohorts and studies [ 5 , 87 , 91 , 114120 ] . the influence of tv and screen time watching on body weight is likely related to the sedentary nature of viewing and displacement of activity , yet also to the quality and quantity of foods consumed while watching tv , particularly during mealtimes . among toddler age children , tv viewing was associated with higher intakes of calories , sugar - sweetened beverages , fast food , and less fruit and vegetable consumption . tv watching during mealtime was also a strong predictor of toddler 's unhealthy food consumption , primarily mediated through their mother 's tv viewing of which affected her own food intake , in turn , influencing the toddler 's food consumption pattern . tv viewing has been associated with foods consumed by school age children , including more pizza , salty snacks , fast foods , and soda , and fewer vegetables or whole grains while watching tv during mealtimes , compared to meals consumed without tv [ 204 , 205 ] . in addition to limits for tv , daily physical activity can prevent a rapid gain in weight which leads to obesity early in life . several studies have documented an inverse association with time spent during active play / physical activity and measures of adiposity or risk of overweight among toddler and preschool age children [ 5 , 78 , 94 , 117 , 118 , 121 ] . although some experts recommend that infants should have supervised tummy time every day when they are awake and that confining infant equipment such as swings , infant seats ( e.g. , bouncers ) , if used , should only be allowed for short periods of time , data to evaluate the prevalence of these behaviors at home , or in infant care settings , and their association to overweight , obesity , or rapid weight gain in infancy are not available . parents may need education and encouragement to provide a least restrictive environment to foster active play time for their young infants and opportunities for gross motor activity [ 207210 ] . early infancy and childhood interventions geared to risk reduction of overweight and obesity should include education on risks associated to screen time , and on physically active alternatives to screen time to encourage motor development in young children . since an early report in 1992 , suggesting that short sleep duration is associated with childhood obesity , multiple observational studies have documented an inverse relationship between sleep duration and measures of adiposity , overweight , or obesity with infant , toddler , and the preschool age children [ 116 , 119 , 122127 ] . recently , shorter sleep duration during infancy ( < 12 hours / day ) has been associated with higher bmi z score , skinfold measures , and increased odds of overweight in 3-year - old children . infants of whom do not sleep for at least 6 hours nightly by age 5 months have a greater risk of short nocturnal sleep duration later in childhood [ 212 , 213 ] and one longitudinal study of sleep in children reported that sleep duration for age , compared with thus , it appears , albeit from limited data , that sleep duration during infancy sets the stage for sleeping patterns throughout childhood . mechanisms to help explain the relationship between sleep and overweight are based on both physiological and behavioral findings . biochemical markers are limited for infants and young children ; however , sleep restriction in adults has been associated with increases in the appetite stimulating peptide , ghrelin , and a reduction in the anorexigenic hormone , leptin [ 215217 ] . although one study with infants identified that lower cord blood ghrelin was associated with slower weight gain from 0-to-3-month - old infants , confirmation from larger studies of infants , addressing the influence of confounding variables , is not currently available . parental feeding practices , related to infant sleeping , may have a strong impact on early and rapid weight gain . the possibility that food , especially bottle feeding and early introduction of complementary food used by parents as an approach to calm an overtired - fussy infant [ 100 , 219221 ] or as a sleep aid for infants may be part of the explanation for these associations . recent interventions that promote the use of alternative approaches to feeding for soothing a fussy infant or increasing nocturnal sleep duration have reported encouraging results [ 222 , 223 ] . given findings of observational studies indicating sleep duration , particularly that nocturnal sleep duration during infancy and early childhood is associated with pediatric obesity , addressing this variable as part of a multicomponent pediatric prevention program for healthy weight has been recommended . families that regularly eat meals together are likely to positively impact the nutritional health and weight in the children . meta - analysis of 17 studies of children ( age 2.8 years and older ) that examined children 's weight status , food consumption , and eating patterns identified a 12% reduction in odds of pediatric overweight ( e.g. , > 85th percentile ) with a family meal frequency of 3 or more meals together per week . the majority of studies included children of adolescent age ; however , when age was tested as a potential moderator , it was found to be non - significant . similarly , cross - sectional analysis of a nationally representative sample of ~8550 4-year - old children reported that children of whom engaged in eating family meals at least 5 or more evenings per week were at a 16% decreased risk of obesity ( > 95th percentile ) , compared to those consuming fewer family meals together . although there is little evidence specifically linking family meal participation of older infants and toddlers to obesity , high rates of infants eating outside the home , in fast food store restaurants , have been reported . family meal time may be a contributor to pediatric overweight for preschool age and older children , and fostering this practice in younger children may have broad benefits , and is unlikely to cause harm . in summary of the aforementioned potentially modifiable parent feeding and related practices that correlate with pediatric obesity , some within the very youngest infants , a fully causal relationship remains to be established . prospective and well - controlled interventional studies , offering a practical and generalizable way to address the feeding and parent related practices associated with childhood obesity are urgently needed to address the obesity epidemic . a great deal is known of the multiple factors associated to obesity , including those associating factors in infancy to obesity , but the documentation of causality remains unclear . the time for additional observational studies is passing us by , and research of interventions that may be efficacious to change the course of the epidemic is overdue . however , to date , few studies have examined the efficacy of interventions for obesity prevention that target infant populations . only three recent published studies with results of the efficacy of assessing specific interventions for prevention of overweight in early infancy could be identified . as described below , results are mixed . , with 110 mother - infant dyads recently reported positive results for their two - component intervention in preventing overweight during infancy among primarily breastfed infants . interventions were provided by home - nurse visits , consisting of educational content addressing alternative strategies to feeding for soothing a fussy baby , complementary feeding information , a feeding demonstration , and guidance on recognizing hunger and fullness cues . in this study , infants who were provided a soothe / sleep education intervention at 2 - 3 weeks of life , and introduction to solid food education between 46 months of life , realized a significantly lower weight - for - length percentile at one year of age . despite study limitations of a relatively small sample size , results indicated that the intervention was effective in helping infants achieve healthy growth , likely through the influence of increases in nocturnal sleep duration , delayed introduction of solid foods , and increases in consumption of vegetable foods . a second recently published pilot study included 80 infants enrolled during the first week of life , and their post - partum mothers , to assess impact of an education program on infant feeding , sleep duration , tv viewing and mothers ' responsiveness to their infants satiety cues . in addition , the intervention aimed to influence the mother 's postpartum diet , activity , tv , and sleep behaviors . after 6 months of brief pediatrician messages , motivational interviewing / coaching by health educators , and group parenting workshops , significantly fewer intervention infants had been introduced to solid foods , compared to the usual care - control group . intervention infants viewed less tv , had larger increases in nocturnal sleep duration , and required less settling time than infants provided usual care . no significant differences in infant weight status were detected ; however , trends indicated a lower change in weight - for - length z scores and fewer infants were found in the highest quartiles of weight - for - length among intervention , compared to control , infants . although this nonrandomized , pediatrician - based , intervention program was not directly effective in influencing maternal postpartum behaviors regarding their own weight , the multicomponent intervention tended to improve infant weight related behaviors employed by the mothers . a third double - blind , randomized educational intervention study enrolled 310-week - old , exclusively formula - fed infants . the educational intervention consisted of one session that focused on recognizing signs of infant satiety and limiting formula volume to no more than 6 oz per bottle . no difference among intervention ( n = 44 ) and control infants ( n = 57 ) with regard to weight gain , formula intake , or parental behavior was realized when assessed at 4 months . although a limited number of potentially modifiable factors associated with early childhood overweight have been identified within the published literature , interventions to examine the impact of these factors on prevention of overweight have not been adequately studied . while comprehensive approaches dealing with all aspects of the global epidemic , at all ages are important and necessary , including pre gestational interventions in fertile women , the first two years of life provide a potential window of opportunity like no other , to establish feeding , dietary , and behavioral patterns that remain with an individual throughout life . randomized controlled trials for obesity prevention in infants that address multiple intervention components are on the horizon , and study protocols have been published by at least three groups in australia [ 7 , 227 , 228 ] , one in london , one in italy , and another in the usa . to date , one study in australia , and another in new zealand targets infants from birth ; participant acceptance of the former intervention is reported as positive , and results of impact on obesity prevention are pending . another exploratory , randomized controlled trial is currently underway in the united kingdom with infants under 18 months , and aims to prevent further obesity in high risk infants ( those with weight > 95th percentile , or a preschool age sibling with obesity , or a mother with obesity ) . preliminary results , published as qualitative pilot data , suggest that this multicomponent intervention may serve as a potentially effective approach for obesity prevention within the high risk weaning age child . causal relationships among potentially modifiable factors and overweight during infancy and young childhood have yet to be firmly established . the potential implementation of a successful intervention strategy to help identify which intervention components are most effective would have meaningful effects on public health and public policy . the prevalence of obesity in infants and toddlers , and its long - term weight status consequences , points to the necessity , as well as the potential for interventions which focus on this age group . clearly , there are genetic , biologic , and prenatal factors that will need to be addressed , some of which may not be modifiable , others which may come about more slowly . obesity prevention strategies encouraging a healthy rate of weight gain in the early months of life , breastfeeding exclusivity , timely introduction of nutrient dense , energy appropriate foods , adequate infant sleep , and attention to activity / screen viewing , provided to infants and young children with responsive parental feeding practices in a family meal setting are suggested . few studies have proposed a comprehensive nutritionally and developmentally appropriate intervention , starting at birth , designed to promote healthy dietary intake , an appropriate early growth trajectory , and a long - term weight status that is consistent with public health recommendations . given the apparent ontogenetic progression , the metabolic programming plasticity , and the behavioral modeling pliability of infancy , this may be the most critical and potentially efficacious window of opportunity available for true prevention of obesity in the general population . interventions within the very early months and years of life are critical times for addressing potentially modifiable factors for overweight and obesity prevention .

childhood obesity is currently one of the most prevailing and challenging public health issues among industrialized countries and of international priority . the global prevalence of obesity poses such a serious concern that the world health organization ( who ) has described it as a global epidemic . recent literature suggests that the genesis of the problem occurs in the first years of life as feeding patterns , dietary habits , and parental feeding practices are established . obesity prevention evidence points to specific dietary factors , such as the promotion of breastfeeding and appropriate introduction of nutritious complementary foods , but also calls for attention to parental feeding practices , awareness of appropriate responses to infant hunger and satiety cues , physical activity / inactivity behaviors , infant sleep duration , and family meals . interventions that begin at birth , targeting multiple factors related to healthy growth , have not been adequately studied . due to the overwhelming importance and global significance of excess weight within pediatric populations , this narrative review was undertaken to summarize factors associated with overweight and obesity among infants and toddlers , with focus on potentially modifiable risk factors beginning at birth , and to address the need for early intervention prevention .

1. Introduction 2. Aims and Methods of Review 3. Literature Search Results 4. Genetic and Biological Factors Associated with Infant and Childhood Overweight 5. Potentially Modifiable Factors Associated with Infant and Childhood Overweight 6. Evidence for Obesity Prevention Strategies with Infants 7. Upcoming Directions 8. Conclusion

worldwide , an estimated twenty - two million children 5 years of age were overweight in 2007 and at present , approximately 1020% of infants and toddlers in the united states ( us ) are overweight , ( 85th body mass index ( bmi ) percentile for age ) , values similar to those reported for young children in multiple countries [ 59 ] . equally concerning is the finding that nearly 10% of infants and toddlers , from birth2 years are also obese , at or above the 95th percentile of the weight for recumbent length growth charts . longitudinal data from 762 infants and children ( age 018 years ) indicated that body weight as early as 2 years of age begins a positive tracking period for adult overweight ; weight status from 26 years was the most critical growth period for prediction and realization of adult overweight . the earlier a child becomes overweight , and the longer excess weight is maintained , the greater the risk that the child 's overweight will follow into adulthood . critical periods for establishing dietary intake patterns , eating habits , and food preferences begin in infancy , and although inconsistent , may be set as early as 2 years of age [ 22 , 23 ] . as the infant progresses to table foods , the family diet exerts a strong influence on food consumption consistent with a general adoption of the eating practices of the family . parental feeding practices , if not causative for weight status in young children , are strongly associated with body weight and healthy food choices throughout childhood . research suggests that interventions to adjust food composition or caloric intake and increase physical activity , especially after infancy , have a small impact on children 's weight or measures of adiposity [ 26 , 27 ] and have been inadequate in curtailing the increased prevalence of overweight affecting young children . the primary objective of this narrative review is to discuss risk factors associated with early childhood overweight , with emphasis on current evidence that supports specific modifiable risk factors for obesity prevention within very young children . a review of the available , but few , interventions designed to prevent overweight and obesity within the infant age population is included . it is hoped that this review will contribute to the development of future strategies , policy , and practices consistent with the public health need for an early life obesity prevention plan . antecedents of early childhood obesity are clearly multifactorial , and associations of varying strength have been documented for genetic , biologic , dietary , environmental , and behavioral factors . to comprehensively identify factors associated with early overweight , the referencing databases of medline , pubmed , cochrane central register of controlled trials , and web of science were searched , through september 15 , 2011 , for studies indicating a relationship between early weight gain , overweight , or obesity and the aforementioned multifactorial categories among infants and toddlers with an average study age up to age 2 years . potentially modifiable factors associated with weight were defined as feeding and related dietary , environmental , or behavioral practices that could be potentially modified with interventions beginning at birth . an a priori exclusion was established for studies not published in the english language , without test statistics or probability levels , with less than 10 subjects completing the study , or the primary study objective was not to specifically evaluate or describe factors associated with weight or adiposity status among full term the search strategy further applied individual and combinations of pertinent key words to the title and abstracts of referencing databases including : infant , toddler , overweight , obesity , prevention , weight , weight change , nutrition , diet , education , behavior change , and parenting practices to assess the pool of studies addressing potentially modifiable factors that may not have been secured with the more broad terms identified above . of the 6255 citations generated , those describing interventions or observational studies with focus on associations with weight , or reducing overweight or obesity in children younger than 2 years were selected and the introduction , discussion , and reference sections of each publication were read . due to the limited number of publications for some of the modifiable risk factor associations that surfaced , the search was subsequently extended to children with an average study age of 5 years . , feeding and related dietary , environmental , and behavioral ) factors associated with overweight within infants and young children is discussed within sections of the narrative review below . however , in the majority children , obesity is attributed to the interaction between multiple genetic factors and an accommodating environment . increasingly , it appears that epigenetic factors , heritable shifts in gene function , that do not involve changes in dna sequence , are gaining more attention as important factors associated with childhood obesity . even racial and ethnic differences in the prevalence of pediatric obesity may be partly explained by differences in potentially modifiable risk factors during early infancy . children of overweight parents are at increased risk for development of obesity and those , up to approximately 5 years of age , with both parents being obese have been associated with a much greater risk ( ~10 fold ) for later obesity than those with one obese parent . recent epidemiological studies , from populations worldwide , have confirmed that indicators of nutritional deficit in pregnancy , such as infant low birth weight , are associated with greater risk of the infant developing adult cardiovascular disease , hypertension , or type 2 diabetes mellitus , all of which have independent and positive association to weight . potential mechanisms for these developmental origins of disease are lacking , but abnormalities in the hypothalamic pituitary axis influencing metabolism , impaired fat oxidation , increased central fat distribution , and abnormal control of energy intake have been suggested [ 46 , 47 ] . prenatal exposure to other environmental toxins that may increase the risk of childhood obesity , such as endocrine disrupting chemicals are gaining attention as potential prenatal obesogenic factors . recent research has brought attention to specific parental feeding practices , commencing at birth , that may interact with genetic predispositions or prenatal covariates to inadvertently promote an obesogenic environment during infancy . these risk factors associated with early overweight or obesity have been identified in observational studies from both prospective and retrospective data review , with various population groups and sample sizes . conclusions from observational studies of potentially modifiable factors associated with pediatric overweight or obesity in children up to age 5 years are presented on table 1 . for example , weight gain during the first 2 months of life , and from 2 to 9 months , measured by dual - energy x - ray absorptiometry , was recently demonstrated in the united states to be associated with fat mass , percentage of fat mass , and fat - free mass ratio in 10-year - old children . similarly , weight gain during the first 3 months of life , and between 312 months , was also positively correlated with who bmi z - scores at age 7 in china , and german infants that gained weight rapidly during the first 2 years of life had greater adiposity during preschool years than those with a normal rate of growth . additionally , increases in weight - for - length or bmi during the first 18 months of life were positively associated with higher bmi , subcutaneous adiposity and obesity at 3 - 4 years of age [ 13 , 14 , 57 ] . effect , rather than a specific obesogenic factor in early life , and the underlying mechanisms of rapid weight gain are unclear , this risk for latter obesity clearly signals the need for intervening in the early period of life . the potential to prevent early excess weight gain , through parental feeding and related practices , beginning at birth , is strong . among the multiple health benefits associated with breastfeeding , worldwide initiation rates of breastfeeding are currently near the who standard , yet globally only 34.6% of infants up to 6 months of age are exclusively breastfed , with values ranging from 43.2% within the who south - east asia region , to 17.7% among babies in the european region . moreover , recent survey data indicate that 8% of mothers offered infants a combination of breastmilk and formula , from birth , during the first week of life . numerous studies , and at least 5 meta - analyses and systematic reviews , have examined the role of breastfeeding in relation to childhood or later obesity [ 141145 ] . not all conclusions are in agreement with regard to the strength of the obesity protective effect of breastfeeding ; or , more correctly expressed , the increased risk of overweight with partial or full infant formula feeding in the first 46 months of life . conclusions vary , primarily due to inconsistency with a definition of breastfeeding , particularly that of exclusive breastfeeding , as well as length of followup , definition of weight status , duration of breastfeeding , and confounding factors , which some analyses considered , and others did not . three of the five meta - analyses of observational studies found that obesity risk at school age was reduced by 1525% with when at least three confounding factors ( such as birth weight , parental weight , parental smoking , dietary factors , physical activity , or socioeconomic status ) were considered , results indicated that the likelihood of obesity was 22% lower among breastfed children , compared to those not breastfed . the importance of breastfeeding , and specifically , exclusive breastfeeding , that is , avoidance of infant formula for the first 6 months of life , is a public health message that can not be over emphasized . breastfeeding is associated with other advantages for decreasing the risk overweight development , such as parent feeding practices of a lower frequency of introducing complementary foods at ages less than 4 months , and less frequently offering high fat or high sucrose foods to infants at 1 year , compared to mothers that formula fed their infants [ 140 , 151 ] . consequently , reading and interpretation of hunger and satiety cues may be significantly affected . any intervention in an infant population aimed at potentially reducing the risk of obesity would by necessity include effective encouragement , establishment , and continuation of exclusive breastfeeding for at least the first 6 months of life . the american academy of pediatrics section on breastfeeding , and the american academy of pediatrics committee on nutrition recommend delaying the introduction of complementary foods to at least the age of 4 months , and preferably 6 months . provision of complementary food that is not synchronized to developmental milestones or to physiologic and immune readiness may be linked to allergies and digestive problems [ 140 , 155 ] , and early introduction of complementary foods ( e.g. , < 4 months ) has been positively associated with rate of weight gain during infancy , increased weight , or measures of adiposity in infants , toddlers , and preschool age children [ 69 , 7581 ] . teaching parents to identify the appropriate developmental readiness milestones , through an anticipatory guidance approach , may be useful in delaying the inappropriate introduction of complementary foods at an early age which has been associated with early or excessive weight gain . although not all studies have employed such sophisticated evaluation techniques , and some have reported neutral results for the association between bmi and energy intake within preschool age children [ 158 , 159 ] , several have reported that total energy intake is positively associated with risk or prevalence of overweight in infants , toddlers , and preschool age children [ 81 , 8385 ] . a higher dietary protein intake during infancy has also been positively associated with early weight gain during the first 2 years of life , although with correlations smaller than that for energy intake . some of the most recent comprehensive assessments of diet quality and quantity among infants and toddlers are the feeding infants and toddlers studies ( fitss ) . high - caloric dessert foods such as cookies , cakes , candies or sweetened beverages were consumed by nearly 20% of the 69-month - old infants and by nearly 45% of 912 month old infants . given the energy density of these foods , and frequency of consumption , they are likely contributing to the caloric intake to a substantial degree . less than 15% of infants and toddlers consumed foods from the micronutrient rich , lower - calorie , dark green vegetable category on the study survey day ; instead , white potato , particularly fried potatoes , were the most frequently consumed vegetable of children aged 1215 months of age ( 18.5% ) and remained a daily dietary habit of many toddlers . for example , simple messages that educate parents and caregivers about healthy feeding and dietary habits for infants and toddlers include those that encourage a wide variety of nutritious foods , especially fruits and vegetables , in forms and times that are developmentally appropriate for the child [ 169 , 170 ] , and with frequency and persistence of offerings to generate acceptance . parental inattention to an infant 's hunger and satiety cues has been associated with weight gain at 4 - 5 months , predictive of weight gain at 612 months , and related to infant and toddler weight - for - length z scores . inadequate feeding practices associated with bottle feeding of infant formula , where hunger and satiety cues may require more attention to be recognized compared to breastfeeding , or the use of bottle feeding as a method for soothing infants , may also contribute to greater than expected energy intake in early life . lack of responsive parent feeding practices , such as inattention to a child 's hunger or satiety cues , has been positively associated with infant overfeeding or overweight [ 100103 ] . in contrast , responsive feeding , where the parents or caregivers recognize and respond to infant cues for hunger and satiety helps foster trust and appears to reduce potential overfeeding . as a consequence , very early attention and appropriate responsiveness by parents to hunger and satiety cues it is unlikely that parent feeding practices of low attention to infant cues for hunger and satiety are transient , or that children do not adapt to such by learning to overeat . parental education about reading appropriate hunger cues presented by the infant and responding appropriately with timing , amount , and pacing of early feeding of nutritious food , as well as a parental acceptance of alternative soothing options , rather than immediate feeding , may be helpful to prevent overfeeding . maternal control during feeding at the weaning period or childhood years , and parental use of food as a solace or reward for their children , have been rather consistently implicated as factors associated with unhealthy eating or weight gain in children . , authoritative parenting style from the child development literature ) , it has led to positive food choices by young children , healthy eating [ 184186 ] , and obesity prevention [ 176 , 187 ] . research to address the directionality of relationships among some parent feeding behaviors with infants and toddlers is needed . although associations between parental feeding practices and subsequent early childhood risk of overweight are well documented , due the nature of the observational research , a causal relation can not be concluded . moreover , as reviewed by ventura and birch , and anzman and colleagues , the majority of research in the arena of parent feeding practices and childhood weight is cross - sectional , or performed in a feeding - laboratory setting . given the above findings , educational interventions on feeding practices with high responsiveness to hunger and satiety cues , and allowing for early self - regulation of food intake by the infant , are needed to curb the potential adverse effects that parent feeding practices can have on children 's innate ability to regulate energy intake . early infancy and childhood interventions geared to risk reduction of overweight and obesity should include education on risks associated to screen time , and on physically active alternatives to screen time to encourage motor development in young children . since an early report in 1992 , suggesting that short sleep duration is associated with childhood obesity , multiple observational studies have documented an inverse relationship between sleep duration and measures of adiposity , overweight , or obesity with infant , toddler , and the preschool age children [ 116 , 119 , 122127 ] . recently , shorter sleep duration during infancy ( < 12 hours / day ) has been associated with higher bmi z score , skinfold measures , and increased odds of overweight in 3-year - old children . biochemical markers are limited for infants and young children ; however , sleep restriction in adults has been associated with increases in the appetite stimulating peptide , ghrelin , and a reduction in the anorexigenic hormone , leptin [ 215217 ] . parental feeding practices , related to infant sleeping , may have a strong impact on early and rapid weight gain . the possibility that food , especially bottle feeding and early introduction of complementary food used by parents as an approach to calm an overtired - fussy infant [ 100 , 219221 ] or as a sleep aid for infants may be part of the explanation for these associations . given findings of observational studies indicating sleep duration , particularly that nocturnal sleep duration during infancy and early childhood is associated with pediatric obesity , addressing this variable as part of a multicomponent pediatric prevention program for healthy weight has been recommended . although there is little evidence specifically linking family meal participation of older infants and toddlers to obesity , high rates of infants eating outside the home , in fast food store restaurants , have been reported . prospective and well - controlled interventional studies , offering a practical and generalizable way to address the feeding and parent related practices associated with childhood obesity are urgently needed to address the obesity epidemic . a great deal is known of the multiple factors associated to obesity , including those associating factors in infancy to obesity , but the documentation of causality remains unclear . the time for additional observational studies is passing us by , and research of interventions that may be efficacious to change the course of the epidemic is overdue . despite study limitations of a relatively small sample size , results indicated that the intervention was effective in helping infants achieve healthy growth , likely through the influence of increases in nocturnal sleep duration , delayed introduction of solid foods , and increases in consumption of vegetable foods . a second recently published pilot study included 80 infants enrolled during the first week of life , and their post - partum mothers , to assess impact of an education program on infant feeding , sleep duration , tv viewing and mothers ' responsiveness to their infants satiety cues . after 6 months of brief pediatrician messages , motivational interviewing / coaching by health educators , and group parenting workshops , significantly fewer intervention infants had been introduced to solid foods , compared to the usual care - control group . intervention infants viewed less tv , had larger increases in nocturnal sleep duration , and required less settling time than infants provided usual care . although a limited number of potentially modifiable factors associated with early childhood overweight have been identified within the published literature , interventions to examine the impact of these factors on prevention of overweight have not been adequately studied . while comprehensive approaches dealing with all aspects of the global epidemic , at all ages are important and necessary , including pre gestational interventions in fertile women , the first two years of life provide a potential window of opportunity like no other , to establish feeding , dietary , and behavioral patterns that remain with an individual throughout life . randomized controlled trials for obesity prevention in infants that address multiple intervention components are on the horizon , and study protocols have been published by at least three groups in australia [ 7 , 227 , 228 ] , one in london , one in italy , and another in the usa . to date , one study in australia , and another in new zealand targets infants from birth ; participant acceptance of the former intervention is reported as positive , and results of impact on obesity prevention are pending . another exploratory , randomized controlled trial is currently underway in the united kingdom with infants under 18 months , and aims to prevent further obesity in high risk infants ( those with weight > 95th percentile , or a preschool age sibling with obesity , or a mother with obesity ) . the prevalence of obesity in infants and toddlers , and its long - term weight status consequences , points to the necessity , as well as the potential for interventions which focus on this age group . obesity prevention strategies encouraging a healthy rate of weight gain in the early months of life , breastfeeding exclusivity , timely introduction of nutrient dense , energy appropriate foods , adequate infant sleep , and attention to activity / screen viewing , provided to infants and young children with responsive parental feeding practices in a family meal setting are suggested . few studies have proposed a comprehensive nutritionally and developmentally appropriate intervention , starting at birth , designed to promote healthy dietary intake , an appropriate early growth trajectory , and a long - term weight status that is consistent with public health recommendations . given the apparent ontogenetic progression , the metabolic programming plasticity , and the behavioral modeling pliability of infancy , this may be the most critical and potentially efficacious window of opportunity available for true prevention of obesity in the general population . interventions within the very early months and years of life are critical times for addressing potentially modifiable factors for overweight and obesity prevention .

diabetic retinopathy ( dr ) , a vision - threatening disease , has classically been regarded as a disease of the retinal microvasculature and a consequence of vascular cell damage . however , recent studies proved that neurodegeneration and impaired visual function are initiated early after the onset of diabetes and progress independently of the vascular lesions [ 14 ] . however , the molecular mechanisms underlying the diabetes - induced retinal neurodegeneration and dysfunction are still not well understood . high - mobility group box-1 ( hmgb1 ) is a nonhistone dna - binding nuclear protein that has been implicated in diverse intracellular functions , including the stabilization of nucleosomal structures and the facilitation of gene transcription . necrotic cell death can result in passive leakage of hmgb1 from the cell as the protein is then no longer bound to dna . in addition , hmgb1 can be actively secreted by different cell types , including activated monocytes and macrophages , mature dendritic cells , natural killer cells , and endothelial cells . extracellular hmgb1 functions as a proinflammatory cytokine and triggers the inflammatory response through the activation of multiple receptors such as the receptor for advanced glycation end products ( rage ) , toll - like receptor-2 ( tlr2 ) , and tlr4 leading to activation of the transcription factors extracellular signal - regulated kinase 1 and 2 ( erk1/2 ) and nuclear factor kappa b ( nf-b ) , which may alter gene transcription and lead to the upregulation of proinflammatory cytokines , chemokines , and adhesion molecules and intensifies cellular oxidative stress [ 510 ] , processes that may play a role in the pathogenesis of diabetic retinal neurodegeneration and dysfunction . strong evidence indicates that chronic , low - grade inflammation is implicated in the pathogenesis of dr [ 11 , 12 ] . recently , it was demonstrated that hmgb1 is the main mediator bridging persistent neuroinflammation and chronic progressive dopaminergic neurodegeneration in neurodegenerative diseases , such as parkinson 's disease . recently , hmgb1 has received particular attention with respect to its pathological role in cerebral ischemia . after ischemic injury induced by transient middle cerebral artery occlusion in mice and rats , hmgb1 was found to be translocated into the cytoplasmic compartment from nuclei and released into the extracellular space from neurons [ 1418 ] . in these studies , extracellular hmgb1 plays a key role in the development of neuronal injury through microglial activation , induction of apoptosis , excitatory amino acid release , and induction of proinflammatory mediators [ 1419 ] . downregulation of hmgb1 or treatment with neutralizing anti - hmgb1 monoclonal antibody remarkably suppressed infarct size , activation of microglia , and induction of proinflammatory markers and inhibited the increased permeability of the blood - brain barrier [ 14 , 18 ] . in previous studies , we demonstrated that hmgb1 and rage were expressed by vascular endothelial cells and stromal cells in fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy ( pdr ) . in addition , we demonstrated increased levels of hmgb1 in the vitreous samples from patients with pdr and that there were significant positive correlations between the vitreous levels of hmgb1 and the levels of the inflammatory biomarkers [ 2022 ] . furthermore , we demonstrated that diabetes induced significant upregulation of the expression of hmgb1 and rage in the retinas of rats and mice and that intravitreal administration of hmgb1 to normal rats induced activation of inflammatory signaling pathways in the retina and increased retinal vascular permeability [ 21 , 23 ] . glycyrrhizin ( ga ) , an ingredient of the licorice roots , has long been known to exhibit glucocorticoid - like anti - inflammatory actions by inhibiting 11-hydroxysteroid dehydrogenase . more recently , ga has also been shown to bind to and inhibit chemoattractant , mitogenic , and cytokine - like activities of hmgb1 . in this study , we explored the hypothesis that hmgb1 plays a pathogenetic role in mediating diabetes - induced retinal neuropathy . to test this hypothesis , we investigated the expression of the neurodegeneration mediators and markers cleaved caspase-3 , synaptophysin , tyrosine hydroxylase ( th ) , glutamine synthetase ( gs ) , glutamate , and glyoxalase 1 ( glo 1 ) in the retinas of diabetic rats and in the retinas of normal rats after intravitreal administration of hmgb1 . in addition , we analyzed whether constant ga intake suppresses retinal neuropathy induced by diabetes in rats . all procedures with animals were performed in accordance with the association for research in vision and ophthalmology ( arvo ) statement for use of animals in ophthalmic and vision research and were approved by the institutional animal care and use committee of the college of pharmacy , king saud university . adult male sprague dawley rats of 8 - 9 weeks of age ( 200220 g ) were overnight fasted and streptozotocin ( stz ) 55 mg / kg in 10 mm sodium citrate buffer , ph 4.5 ( sigma , st . rats were considered diabetic if their blood glucose was greater than 250 mg / dl . diabetic rats were divided into 2 groups : the rats in group i received normal drinking water without any supplementation , and group ii received drinking water supplemented with glycyrrhizic acid ( 150 mg / kg / day , santa cruz biotechnology , inc . , santa cruz , ca ) immediately after establishment of diabetes throughout the course of the experiment . after 4 weeks of diabetes , the rats were euthanized by an overdose of chloral hydrate , the eyes were removed , and retina was isolated and frozen immediately in liquid nitrogen and stored at 80c to be analyzed by western blot analysis or biochemical assay . sprague dawley rats ( 200220 g ) were kept under deep anesthesia , and sterilized solution of recombinant hmgb1 ( 5 ng/5 l ; r&d systems , minneapolis , mn ) was injected into the vitreous of the right eye as previously described by us . for the control , the left eye received 5 l of sterile phosphate buffer saline ( pbs ) . the animals were sacrificed 4 days after intravitreal administration , and the retinas were carefully dissected , snap - frozen in liquid nitrogen , and stored at 80c to be analyzed by western blot analysis or biochemical assay . the breakdown of blood - retina barrier induced by intravitreal injection of hmgb1 might lead to raised levels of hmgb1 in the serum . therefore , we determined the levels of hmgb1 in equal amounts of serum from intravitreally injected rats and normal rats using western blot analysis . retinas were homogenized in a western lysis buffer ( 30 mm tris - hcl ; ph 7.4 , 250 mm na3vo4 , 5 mm edta , 250 mm sucrose , 1% triton x-100 with protease inhibitor ) . the lysate was centrifuged at 14,000 g for 10 min at 4c , and the supernatant was collected . protein content was assayed by dc protein assay ( bio - rad laboratories , hercules , ca ) . the tissue lysate containing 4050 g of protein was separated on 10% or 12% sds - polyacrylamide gels and was transferred onto nitrocellulose membranes . the blots were blocked with 5% nonfat milk in tbst ( 20 mm tris - hcl , ph 7.6 , 136 mm nacl , and 0.1% tween-20 ) . for detection of hmgb1 , phospho - erk1/2 , synaptophysin , cleaved caspase-3 , th , glo1 , and gs , the membrane was incubated overnight at 4c with rabbit polyclonal anti - hmgb1 ( 1 : 1000 , cat . number ab18256 , abcam , uk ) , rabbit monoclonal anti - phospho - erk1/2 ( 0.5 g / ml , cat . number mab1018 , r&d systems ) , mouse monoclonal anti - erk1/2 ( 0.5 g / ml , cat . number mab1576 , r&d systems ) , goat polyclonal anti - synaptophysin ( 1 g / ml , cat . number af-5555 , r&d systems ) , rabbit polyclonal anti - cleaved caspase-3 ( 1 : 300 , cat . number sc-7148 , santa cruz ) , mouse monoclonal anti - th ( 0.5 g / ml , cat . number mab7566 , r&d systems ) , rabbit polyclonal anti - glo1 ( 1 : 200 , cat . number ab96032 , abcam ) , and goat polyclonal anti - gs ( 1 : 500 , sc-6640 , santa cruz ) . after overnight incubation with primary antibodies , the membranes were washed four times with tbs - t ( 5 min each ) . for synaptophysin and gs , the membrane was incubated at room temperature for 1.5 h with anti - goat secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2768 , santa cruz ) , for hmgb1 , phospo - erk1/2 cleaved caspase-3 and glo1 , with anti - rabbit secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2004 , santa cruz ) , and for erk1/2 and th with anti - mouse secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2005 , santa cruz ) . after incubations with secondary antibodies , membranes were washed four times with tbs - t ( 5 min each ) and the immunoreactivity of bands was visualized on a high - performance chemiluminescence machine ( g : box chemi - xx8 from syngene , synoptic ltd . cambridge , uk ) by using enhanced chemiluminescence plus luminol ( sc-2048 , santa cruz ) and quantified by densitometric analysis using image processing and analysis in genetools ( syngene by synoptic ltd . for loading control , the blots were stripped and detected with a mouse monoclonal anti--actin ( 1 : 2000 , sc-2048 , santa cruz ) antibody . all data from the three independent experiments were expressed as a ratio to optical density . total rna was extracted from retina using tri reagent ( ambion , tx ) , according to manufacturer 's protocol . cdna was synthesized from 1 g rna , using a high capacity cdna reverse transcription kit ( applied biosystem , ca ) following manufacturer 's instruction . forward 5-cctctatgccaacacagtgc-3 reverse 5-catcgtactcctgcttgctg-3. the standard pcr conditions included 2 minutes at 50c and 10 min at 95c followed by 40 cycles of extension at 95c for 15 seconds and one minute at 60c . threshold lines were automatically adjusted to intersect amplification lines in the linear portion of the amplification curves and cycle to threshold ( ct ) was recorded automatically . data were normalized with -actin mrna level ( housekeeping gene ) and the fold change in gene expression relative to normal was calculated using the ddct method . briefly , 50 l of standards and retinal homogenate ( equal amount ) was loaded in a clear 96-well plate , followed by addition of 100 l reaction mix solution having assay buffer , glutamate developer , and glutamate enzyme mix . the plate was incubated for 30 minutes at 37c ( protected from light ) and was read at 450 nm in microplate reader ( stat fax 4200 microplate reader , awareness technology , palm city , fl ) . the body weights of the diabetic rats were lower and their blood glucose levels were more than fourfold higher compared with age - matched normal control rats ( 178 22 versus 287 28 g and 475 32 versus 111 12 mg / dl , resp . ) . treatment of the diabetic rats with ga for one month did not change these metabolic variables in the diabetic rats ( 167 25 versus 178 22 g and 449 36 versus 475 32 mg / dl , resp . ) . western blot analysis demonstrated significant upregulation of hmgb1 expression in diabetic retinas compared to nondiabetic retinas . the expression of hmgb1 protein in the retinas of diabetic rats was upregulated by about 66% as compared to the retinas of nondiabetic rats ( figure 1(a ) ) . diabetes significantly increased erk1/2 activation in the retinas by about 77% compared to nondiabetic controls ( figure 1(b ) ) . cleaved caspase-3 , the apoptosis executer enzyme , was significantly upregulated in diabetic retinas compared to nondiabetic controls . cleaved caspase-3 levels in the retinas of diabetic rats were increased by about 70% compared to nondiabetic controls ( figure 1(c ) ) . the synaptic vesicle protein synaptophysin and the dopaminergic amacrine cell marker th levels obtained in diabetic animals were significantly lower than those of nondiabetic animals . the levels decreased by about 68% and 46% , respectively ( figures 1(d ) and 1(e ) ) . gs , an enzyme that converts glutamate into glutamine , protein expression was significantly decreased in diabetic retinas compared to nondiabetic retinas . glo1 , an enzyme critical for the detoxification of advanced glycation end products ( ages ) , protein expression was significantly decreased in diabetic retinas compared to nondiabetic rats . glutamate assay revealed that glutamate levels in the retinas of diabetic animals ( 0.04 0.015 nmol/l/g protein ) were significantly higher than those in nondiabetic controls ( 0.022 0.004 nmol/l/g protein ) ( p = 0.036 ) ( figure 1(h ) ) . there was no significant difference in serum levels of hmgb1 between rats intravitreally injected with hmgb1 ( 135.64 14.38 ) and normal rats ( 134.88 11.01 ) ( p = 0.225 ) . intravitreal injection of hmgb1 resulted in increased hmgb1 expression by about 72% compared to the values obtained from the contralateral eye that received pbs alone ( figure 2(a ) ) . in the same retinal samples , hmgb1 injection resulted in a 76% increase in erk1/2 activation ( figure 2(b ) ) , 31% increase in cleaved caspase-3 expression ( figure 2(c ) ) , 65% decrease in synaptophysin expression ( figure 2(d ) ) , 65% decrease in th expression ( figure 2(e ) ) , 70% decrease in gs expression ( figure 2(f ) ) , and 71% decrease in glo1 expression ( figure 2(g ) ) . injection of hmgb1 tended to increase retinal glutamate expression , but this was not significant ( 0.041 0.025 versus 0.021 0.004 nmol/l/g protein ; p = 0.07 ) ( figure 2(h ) ) . western blot analysis was used to assess the effect of ga on diabetes - induced alterations of hmgb1 , erk1/2 activation , cleaved caspase-3 , synaptophysin , th , gs , and glo1 . constant ga intake from the onset of diabetes significantly attenuated diabetes - induced upregulation of hmgb1 , erk1/2 activation , and cleaved caspase-3 by about 73% , 55% , and 78% , respectively ( figures 1(a ) , 1(b ) , and 1(c ) ) . in the ga - fed diabetic rats , the decrease in synaptophysin , th , gs , and glo1 caused by diabetes was attenuated by about 58% , 55% , 79% , and 53% , respectively ( figures 1(d ) , 1(e ) , 1(f ) , and 1(g ) ) . the levels of glutamate in the ga - treated diabetic rats ( 0.024 0.002 nmol/l/g protein ) were significantly less than those in the untreated diabetic rats ( 0.04 0.015 nmol/l/g protein ) ( p = 0.045 ) ( figure 1(h ) ) . the expression of hmgb1 mrna in the retinas of diabetic rats was increased by about 4-fold compared to the retinas of nondiabetic rats . intravitreal injection of hmgb1 resulted in increased hmgb1 mrna expression by about 5-fold compared to the values obtained from the contralateral eye that received pbs alone . ga intake significantly attenuated diabetes - induced upregulation of hmgb1 mrna by about 3.5-fold compared to untreated diabetic rats ( figure 3 ) . in the present study , we investigated the pathological role of hmgb1 in diabetes - induced retinal neuropathy . our previous studies showed that diabetes upregulates hmgb1 expression in the vitreous fluid and preretinal membranes from patients with pdr and its expression correlated with the activity of the disease and the levels of inflammatory biomarkers [ 2022 ] . in addition , we demonstrated that hmgb1 expression was upregulated in the retinas of diabetic mice and rats [ 21 , 23 ] . in the current study , we demonstrated that diabetes induced significant upregulation of cleaved caspase-3 and glutamate expression in the retinas of rats . on the other hand , diabetes induced significant downregulation of synaptophysin , th , gs , and glo1 in the retinas of rats . furthermore , our data show that intravitreal injection of hmgb1 in normal rats mimics the effect of diabetes . the hmgb1 inhibitor ga attenuated diabetes - induced upregulation of hmgb1 protein and mrna ; cleaved caspase-3 and glutamate ; and downregulation of synaptophysin , th , gs , and glo1 in the retinas of rats . taken together , these findings suggest that hmgb1 contributes to retinal neuropathy induced by diabetes . diabetes induces retinal neurodegeneration as evidenced by the presence of apoptotic cells in all retinal layers [ 4 , 25 ] . several studies demonstrated that expression of active caspase-3 , an indication of apoptosis , was upregulated in the diabetic retinas of human subjects and rats . in stz - diabetic rat retinas , however , feit - leichman et al . demonstrated , in stz - diabetic mice , upregulation of retinal caspase-3 activity at 1 month after induction of diabetes , which diminished to normal and began to increase again after approximately 6 months . they concluded that diabetes - induced degeneration of retinal capillaries can develop independent of neuronal loss . it possibly serves multiple functions in synaptic vesicle formation and exocytosis , playing an important role in neurotransmitter delivery . it is widely used as one of the synaptic function markers and is also thought to be closely related to synaptogenesis and synaptic plasticity during neural tissue development . synaptophysin knockout mice exhibited a significant decrease in synaptic vesicles in retinal rod photoreceptors which disturbs neurotransmitter release and synaptic network activity . previous studies demonstrated that 1 month of diabetes decreases retinal expression of synaptophysin [ 3 , 2830 ] . therefore , the th protein level is a marker of dopaminergic amacrine cells in the retina [ 1 , 25 , 31 ] . several studies showed decreased th protein levels in the diabetic retinas , reflecting reductions in the density of dopaminergic amacrine cells [ 1 , 3133 ] . the synaptically released glutamate is taken up by mller cells where gs converts it into glutamine . several studies found that the expression of gs was significantly decreased in the diabetic rat retinas [ 34 , 35 ] . these dysfunctions resulted in elevated glutamate levels in the diabetic retinas [ 34 , 36 , 37 ] , which might induce retinal neurodegeneration via glutamate excitotoxicity . in diabetes , there is accumulation of the ages precursor methylglyoxal ( mg ) . in diabetic retinopathy , mg - derived ages are elevated in retina and are viewed to be causative in retinal injury and neurodegeneration [ 3840 ] . normally , mg is detoxified by the glyoxalase ( glo ) enzyme system , composed of glo1 and glo2 . it was also shown that glo1 overexpression in diabetic rats prevents hyperglycemia - induced formation of mg - derived ages in the neural retina , prevents mller glia dysfunction , and protects against capillary degenerative pathology . in the present study , we demonstrated that , similar to diabetes , intravitreal injection of hmgb1 caused a significant upregulation of hmgb1 protein and mrna and activated cleaved caspase-3 in the retina of normal rats . in addition , injection of hmgb1 tended to increase retinal glutamate expression , but this was not significant . in this regard , the recent finding that hmgb1 promotes glutamate release from gliosomes suggests that its neurotoxicity is mediated , at least in part , by increased release of glutamate and enhanced excitotoxic neuronal death . on the other hand , in vitro studies demonstrated that glutamate can induce the release of hmgb1 from neuronal cells [ 14 , 15 ] . furthermore , intravitreal administration of hmgb1 induced downregulation of synaptophysin , th , gs , and glo1 . these findings suggest that the early retinal neuropathy of diabetes involves the upregulated expression of hmgb1 . several studies showed that hmgb1 is massively released immediately after an ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain [ 14 , 15 , 17 ] . to exert these activities , hmgb1 must transit from the nucleus , through the cytoplasm , to the outside of the cell . several studies demonstrated that the inflammatory marker c - reactive protein and the proinflammatory cytokines tumor necrosis factor- ( tnf- ) and interleukin-1 ( il-1 ) induce translocation of hmgb1 from nucleus to the cytoplasm and its subsequent extracellular release from the cell . in vitro studies demonstrated that extracellular hmgb1 induced the proinflammatory biomarkers tnf- , il-1 , interferon- , intercellular adhesion molecule-1 ( icam-1 ) , inducible nitric oxide synthase ( inos ) , and cyclooxgenase-2 in cultured neurons , astrocytes , microglia , and endothelial cells moreover , cerebral microinjection of exogenous hmgb1 increased postischemic brain injury and upregulated the expression of inos and il-1 . these findings indicate that hmgb1 functions as a novel proinflammatory cytokine - like factor linking very early stages of cerebral ischemic injury with the activation of local neuroinflammatory response in the postischemic brain [ 14 , 15 ] . furthermore , it was also shown that extracellular hmgb1 released from the damaged brain neurons induces neuronal apoptosis and that this involves hmgb1-rage interaction . in our laboratory , we recently demonstrated that diabetes induced significant upregulation of retinal expression of hmgb1 , rage , activated nf-b , activated erk1/2 , and icam-1 and that intravitreal administration of hmgb1 in normal rats mimics the effect of diabetes . we also showed that the hmgb1 inhibitor ga was effective in preventing diabetes - induced nf-b activation . in addition , coimmunoprecipitation studies showed that diabetes increases the interaction between hmgb1 and rage in the retina . these findings suggest that activation of hmgb1/rage signaling axis with subsequent activation of nf-b and erk1/2 is important in promoting diabetes - induced retinal neuropathy . recently , it was reported that chronic neuroinflammation may be a driving force of progressive neurodegeneration and that hmgb1 provides the link between chronic neuroinflammation and progressive neurodegeneration in neurodegenerative diseases , such as parkinson 's disease . to confirm the neuropathological implications of hmgb1 in diabetic retinopathy , ga is known to bind directly to hmgb1 and inhibit its chemoattractant and mitogenic activities . interestingly , constant intake of ga significantly reduced retinal hmgb1 protein and mrna expression induced by diabetes . in addition , ga attenuated diabetes - induced upregulation of cleaved caspase-3 and glutamate and counteracted the downregulation of synaptophysin , th , gs , and glo1 induced by diabetes without changing body weight or blood glucose levels . our results are consistent with previous reports that demonstrated a neuroprotective effect of ga in animal models of brain ischemia , intracerebral hemorrhage , and spinal cord ischemia . ga is known to possess glucocorticoid - like anti - inflammatory properties , due to its inhibitory activity on 11-hydroxysteroid dehydrogenase . . demonstrated that the neuroprotective effect of ga on intracerebral hemorrhage - related pathogenic events was not mediated by glucocorticoid receptors or modulation of nitric oxide production and was reversed by exogenous hmgb1 application . these findings suggest that the protective effect of ga is mediated by its anti - hmgb1 activity and is irrelevant to the glucocorticoid system . it was demonstrated that the neuroprotective effect of ga was partly attributable to its inhibitory effect on hmgb1 release [ 4749 ] . furthermore , it was shown that ga affords protection in postischemic brain and spinal cord via its anti - inflammatory , antiapoptotic , antiexcitotoxic , and antioxidative effects [ 47 , 49 ] . together , these findings suggest that early retinal neuropathy induced by diabetes is , at least in part , attributable to diabetes - induced upregulation of hmgb1 expression and that inhibiting the release of hmgb1 with constant intake of ga results in less diabetes - induced retinal neuropathy . it is important to note that excessive intake of licorice may cause hypermineralocorticoidism - like syndrome characterized by sodium and water retention , potassium loss , edema , increased blood pressure , metabolic alkalosis , and depression of rennin - angiotensin - aldosterone system [ 50 , 51 ] . in conclusion , our data point to a potential novel and pivotal role for hmgb1 as a mediator of diabetes - induced neuropathy in the retina . the hmgb1 inhibitor ga attenuated diabetes - induced upregulation of hmgb1 and diabetes - induced retinal neuropathy . therefore , ga and other agents targeted to hmgb1 may provide novel therapeutic options for diabetic retinopathy .

to test the hypothesis that increased expression of proinflammatory cytokine high - mobility group box-1 ( hmgb1 ) in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in retinas of diabetic rats plays a pathogenetic role in mediating diabetes - induced retinal neuropathy . retinas of 1-month diabetic rats and hmgb1 intravitreally injected normal rats were studied using western blot analysis , rt - pcr and glutamate assay . in addition , we studied the effect of the hmgb1 inhibitor glycyrrhizin on diabetes - induced biochemical changes in the retina . diabetes and intravitreal injection of hmgb1 in normal rats induced significant upregulation of hmgb1 protein and mrna , activated extracellular signal - regulated kinase 1 and 2 ( erk1/2 ) , cleaved caspase-3 and glutamate ; and significant downregulation of synaptophysin , tyrosine hydroxylase , glutamine synthetase , and glyoxalase 1 . constant glycyrrhizin intake from the onset of diabetes did not affect the metabolic status of the diabetic rats , but it significantly attenuated diabetes - induced upregulation of hmgb1 protein and mrna , activated erk1/2 , cleaved caspase-3 , and glutamate . in the glycyrrhizin - fed diabetic rats , the decrease in synaptophysin , tyrosine hydroxylase , and glyoxalase 1 caused by diabetes was significantly attenuated . these findings suggest that early retinal neuropathy of diabetes involves upregulated expression of hmgb1 and can be ameliorated by inhibition of hmgb1 .

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

diabetic retinopathy ( dr ) , a vision - threatening disease , has classically been regarded as a disease of the retinal microvasculature and a consequence of vascular cell damage . however , recent studies proved that neurodegeneration and impaired visual function are initiated early after the onset of diabetes and progress independently of the vascular lesions [ 14 ] . however , the molecular mechanisms underlying the diabetes - induced retinal neurodegeneration and dysfunction are still not well understood . high - mobility group box-1 ( hmgb1 ) is a nonhistone dna - binding nuclear protein that has been implicated in diverse intracellular functions , including the stabilization of nucleosomal structures and the facilitation of gene transcription . necrotic cell death can result in passive leakage of hmgb1 from the cell as the protein is then no longer bound to dna . in addition , hmgb1 can be actively secreted by different cell types , including activated monocytes and macrophages , mature dendritic cells , natural killer cells , and endothelial cells . extracellular hmgb1 functions as a proinflammatory cytokine and triggers the inflammatory response through the activation of multiple receptors such as the receptor for advanced glycation end products ( rage ) , toll - like receptor-2 ( tlr2 ) , and tlr4 leading to activation of the transcription factors extracellular signal - regulated kinase 1 and 2 ( erk1/2 ) and nuclear factor kappa b ( nf-b ) , which may alter gene transcription and lead to the upregulation of proinflammatory cytokines , chemokines , and adhesion molecules and intensifies cellular oxidative stress [ 510 ] , processes that may play a role in the pathogenesis of diabetic retinal neurodegeneration and dysfunction . in these studies , extracellular hmgb1 plays a key role in the development of neuronal injury through microglial activation , induction of apoptosis , excitatory amino acid release , and induction of proinflammatory mediators [ 1419 ] . downregulation of hmgb1 or treatment with neutralizing anti - hmgb1 monoclonal antibody remarkably suppressed infarct size , activation of microglia , and induction of proinflammatory markers and inhibited the increased permeability of the blood - brain barrier [ 14 , 18 ] . in previous studies , we demonstrated that hmgb1 and rage were expressed by vascular endothelial cells and stromal cells in fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy ( pdr ) . in addition , we demonstrated increased levels of hmgb1 in the vitreous samples from patients with pdr and that there were significant positive correlations between the vitreous levels of hmgb1 and the levels of the inflammatory biomarkers [ 2022 ] . furthermore , we demonstrated that diabetes induced significant upregulation of the expression of hmgb1 and rage in the retinas of rats and mice and that intravitreal administration of hmgb1 to normal rats induced activation of inflammatory signaling pathways in the retina and increased retinal vascular permeability [ 21 , 23 ] . glycyrrhizin ( ga ) , an ingredient of the licorice roots , has long been known to exhibit glucocorticoid - like anti - inflammatory actions by inhibiting 11-hydroxysteroid dehydrogenase . more recently , ga has also been shown to bind to and inhibit chemoattractant , mitogenic , and cytokine - like activities of hmgb1 . in this study , we explored the hypothesis that hmgb1 plays a pathogenetic role in mediating diabetes - induced retinal neuropathy . to test this hypothesis , we investigated the expression of the neurodegeneration mediators and markers cleaved caspase-3 , synaptophysin , tyrosine hydroxylase ( th ) , glutamine synthetase ( gs ) , glutamate , and glyoxalase 1 ( glo 1 ) in the retinas of diabetic rats and in the retinas of normal rats after intravitreal administration of hmgb1 . in addition , we analyzed whether constant ga intake suppresses retinal neuropathy induced by diabetes in rats . diabetic rats were divided into 2 groups : the rats in group i received normal drinking water without any supplementation , and group ii received drinking water supplemented with glycyrrhizic acid ( 150 mg / kg / day , santa cruz biotechnology , inc . , santa cruz , ca ) immediately after establishment of diabetes throughout the course of the experiment . after 4 weeks of diabetes , the rats were euthanized by an overdose of chloral hydrate , the eyes were removed , and retina was isolated and frozen immediately in liquid nitrogen and stored at 80c to be analyzed by western blot analysis or biochemical assay . sprague dawley rats ( 200220 g ) were kept under deep anesthesia , and sterilized solution of recombinant hmgb1 ( 5 ng/5 l ; r&d systems , minneapolis , mn ) was injected into the vitreous of the right eye as previously described by us . the animals were sacrificed 4 days after intravitreal administration , and the retinas were carefully dissected , snap - frozen in liquid nitrogen , and stored at 80c to be analyzed by western blot analysis or biochemical assay . the breakdown of blood - retina barrier induced by intravitreal injection of hmgb1 might lead to raised levels of hmgb1 in the serum . therefore , we determined the levels of hmgb1 in equal amounts of serum from intravitreally injected rats and normal rats using western blot analysis . the lysate was centrifuged at 14,000 g for 10 min at 4c , and the supernatant was collected . for detection of hmgb1 , phospho - erk1/2 , synaptophysin , cleaved caspase-3 , th , glo1 , and gs , the membrane was incubated overnight at 4c with rabbit polyclonal anti - hmgb1 ( 1 : 1000 , cat . number af-5555 , r&d systems ) , rabbit polyclonal anti - cleaved caspase-3 ( 1 : 300 , cat . number ab96032 , abcam ) , and goat polyclonal anti - gs ( 1 : 500 , sc-6640 , santa cruz ) . for synaptophysin and gs , the membrane was incubated at room temperature for 1.5 h with anti - goat secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2768 , santa cruz ) , for hmgb1 , phospo - erk1/2 cleaved caspase-3 and glo1 , with anti - rabbit secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2004 , santa cruz ) , and for erk1/2 and th with anti - mouse secondary horseradish peroxidase - conjugated antibody ( 1 : 2000 , sc-2005 , santa cruz ) . threshold lines were automatically adjusted to intersect amplification lines in the linear portion of the amplification curves and cycle to threshold ( ct ) was recorded automatically . briefly , 50 l of standards and retinal homogenate ( equal amount ) was loaded in a clear 96-well plate , followed by addition of 100 l reaction mix solution having assay buffer , glutamate developer , and glutamate enzyme mix . the body weights of the diabetic rats were lower and their blood glucose levels were more than fourfold higher compared with age - matched normal control rats ( 178 22 versus 287 28 g and 475 32 versus 111 12 mg / dl , resp . ) treatment of the diabetic rats with ga for one month did not change these metabolic variables in the diabetic rats ( 167 25 versus 178 22 g and 449 36 versus 475 32 mg / dl , resp . ) western blot analysis demonstrated significant upregulation of hmgb1 expression in diabetic retinas compared to nondiabetic retinas . the expression of hmgb1 protein in the retinas of diabetic rats was upregulated by about 66% as compared to the retinas of nondiabetic rats ( figure 1(a ) ) . cleaved caspase-3 , the apoptosis executer enzyme , was significantly upregulated in diabetic retinas compared to nondiabetic controls . cleaved caspase-3 levels in the retinas of diabetic rats were increased by about 70% compared to nondiabetic controls ( figure 1(c ) ) . glo1 , an enzyme critical for the detoxification of advanced glycation end products ( ages ) , protein expression was significantly decreased in diabetic retinas compared to nondiabetic rats . glutamate assay revealed that glutamate levels in the retinas of diabetic animals ( 0.04 0.015 nmol/l/g protein ) were significantly higher than those in nondiabetic controls ( 0.022 0.004 nmol/l/g protein ) ( p = 0.036 ) ( figure 1(h ) ) . there was no significant difference in serum levels of hmgb1 between rats intravitreally injected with hmgb1 ( 135.64 14.38 ) and normal rats ( 134.88 11.01 ) ( p = 0.225 ) . intravitreal injection of hmgb1 resulted in increased hmgb1 expression by about 72% compared to the values obtained from the contralateral eye that received pbs alone ( figure 2(a ) ) . in the same retinal samples , hmgb1 injection resulted in a 76% increase in erk1/2 activation ( figure 2(b ) ) , 31% increase in cleaved caspase-3 expression ( figure 2(c ) ) , 65% decrease in synaptophysin expression ( figure 2(d ) ) , 65% decrease in th expression ( figure 2(e ) ) , 70% decrease in gs expression ( figure 2(f ) ) , and 71% decrease in glo1 expression ( figure 2(g ) ) . injection of hmgb1 tended to increase retinal glutamate expression , but this was not significant ( 0.041 0.025 versus 0.021 0.004 nmol/l/g protein ; p = 0.07 ) ( figure 2(h ) ) . western blot analysis was used to assess the effect of ga on diabetes - induced alterations of hmgb1 , erk1/2 activation , cleaved caspase-3 , synaptophysin , th , gs , and glo1 . constant ga intake from the onset of diabetes significantly attenuated diabetes - induced upregulation of hmgb1 , erk1/2 activation , and cleaved caspase-3 by about 73% , 55% , and 78% , respectively ( figures 1(a ) , 1(b ) , and 1(c ) ) . in the ga - fed diabetic rats , the decrease in synaptophysin , th , gs , and glo1 caused by diabetes was attenuated by about 58% , 55% , 79% , and 53% , respectively ( figures 1(d ) , 1(e ) , 1(f ) , and 1(g ) ) . the levels of glutamate in the ga - treated diabetic rats ( 0.024 0.002 nmol/l/g protein ) were significantly less than those in the untreated diabetic rats ( 0.04 0.015 nmol/l/g protein ) ( p = 0.045 ) ( figure 1(h ) ) . the expression of hmgb1 mrna in the retinas of diabetic rats was increased by about 4-fold compared to the retinas of nondiabetic rats . intravitreal injection of hmgb1 resulted in increased hmgb1 mrna expression by about 5-fold compared to the values obtained from the contralateral eye that received pbs alone . ga intake significantly attenuated diabetes - induced upregulation of hmgb1 mrna by about 3.5-fold compared to untreated diabetic rats ( figure 3 ) . in the present study , we investigated the pathological role of hmgb1 in diabetes - induced retinal neuropathy . our previous studies showed that diabetes upregulates hmgb1 expression in the vitreous fluid and preretinal membranes from patients with pdr and its expression correlated with the activity of the disease and the levels of inflammatory biomarkers [ 2022 ] . in addition , we demonstrated that hmgb1 expression was upregulated in the retinas of diabetic mice and rats [ 21 , 23 ] . in the current study , we demonstrated that diabetes induced significant upregulation of cleaved caspase-3 and glutamate expression in the retinas of rats . on the other hand , diabetes induced significant downregulation of synaptophysin , th , gs , and glo1 in the retinas of rats . furthermore , our data show that intravitreal injection of hmgb1 in normal rats mimics the effect of diabetes . the hmgb1 inhibitor ga attenuated diabetes - induced upregulation of hmgb1 protein and mrna ; cleaved caspase-3 and glutamate ; and downregulation of synaptophysin , th , gs , and glo1 in the retinas of rats . taken together , these findings suggest that hmgb1 contributes to retinal neuropathy induced by diabetes . several studies demonstrated that expression of active caspase-3 , an indication of apoptosis , was upregulated in the diabetic retinas of human subjects and rats . demonstrated , in stz - diabetic mice , upregulation of retinal caspase-3 activity at 1 month after induction of diabetes , which diminished to normal and began to increase again after approximately 6 months . they concluded that diabetes - induced degeneration of retinal capillaries can develop independent of neuronal loss . previous studies demonstrated that 1 month of diabetes decreases retinal expression of synaptophysin [ 3 , 2830 ] . therefore , the th protein level is a marker of dopaminergic amacrine cells in the retina [ 1 , 25 , 31 ] . several studies showed decreased th protein levels in the diabetic retinas , reflecting reductions in the density of dopaminergic amacrine cells [ 1 , 3133 ] . several studies found that the expression of gs was significantly decreased in the diabetic rat retinas [ 34 , 35 ] . these dysfunctions resulted in elevated glutamate levels in the diabetic retinas [ 34 , 36 , 37 ] , which might induce retinal neurodegeneration via glutamate excitotoxicity . it was also shown that glo1 overexpression in diabetic rats prevents hyperglycemia - induced formation of mg - derived ages in the neural retina , prevents mller glia dysfunction , and protects against capillary degenerative pathology . in the present study , we demonstrated that , similar to diabetes , intravitreal injection of hmgb1 caused a significant upregulation of hmgb1 protein and mrna and activated cleaved caspase-3 in the retina of normal rats . in addition , injection of hmgb1 tended to increase retinal glutamate expression , but this was not significant . furthermore , intravitreal administration of hmgb1 induced downregulation of synaptophysin , th , gs , and glo1 . these findings suggest that the early retinal neuropathy of diabetes involves the upregulated expression of hmgb1 . to exert these activities , hmgb1 must transit from the nucleus , through the cytoplasm , to the outside of the cell . several studies demonstrated that the inflammatory marker c - reactive protein and the proinflammatory cytokines tumor necrosis factor- ( tnf- ) and interleukin-1 ( il-1 ) induce translocation of hmgb1 from nucleus to the cytoplasm and its subsequent extracellular release from the cell . in vitro studies demonstrated that extracellular hmgb1 induced the proinflammatory biomarkers tnf- , il-1 , interferon- , intercellular adhesion molecule-1 ( icam-1 ) , inducible nitric oxide synthase ( inos ) , and cyclooxgenase-2 in cultured neurons , astrocytes , microglia , and endothelial cells moreover , cerebral microinjection of exogenous hmgb1 increased postischemic brain injury and upregulated the expression of inos and il-1 . these findings indicate that hmgb1 functions as a novel proinflammatory cytokine - like factor linking very early stages of cerebral ischemic injury with the activation of local neuroinflammatory response in the postischemic brain [ 14 , 15 ] . in our laboratory , we recently demonstrated that diabetes induced significant upregulation of retinal expression of hmgb1 , rage , activated nf-b , activated erk1/2 , and icam-1 and that intravitreal administration of hmgb1 in normal rats mimics the effect of diabetes . we also showed that the hmgb1 inhibitor ga was effective in preventing diabetes - induced nf-b activation . in addition , coimmunoprecipitation studies showed that diabetes increases the interaction between hmgb1 and rage in the retina . these findings suggest that activation of hmgb1/rage signaling axis with subsequent activation of nf-b and erk1/2 is important in promoting diabetes - induced retinal neuropathy . to confirm the neuropathological implications of hmgb1 in diabetic retinopathy , ga is known to bind directly to hmgb1 and inhibit its chemoattractant and mitogenic activities . interestingly , constant intake of ga significantly reduced retinal hmgb1 protein and mrna expression induced by diabetes . in addition , ga attenuated diabetes - induced upregulation of cleaved caspase-3 and glutamate and counteracted the downregulation of synaptophysin , th , gs , and glo1 induced by diabetes without changing body weight or blood glucose levels . our results are consistent with previous reports that demonstrated a neuroprotective effect of ga in animal models of brain ischemia , intracerebral hemorrhage , and spinal cord ischemia . these findings suggest that the protective effect of ga is mediated by its anti - hmgb1 activity and is irrelevant to the glucocorticoid system . together , these findings suggest that early retinal neuropathy induced by diabetes is , at least in part , attributable to diabetes - induced upregulation of hmgb1 expression and that inhibiting the release of hmgb1 with constant intake of ga results in less diabetes - induced retinal neuropathy . it is important to note that excessive intake of licorice may cause hypermineralocorticoidism - like syndrome characterized by sodium and water retention , potassium loss , edema , increased blood pressure , metabolic alkalosis , and depression of rennin - angiotensin - aldosterone system [ 50 , 51 ] . in conclusion , our data point to a potential novel and pivotal role for hmgb1 as a mediator of diabetes - induced neuropathy in the retina . the hmgb1 inhibitor ga attenuated diabetes - induced upregulation of hmgb1 and diabetes - induced retinal neuropathy .

embryonic stem cell ( esc ) lines are derived from the inner cell mass ( icm ) of blastocysts , and the defining feature of these cells is their ability to differentiate into a variety of cell types that encompass all three embryonic germ layers . embryonic stem cells , particularly human stem cells hold tremendous potential in the field of regenerative medicine , in addition to being a useful tool in basic scientific research and for pharmacological and cytotoxicity screening [ 4 , 5 ] . methods for efficient and reliable preservation of embryonic stem cells are necessary to preserve these pluripotent cells for therapeutic and experimental use . the traditional method for embryonic stem cell cryopreservation has been slow cooling at rates of approximately 1c per minute [ reviewed in ] . this technique has been successfully applied to the cryopreservation of embryos at a variety of stages [ 712 ] as well as oocytes [ 1315 ] . vitrification involves ultra - rapid cooling of the cell at rates as high as 20,000c / min and the use of high concentrations of cryoprotectant agents ( cpa ) . to achieve these high cooling rates the cell is frozen in miniscule volumes of fluid in vessels that allow almost instantaneous cooling to 196c . vitrification protocols have also been successfully used for cryopreservation of human embryonic stem cell lines [ 16 , 17 ] . numerous publications suggest that cell preservation by vitrification may be superior to slow cooling methodologies [ reviewed in [ 19 , 20 ] ] . to date there have been no reports on the cryopreservation of icm cells extracted from the blastocyst stage embryo . preservation of isolated embryo - derived stem cells without first establishing a viable es cell line offers a new avenue for banking the stem cell source material . the small number of cells in the icm of day 5 or day 6 blastocysts poses a special challenge during the cryopreservation process . in this investigation we explore the feasibility of icm vitrification and subsequent establishment of esc - like colonies . icm isolation methodology and its influence on cryosurvival and subsequent retention of es phenotype were looked at . mouse one - cell zygotes ( charles river laboratory ; wilmington , ma ) were used for all icm isolation experiments . the zygotes were thawed as per manufacturer s instructions and cultured overnight in global blastocyst medium ( lifeglobal ; guilford , ct , usa ) supplemented with 10% serum protein substitute ( sps , cooper / sage ; pasadena , ca , usa ) . icms from mouse embryos were isolated by immunosurgery , micro - dissection or laser with micro - dissection . zonae were removed from any unhatched blastocysts using acid tyrodes solution . for immunosurgical isolation , zona free blastocysts were incubated with 20% rat anti - mouse serum for 1 h followed by 1 h incubation with 20% guinea pig complement . lysed trophectodermal cells were removed from icms by repeated pipetting with finely drawn glass micropipettes . for both of the micro - dissection techniques , zona - free blastocysts were first plated and allowed to attach to the dish . after 48 h , the icm outgrowth was either micro- dissected free from surrounding trophectodermal cells or treated first with laser to destroy any residual trophectoderm cells before micro - dissection . vitrification of isolated icms was carried out using a two step equilibration protocol : ( 1 ) 7.5% dmso/7.5% ethylene glycol ( eg ) for 2 min , ( 2 ) 15% dmso/15% eg + 10 g / ml ficoll + 0.65 mol / l sucrose for 45 s. the basal medium used to constitute vitrification solutions was global blast medium with 20% sps . after the final equilibration step , individual icms were loaded on to the vitrification carrier and immersed in liquid nitrogen ( ln2 ) . in this study we tested two different carrier systems ; the cryoloop ( hampton research ; laguna , ca , usa ) and the hsv straw ( mid - atlantic diagnostics ; laurrel , nj , usa ) . the cryoloop is a microscopic nylon loop measuring ~1.0 mm in diameter . for cryoloop vitrification , the icm was loaded on to a thin film of fluid applied to the loop , before plunging into a vial containing ln2 . the hsv straw consists of a capillary tube with a cut out region , creating a narrow gutter and an outer straw . the outer straw was then drawn over it and sealed to create a closed system . the cryoloop or hsv straw was immersed directly in basal culture medium containing 0.25 m sucrose . the icm cells displaced from the carriers were readily visualized using the dissecting scope . after 2 min , recovered icms were moved to the second solution containing 0.125 m sucrose for 3 min and then placed in culture . embryonic stem cell medium ( esc - sure dmem ; applied stem cell , sunnyvale , ca , usa ) supplemented with 20% fbs , 200 mm non - essential amino acids , 100 u / ml penicillin , 10 g / ml streptomycin , 100 mm sodium pyruvate and mercaptoethanol and 10 ng / ml of mouse leukemia inhibitory factor ( lif ; chemicon ; temecula , ca , usa ) was used for culture of icms . icm - derived es cells were cultivated on inactivated mouse embryonic feeder layers ( mef ) . mef cells were purchased ( applied stem cell ; sunnyvale , ca , usa ) and prepared as per manufacturer s instructions . alternatively , icms were grown without feeder cells on 0.1% gelatin coated eight or 16 well chamber slides with 25% mef conditioned medium/75% esc complete medium . these cell clumps were individually picked off of the culture surface and disaggregated by treatment with trypsin : edta solution for 35 min at room temperature . culture medium was exchanged every 48 h. icms and icm - derived esc colonies were tested during the culture interval for expression of stem cell specific markers ; ssea-1 , oct-4 and sox-2 and alkaline phospatase(ap ) activity using the mouse es / ips cell characterization kit ( applied stem cell , sunnyvale , ca , usa ) . cell fixation and permeabilization was carried out using kit reagents as per manufacturer s instructions . after blocking , cells were incubated with primary antibody against ssea-1 , sox-2 or oct-4 overnight at 4c . cells were subsequently washed twice with pbs and incubated with corresponding secondary antibodies conjugated with alexa fluor 594 for 1 h at room temperature . immunoflourescent staining was visualized using confocal laser microscopy and optical sectioning with image analysis software . alkaline phospatase ( ap ) staining was performed by incubating fixed cells with ap test solution ( provided in kit ) for 2 h at room temperature . the main outcome parameters measured were icm survival , recovery and ability to attach within 24 h of warming . mouse one - cell zygotes ( charles river laboratory ; wilmington , ma ) were used for all icm isolation experiments . the zygotes were thawed as per manufacturer s instructions and cultured overnight in global blastocyst medium ( lifeglobal ; guilford , ct , usa ) supplemented with 10% serum protein substitute ( sps , cooper / sage ; pasadena , ca , usa ) . icms from mouse embryos were isolated by immunosurgery , micro - dissection or laser with micro - dissection . zonae were removed from any unhatched blastocysts using acid tyrodes solution . for immunosurgical isolation , zona free blastocysts were incubated with 20% rat anti - mouse serum for 1 h followed by 1 h incubation with 20% guinea pig complement . lysed trophectodermal cells were removed from icms by repeated pipetting with finely drawn glass micropipettes . for both of the micro - dissection techniques , zona - free blastocysts were first plated and allowed to attach to the dish . after 48 h , the icm outgrowth was either micro- dissected free from surrounding trophectodermal cells or treated first with laser to destroy any residual trophectoderm cells before micro - dissection . vitrification of isolated icms was carried out using a two step equilibration protocol : ( 1 ) 7.5% dmso/7.5% ethylene glycol ( eg ) for 2 min , ( 2 ) 15% dmso/15% eg + 10 g / ml ficoll + 0.65 mol / l sucrose for 45 s. the basal medium used to constitute vitrification solutions was global blast medium with 20% sps . after the final equilibration step , individual icms were loaded on to the vitrification carrier and immersed in liquid nitrogen ( ln2 ) . in this study we tested two different carrier systems ; the cryoloop ( hampton research ; laguna , ca , usa ) and the hsv straw ( mid - atlantic diagnostics ; laurrel , nj , usa ) . the cryoloop is a microscopic nylon loop measuring ~1.0 mm in diameter . for cryoloop vitrification , the icm was loaded on to a thin film of fluid applied to the loop , before plunging into a vial containing ln2 . the hsv straw consists of a capillary tube with a cut out region , creating a narrow gutter and an outer straw . the outer straw was then drawn over it and sealed to create a closed system . the cryoloop or hsv straw was immersed directly in basal culture medium containing 0.25 m sucrose . the icm cells displaced from the carriers were readily visualized using the dissecting scope . after 2 min , recovered icms were moved to the second solution containing 0.125 m sucrose for 3 min and then placed in culture vitrification of isolated icms was carried out using a two step equilibration protocol : ( 1 ) 7.5% dmso/7.5% ethylene glycol ( eg ) for 2 min , ( 2 ) 15% dmso/15% eg + 10 g / ml ficoll + 0.65 mol / l sucrose for 45 s. the basal medium used to constitute vitrification solutions was global blast medium with 20% sps . after the final equilibration step , individual icms were loaded on to the vitrification carrier and immersed in liquid nitrogen ( ln2 ) . in this study we tested two different carrier systems ; the cryoloop ( hampton research ; laguna , ca , usa ) and the hsv straw ( mid - atlantic diagnostics ; laurrel , nj , usa ) . the cryoloop is a microscopic nylon loop measuring ~1.0 mm in diameter . for cryoloop vitrification , the icm was loaded on to a thin film of fluid applied to the loop , before plunging into a vial containing ln2 . the hsv straw consists of a capillary tube with a cut out region , creating a narrow gutter and an outer straw . the outer straw was then drawn over it and sealed to create a closed system . the cryoloop or hsv straw was immersed directly in basal culture medium containing 0.25 m sucrose . the icm cells displaced from the carriers were readily visualized using the dissecting scope . after 2 min , recovered icms were moved to the second solution containing 0.125 m sucrose for 3 min and then placed in culture embryonic stem cell medium ( esc - sure dmem ; applied stem cell , sunnyvale , ca , usa ) supplemented with 20% fbs , 200 mm non - essential amino acids , 100 u / ml penicillin , 10 g / ml streptomycin , 100 mm sodium pyruvate and mercaptoethanol and 10 ng / ml of mouse leukemia inhibitory factor ( lif ; chemicon ; temecula , ca , usa ) was used for culture of icms . icm - derived es cells were cultivated on inactivated mouse embryonic feeder layers ( mef ) . mef cells were purchased ( applied stem cell ; sunnyvale , ca , usa ) and prepared as per manufacturer s instructions . these cell clumps were individually picked off of the culture surface and disaggregated by treatment with trypsin : edta solution for 35 min at room temperature . culture medium was exchanged every 48 h. icms and icm - derived esc colonies were tested during the culture interval for expression of stem cell specific markers ; ssea-1 , oct-4 and sox-2 and alkaline phospatase(ap ) activity using the mouse es / ips cell characterization kit ( applied stem cell , sunnyvale , ca , usa ) . cell fixation and permeabilization was carried out using kit reagents as per manufacturer s instructions . after blocking , cells were incubated with primary antibody against ssea-1 , sox-2 or oct-4 overnight at 4c . cells were subsequently washed twice with pbs and incubated with corresponding secondary antibodies conjugated with alexa fluor 594 for 1 h at room temperature . immunoflourescent staining was visualized using confocal laser microscopy and optical sectioning with image analysis software . alkaline phospatase ( ap ) staining was performed by incubating fixed cells with ap test solution ( provided in kit ) for 2 h at room temperature . the main outcome parameters measured were icm survival , recovery and ability to attach within 24 h of warming . icms and icm - derived esc colonies were tested during the culture interval for expression of stem cell specific markers ; ssea-1 , oct-4 and sox-2 and alkaline phospatase(ap ) activity using the mouse es / ips cell characterization kit ( applied stem cell , sunnyvale , ca , usa ) . cell fixation and permeabilization was carried out using kit reagents as per manufacturer s instructions . after blocking , cells were incubated with primary antibody against ssea-1 , sox-2 or oct-4 overnight at 4c . cells were subsequently washed twice with pbs and incubated with corresponding secondary antibodies conjugated with alexa fluor 594 for 1 h at room temperature . immunoflourescent staining was visualized using confocal laser microscopy and optical sectioning with image analysis software . alkaline phospatase ( ap ) staining was performed by incubating fixed cells with ap test solution ( provided in kit ) for 2 h at room temperature . the main outcome parameters measured were icm survival , recovery and ability to attach within 24 h of warming . isolated icms were easily vitrified using a two step ethylene - glycol / dmso protocol . after plating , 95% ( 74/78 ) of icms were able to attach to mef feeder layers and proliferate in culture . this was similar to the 95% attachment rate observed with non - vitrified control icms after 24 h of culture . cryopreserved icms exhibited similar proliferation patterns as fresh control icm s and could be passaged and expanded . figure 1 depicts the morphology of vitrified - warmed icms and the rapid increase in icm outgrowth within 72 h of plating . morphologically the cells displayed characteristics typical of pluripotent stem cells [ 3 , 16 ] i.e. high ratio of nucleus to cytoplasm and prominent nucleoli . alkaline phosphatase activity was also high in the icm - derived cells , another indication of stem cell origin ( fig . immunocytochemical analysis further confirmed that icm - derived es - like cells from both fresh and vitrified icms expressed markers characteristic of pluripotent stem cells . colonies were positive for ssea-1 , sox-2 and oct-4 ( fig . 2 ) . ( b - e ) morphology of vitrified warmed icms after plating on mef feeder layer . ( b ) 3 hours after warming ( c ) 24 hours ( d ) 48 hours ( e ) 72 hours . 2expression and localization of stem cell specific markers in vitrified - warmed icms after 48 hours in culture . ( a ) icm stained with ssea 1 ( red signal ) and dapi ( blue signal ) . ( c ) sox-2 staining ( red ) , detected in both nucleus and cytoplasm . ( e ) oct-4 expression on day 16 of culture icms were isolated from mouse blastocysts . ( b - e ) morphology of vitrified warmed icms after plating on mef feeder layer . ( b ) 3 hours after warming ( c ) 24 hours ( d ) 48 hours ( e ) 72 hours . trophectodermal and mef cells adjacent to icm were negative for ap expression and localization of stem cell specific markers in vitrified - warmed icms after 48 hours in culture . ( a ) icm stained with ssea 1 ( red signal ) and dapi ( blue signal ) . ( c ) sox-2 staining ( red ) , detected in both nucleus and cytoplasm . ( e ) oct-4 expression on day 16 of culture approximately 57 days after the first passage , numerous esc - like colonies could be seen . diameters ranged from 50100 m in small colonies , 120160 m in medium colonies and over > 180 m in the larger colonies . cultures were trypsinized and re - plated when most colonies fell in the mid - size range . colonies could be dissociated in to individual cells and re - seeded on to fresh mef feeder layers . to date the embryonic stem cell characteristics and colony - forming ability of the vitrified - warmed icm cells have been maintained through three passages and up to 16 days in culture . we saw no indication of premature differentiation in the vitrified - warmed icms during the 16 days of culture . no statistical difference was found in survival , recovery or plating efficiency between the two carriers . the one distinct advantage of the hsv straw was that it allowed aseptic vitrification with no direct contact between icm cells and ln2 . icm - derived es cells from both carriers retained their pluripotency and stem cell characteristics for the 16 day culture duration tested . table 1effect of carrier on icm vitrificationrecoverysurvivalattachmentcryoloop(open)21/21 ( 100%)21/21 ( 100%)20/21 ( 95%)hsv ( closed)57/58 ( 98%)57/57 ( 100%)54/57 ( 95%)*no significant differences were found between carriers for all parameters ( p > 0.05 ) effect of carrier on icm vitrification * no significant differences were found between carriers for all parameters ( p > 0.05 ) separation of the icm from the whole blastocyst is an important step if icm banking is to be successfully applied . to this end we tried three different techniques for icm isolation but none appeared to influence overall survival after warming or subsequent retention of es phenotype during culture . survival and attachment of cryopreserved icms was 85% ( 6/7 ) with immunosurgically isolated icms , as compared to 92% ( 47/51 ) and 100% ( 21/21 ) with the micro - dissection or micro - dissection / laser ablation technique , respectively . the immunosurgical method involved lysis of trophectodermal cells surrounding the icm and thus required more hands - on manipulation of the embryo , as compared to the two micro - dissection methods . with the two microdissection methods , the icm cluster was distinctly larger , owing to the additional growth time in vitro before actual icm isolation . we observed that laser ablation decreased residual contamination of icms with trophectodermal cells before vitrification . icm - derived stem cell populations were evident and enriched with each cell passage , regardless of initial amount of trophectodermal cells contamination . establishment of stable pluripotent stem cell lines from embryos is a fairly labor - intensive process , involving numerous cell passages . the efficiency of stem cell derivation from the icm of human embryos is between 30% and 50% , depending on characteristics of the icm . vitrification of isolated icms is a novel approach for preservation of the stem cell source material , without first establishing an esc line . this may be especially valuable for human esc banking , eliminating the ethical complications of viable human embryos being kept in long term storage for future stem cell derivation . current studies with embryos indicate that cryopreservation by vitrification may result in higher cell survival rates than with traditional slow cooling [ 19 , 20 ] . successfully applied vitrification to human embryonic cell lines hes1 and hes2 in an open pulled straw . vitrification resulted in 100% recovery of es clumps , subsequent attachment and colony formation . with slow cooling , they noted that only 70% of es cell clumps were recovered after thawing and only 16% attached . moreover , developing colonies were undersized and only a minority retained their pluripotent stem cell characteristics . higher attachment and recovery rates after vitrification as compared to slow cooling were also reported by li et al . . . also observed higher post - thaw es cell differentiation and cell death with the conventional slow freezing method as compared to vitrification in either open pulled straws or sealed straws . the scope of this investigation was to demonstrate the feasibility of icm vitrification in a safe and effective manner and to provide evidence that the pluripotency of icm cells was retained for several passages . vitrification requires the use of high concentrations of cpas and ultra - rapid reduction in temperature to minimize the toxicity of cryoprotectant agents . to achieve this , special vessels or carriers are used that permit loading of cells in volumes of < 2 ul . the type of carrier can affect the rate of cooling . direct exposure of the cellular material to ln2 as with the cryoloop results in cooling rates of close to 20,000c / min . in contrast , with closed sealed systems like the hsv straw , no direct contact is made with ln2 and the insulating effect of the outer sheath may reduce the cooling rate . the current data however establish that with either the open cryoloop or closed hsv carrier , a sufficiently high rate of temperature reduction was achieved to ensure icm stem cell survival and capacity to propagate as pluripotent stem cell colonies . concerns about cross - contamination in ln2 storage containers may however make the closed hsv system more practical for long term storage of icms . all of the icms in this investigation were isolated from blastocysts derived from frozen one - cell mouse embryos . the fact that icms from embryos that had already been cryopreserved once could survive a second cryopreservaton procedure attests to the effectiveness of our vitrification technique . regardless of the initial method of icm isolation , stem cell characteristics were retained by icms through at least three cell passages and 16 days of culture . oct-4 is highly expressed in es cells , and its expression is down regulated when cells differentiate . increased expression of this marker along with continued expression of ssea-1 and sox 2 are all indicative of icm - derived cells generating pluripotent stem cells in culture . stem cell research holds great promise for the treatment of numerous diseases and for advancing the field of regenerative medicine . the demand for human embryonic stem cells is expected to increase as es cells and derived tissue are used in therapeutic trials . currently from 64 es cell lines available from nih registry , only one cell line , h1 has been approved for clinical trials . clearly , additional therapeutic grade stem cell lines derived in xeno - free culture systems and meeting stringent gmp standards will ultimately be needed . the source material for deriving such cell lines comes from embryos derived from in vitro fertilization . cryopreservaton and storage of embryonic stem cells may be more socially acceptable than establishing banks of embryos exclusively for the purpose of future embryonic stem cell derivation . currently , there are nearly 400,000 ivf - produced embryos in frozen storage in the united states alone . most of these will be used to treat infertility , but approximately 2.8% are destined to be discarded . icm banking is a novel approach to the problem of storing stem cell source material of varying genotypes without long term banking of the whole embryo and/or months of cell passaging of each donated embryo to derive a unique stem cell line . studies are underway to further expand the esc colonies , derived from vitrified mouse icms to further document pluripotent functionality and retention of stem cell characteristics over extended in vitro culture intervals . further validation of the concept of icm banking requires the establishment of an es cell line from a vitrified icm that is capable of differentiating into the three embryonic germ cell lines both in vivo , after transplantation and in vitro thru directed differentiation using defined culture conditions . long term studies are needed to determine its efficiency for establishing stable es cell lines . expansion of this work to human embryos is needed to determine if human icms are as easily vitrified and expanded as those in the mouse model . immunosurgical isolation methods for human icm are less desirable due to the use of animal - derived reagents . laser ablation combined with micro - dissection and improved culture methodology for expansion of human icm - derived stem cells without exposure to animal feeder layers or sera are still clearly needed to produce clinical grade

purposevitrification technology presents new opportunities for preservation of embryo derived stem cells without first establishing a viable esc line . this study tests the feasibility of cryopreserving icm cells using vitrification.materials and methodsicms from mouse embryos were isolated and vitrified in hsv straws or on cryoloops . upon warming , the vitrified icms were cultured and observed for attachment and morphology . colonies were passaged every 36 days . icms and icm - derived esc colonies were tested for expression of stem cell specific markers.resultsicms vitrified on both the cryoloop and the hsv straw had high survival rates . icm derived escs remained undifferentiated for several passages and demonstrated expression of typical stem cell markers ; ssea-1 , sox-2 , oct 4 and alkaline phosphatase.conclusionthis is the first report on successful vitrification of isolated icms and the subsequent derivation of esc colonies . vitrification of isolated icms is a novel approach for preservation of the stem cell source material .

Introduction Materials and methods ICM isolation ICM vitrification/warming ICM culture Stem cell characterization Data analysis Results Discussion Conclusions

embryonic stem cell ( esc ) lines are derived from the inner cell mass ( icm ) of blastocysts , and the defining feature of these cells is their ability to differentiate into a variety of cell types that encompass all three embryonic germ layers . embryonic stem cells , particularly human stem cells hold tremendous potential in the field of regenerative medicine , in addition to being a useful tool in basic scientific research and for pharmacological and cytotoxicity screening [ 4 , 5 ] . methods for efficient and reliable preservation of embryonic stem cells are necessary to preserve these pluripotent cells for therapeutic and experimental use . the traditional method for embryonic stem cell cryopreservation has been slow cooling at rates of approximately 1c per minute [ reviewed in ] . vitrification involves ultra - rapid cooling of the cell at rates as high as 20,000c / min and the use of high concentrations of cryoprotectant agents ( cpa ) . vitrification protocols have also been successfully used for cryopreservation of human embryonic stem cell lines [ 16 , 17 ] . to date there have been no reports on the cryopreservation of icm cells extracted from the blastocyst stage embryo . preservation of isolated embryo - derived stem cells without first establishing a viable es cell line offers a new avenue for banking the stem cell source material . in this investigation we explore the feasibility of icm vitrification and subsequent establishment of esc - like colonies . the zygotes were thawed as per manufacturer s instructions and cultured overnight in global blastocyst medium ( lifeglobal ; guilford , ct , usa ) supplemented with 10% serum protein substitute ( sps , cooper / sage ; pasadena , ca , usa ) . icms from mouse embryos were isolated by immunosurgery , micro - dissection or laser with micro - dissection . for both of the micro - dissection techniques , zona - free blastocysts were first plated and allowed to attach to the dish . after 48 h , the icm outgrowth was either micro- dissected free from surrounding trophectodermal cells or treated first with laser to destroy any residual trophectoderm cells before micro - dissection . vitrification of isolated icms was carried out using a two step equilibration protocol : ( 1 ) 7.5% dmso/7.5% ethylene glycol ( eg ) for 2 min , ( 2 ) 15% dmso/15% eg + 10 g / ml ficoll + 0.65 mol / l sucrose for 45 s. the basal medium used to constitute vitrification solutions was global blast medium with 20% sps . after the final equilibration step , individual icms were loaded on to the vitrification carrier and immersed in liquid nitrogen ( ln2 ) . in this study we tested two different carrier systems ; the cryoloop ( hampton research ; laguna , ca , usa ) and the hsv straw ( mid - atlantic diagnostics ; laurrel , nj , usa ) . the cryoloop is a microscopic nylon loop measuring ~1.0 mm in diameter . for cryoloop vitrification , the icm was loaded on to a thin film of fluid applied to the loop , before plunging into a vial containing ln2 . the hsv straw consists of a capillary tube with a cut out region , creating a narrow gutter and an outer straw . the cryoloop or hsv straw was immersed directly in basal culture medium containing 0.25 m sucrose . the icm cells displaced from the carriers were readily visualized using the dissecting scope . after 2 min , recovered icms were moved to the second solution containing 0.125 m sucrose for 3 min and then placed in culture . embryonic stem cell medium ( esc - sure dmem ; applied stem cell , sunnyvale , ca , usa ) supplemented with 20% fbs , 200 mm non - essential amino acids , 100 u / ml penicillin , 10 g / ml streptomycin , 100 mm sodium pyruvate and mercaptoethanol and 10 ng / ml of mouse leukemia inhibitory factor ( lif ; chemicon ; temecula , ca , usa ) was used for culture of icms . icm - derived es cells were cultivated on inactivated mouse embryonic feeder layers ( mef ) . mef cells were purchased ( applied stem cell ; sunnyvale , ca , usa ) and prepared as per manufacturer s instructions . alternatively , icms were grown without feeder cells on 0.1% gelatin coated eight or 16 well chamber slides with 25% mef conditioned medium/75% esc complete medium . these cell clumps were individually picked off of the culture surface and disaggregated by treatment with trypsin : edta solution for 35 min at room temperature . culture medium was exchanged every 48 h. icms and icm - derived esc colonies were tested during the culture interval for expression of stem cell specific markers ; ssea-1 , oct-4 and sox-2 and alkaline phospatase(ap ) activity using the mouse es / ips cell characterization kit ( applied stem cell , sunnyvale , ca , usa ) . after blocking , cells were incubated with primary antibody against ssea-1 , sox-2 or oct-4 overnight at 4c . immunoflourescent staining was visualized using confocal laser microscopy and optical sectioning with image analysis software . the main outcome parameters measured were icm survival , recovery and ability to attach within 24 h of warming . icms from mouse embryos were isolated by immunosurgery , micro - dissection or laser with micro - dissection . lysed trophectodermal cells were removed from icms by repeated pipetting with finely drawn glass micropipettes . for both of the micro - dissection techniques , zona - free blastocysts were first plated and allowed to attach to the dish . after 48 h , the icm outgrowth was either micro- dissected free from surrounding trophectodermal cells or treated first with laser to destroy any residual trophectoderm cells before micro - dissection . vitrification of isolated icms was carried out using a two step equilibration protocol : ( 1 ) 7.5% dmso/7.5% ethylene glycol ( eg ) for 2 min , ( 2 ) 15% dmso/15% eg + 10 g / ml ficoll + 0.65 mol / l sucrose for 45 s. the basal medium used to constitute vitrification solutions was global blast medium with 20% sps . after the final equilibration step , individual icms were loaded on to the vitrification carrier and immersed in liquid nitrogen ( ln2 ) . in this study we tested two different carrier systems ; the cryoloop ( hampton research ; laguna , ca , usa ) and the hsv straw ( mid - atlantic diagnostics ; laurrel , nj , usa ) . the cryoloop is a microscopic nylon loop measuring ~1.0 mm in diameter . for cryoloop vitrification , the icm was loaded on to a thin film of fluid applied to the loop , before plunging into a vial containing ln2 . the hsv straw consists of a capillary tube with a cut out region , creating a narrow gutter and an outer straw . the cryoloop or hsv straw was immersed directly in basal culture medium containing 0.25 m sucrose . the icm cells displaced from the carriers were readily visualized using the dissecting scope . after 2 min , recovered icms were moved to the second solution containing 0.125 m sucrose for 3 min and then placed in culture vitrification of isolated icms was carried out using a two step equilibration protocol : ( 1 ) 7.5% dmso/7.5% ethylene glycol ( eg ) for 2 min , ( 2 ) 15% dmso/15% eg + 10 g / ml ficoll + 0.65 mol / l sucrose for 45 s. the basal medium used to constitute vitrification solutions was global blast medium with 20% sps . after the final equilibration step , individual icms were loaded on to the vitrification carrier and immersed in liquid nitrogen ( ln2 ) . in this study we tested two different carrier systems ; the cryoloop ( hampton research ; laguna , ca , usa ) and the hsv straw ( mid - atlantic diagnostics ; laurrel , nj , usa ) . the cryoloop is a microscopic nylon loop measuring ~1.0 mm in diameter . for cryoloop vitrification , the icm was loaded on to a thin film of fluid applied to the loop , before plunging into a vial containing ln2 . the hsv straw consists of a capillary tube with a cut out region , creating a narrow gutter and an outer straw . the cryoloop or hsv straw was immersed directly in basal culture medium containing 0.25 m sucrose . the icm cells displaced from the carriers were readily visualized using the dissecting scope . after 2 min , recovered icms were moved to the second solution containing 0.125 m sucrose for 3 min and then placed in culture embryonic stem cell medium ( esc - sure dmem ; applied stem cell , sunnyvale , ca , usa ) supplemented with 20% fbs , 200 mm non - essential amino acids , 100 u / ml penicillin , 10 g / ml streptomycin , 100 mm sodium pyruvate and mercaptoethanol and 10 ng / ml of mouse leukemia inhibitory factor ( lif ; chemicon ; temecula , ca , usa ) was used for culture of icms . icm - derived es cells were cultivated on inactivated mouse embryonic feeder layers ( mef ) . mef cells were purchased ( applied stem cell ; sunnyvale , ca , usa ) and prepared as per manufacturer s instructions . these cell clumps were individually picked off of the culture surface and disaggregated by treatment with trypsin : edta solution for 35 min at room temperature . culture medium was exchanged every 48 h. icms and icm - derived esc colonies were tested during the culture interval for expression of stem cell specific markers ; ssea-1 , oct-4 and sox-2 and alkaline phospatase(ap ) activity using the mouse es / ips cell characterization kit ( applied stem cell , sunnyvale , ca , usa ) . after blocking , cells were incubated with primary antibody against ssea-1 , sox-2 or oct-4 overnight at 4c . cells were subsequently washed twice with pbs and incubated with corresponding secondary antibodies conjugated with alexa fluor 594 for 1 h at room temperature . icms and icm - derived esc colonies were tested during the culture interval for expression of stem cell specific markers ; ssea-1 , oct-4 and sox-2 and alkaline phospatase(ap ) activity using the mouse es / ips cell characterization kit ( applied stem cell , sunnyvale , ca , usa ) . cell fixation and permeabilization was carried out using kit reagents as per manufacturer s instructions . after blocking , cells were incubated with primary antibody against ssea-1 , sox-2 or oct-4 overnight at 4c . cells were subsequently washed twice with pbs and incubated with corresponding secondary antibodies conjugated with alexa fluor 594 for 1 h at room temperature . isolated icms were easily vitrified using a two step ethylene - glycol / dmso protocol . after plating , 95% ( 74/78 ) of icms were able to attach to mef feeder layers and proliferate in culture . figure 1 depicts the morphology of vitrified - warmed icms and the rapid increase in icm outgrowth within 72 h of plating . morphologically the cells displayed characteristics typical of pluripotent stem cells [ 3 , 16 ] i.e. alkaline phosphatase activity was also high in the icm - derived cells , another indication of stem cell origin ( fig . immunocytochemical analysis further confirmed that icm - derived es - like cells from both fresh and vitrified icms expressed markers characteristic of pluripotent stem cells . colonies were positive for ssea-1 , sox-2 and oct-4 ( fig . 2expression and localization of stem cell specific markers in vitrified - warmed icms after 48 hours in culture . ( c ) sox-2 staining ( red ) , detected in both nucleus and cytoplasm . ( e ) oct-4 expression on day 16 of culture icms were isolated from mouse blastocysts . trophectodermal and mef cells adjacent to icm were negative for ap expression and localization of stem cell specific markers in vitrified - warmed icms after 48 hours in culture . ( e ) oct-4 expression on day 16 of culture approximately 57 days after the first passage , numerous esc - like colonies could be seen . colonies could be dissociated in to individual cells and re - seeded on to fresh mef feeder layers . to date the embryonic stem cell characteristics and colony - forming ability of the vitrified - warmed icm cells have been maintained through three passages and up to 16 days in culture . we saw no indication of premature differentiation in the vitrified - warmed icms during the 16 days of culture . the one distinct advantage of the hsv straw was that it allowed aseptic vitrification with no direct contact between icm cells and ln2 . icm - derived es cells from both carriers retained their pluripotency and stem cell characteristics for the 16 day culture duration tested . table 1effect of carrier on icm vitrificationrecoverysurvivalattachmentcryoloop(open)21/21 ( 100%)21/21 ( 100%)20/21 ( 95%)hsv ( closed)57/58 ( 98%)57/57 ( 100%)54/57 ( 95%)*no significant differences were found between carriers for all parameters ( p > 0.05 ) effect of carrier on icm vitrification * no significant differences were found between carriers for all parameters ( p > 0.05 ) separation of the icm from the whole blastocyst is an important step if icm banking is to be successfully applied . to this end we tried three different techniques for icm isolation but none appeared to influence overall survival after warming or subsequent retention of es phenotype during culture . survival and attachment of cryopreserved icms was 85% ( 6/7 ) with immunosurgically isolated icms , as compared to 92% ( 47/51 ) and 100% ( 21/21 ) with the micro - dissection or micro - dissection / laser ablation technique , respectively . the immunosurgical method involved lysis of trophectodermal cells surrounding the icm and thus required more hands - on manipulation of the embryo , as compared to the two micro - dissection methods . with the two microdissection methods , the icm cluster was distinctly larger , owing to the additional growth time in vitro before actual icm isolation . we observed that laser ablation decreased residual contamination of icms with trophectodermal cells before vitrification . icm - derived stem cell populations were evident and enriched with each cell passage , regardless of initial amount of trophectodermal cells contamination . establishment of stable pluripotent stem cell lines from embryos is a fairly labor - intensive process , involving numerous cell passages . the efficiency of stem cell derivation from the icm of human embryos is between 30% and 50% , depending on characteristics of the icm . vitrification of isolated icms is a novel approach for preservation of the stem cell source material , without first establishing an esc line . this may be especially valuable for human esc banking , eliminating the ethical complications of viable human embryos being kept in long term storage for future stem cell derivation . current studies with embryos indicate that cryopreservation by vitrification may result in higher cell survival rates than with traditional slow cooling [ 19 , 20 ] . vitrification resulted in 100% recovery of es clumps , subsequent attachment and colony formation . moreover , developing colonies were undersized and only a minority retained their pluripotent stem cell characteristics . higher attachment and recovery rates after vitrification as compared to slow cooling were also reported by li et al . also observed higher post - thaw es cell differentiation and cell death with the conventional slow freezing method as compared to vitrification in either open pulled straws or sealed straws . the scope of this investigation was to demonstrate the feasibility of icm vitrification in a safe and effective manner and to provide evidence that the pluripotency of icm cells was retained for several passages . to achieve this , special vessels or carriers are used that permit loading of cells in volumes of < 2 ul . direct exposure of the cellular material to ln2 as with the cryoloop results in cooling rates of close to 20,000c / min . in contrast , with closed sealed systems like the hsv straw , no direct contact is made with ln2 and the insulating effect of the outer sheath may reduce the cooling rate . the current data however establish that with either the open cryoloop or closed hsv carrier , a sufficiently high rate of temperature reduction was achieved to ensure icm stem cell survival and capacity to propagate as pluripotent stem cell colonies . all of the icms in this investigation were isolated from blastocysts derived from frozen one - cell mouse embryos . regardless of the initial method of icm isolation , stem cell characteristics were retained by icms through at least three cell passages and 16 days of culture . increased expression of this marker along with continued expression of ssea-1 and sox 2 are all indicative of icm - derived cells generating pluripotent stem cells in culture . stem cell research holds great promise for the treatment of numerous diseases and for advancing the field of regenerative medicine . the demand for human embryonic stem cells is expected to increase as es cells and derived tissue are used in therapeutic trials . clearly , additional therapeutic grade stem cell lines derived in xeno - free culture systems and meeting stringent gmp standards will ultimately be needed . the source material for deriving such cell lines comes from embryos derived from in vitro fertilization . cryopreservaton and storage of embryonic stem cells may be more socially acceptable than establishing banks of embryos exclusively for the purpose of future embryonic stem cell derivation . icm banking is a novel approach to the problem of storing stem cell source material of varying genotypes without long term banking of the whole embryo and/or months of cell passaging of each donated embryo to derive a unique stem cell line . studies are underway to further expand the esc colonies , derived from vitrified mouse icms to further document pluripotent functionality and retention of stem cell characteristics over extended in vitro culture intervals . further validation of the concept of icm banking requires the establishment of an es cell line from a vitrified icm that is capable of differentiating into the three embryonic germ cell lines both in vivo , after transplantation and in vitro thru directed differentiation using defined culture conditions . immunosurgical isolation methods for human icm are less desirable due to the use of animal - derived reagents . laser ablation combined with micro - dissection and improved culture methodology for expansion of human icm - derived stem cells without exposure to animal feeder layers or sera are still clearly needed to produce clinical grade

my laboratory has been studying longitudinally a large group of healthy , caucasian children from middle - class families who have been followed from 4 months to 11 years : each infant was classified at 4 months of age into one of four temperamental groups based on their behavior to a standard battery of visual , auditory , and olfactory stimuli . infants who showed a combination of frequent , vigorous motor activity combined with frequent crying were classified as high reactive ( 22% of the sample ) . infants who showed the opposite profile of infrequent motor activity and minimal crying were classified as low reactive ( 40% ) . infants who showed infrequent , motor activity , but frequent , crying were classified as distressed ( 25% ) , and infants who showed frequent , motor activity , but minimal distress were classified as aroused ( 10% ) . it is assumed , but not yet proven , that the high- and lowreactive groups inherit different profiles of excitability in the amygdala and/or bed nucleus and their projections . the children from these temperamental groups were evaluated twice in the second year for their reaction to unfamiliar people , situations , and procedures . the 14and 21 -month - old children who had been categorized as high reactive as infants were more likely than the low reactives to display high levels of fear to unfamiliar people , rooms , and events . this relationship has been verified by fox and colleagues , who also found that 1-yearolds who had been classified as high - reactive infants at 4 months were more fearful than others when they encountered unfamiliar events . these children were observed when they were four and a half years old in a play session with two other unfamiliar children of the same sex and age , while the three mothers sat on a couch in the playroom . each child was classified reliably as inhibited , uninhibited , or neither , based on their behavior with the other children and their reactions to two unfamiliar events that occurred after the play session . they were quiet , spent long times close to their mother , and did not initiate social interaction with the other children . when the children were seven and a half years old , we evaluated the prevalence of anxious symptoms in 51 high ractives , 60 low reactives , and 53 children from the other two temperamental groups . the classification of anxious symptoms , which included extreme shyness , worry about , the future , fear of thunderstorms , animals , or loud noises , recurrent , nightmares , and occasional reluctance to go to school , was based on questionnaire and interview data with the mother and the child 's teacher . a total of 43 of the 164 children met criteria for possession of anxious symptoms . forty - five percent , of the children who had been high - reactive infants , compared with 15% of low ractives , had anxious symptoms ( chi - square=12.8 , p<0.01 ) high reactives who had anxious symptoms were more fearful in the second year and had higher sitting diastolic blood pressures and a greater magnitude of cooling of the temperature of the fingertips across a series of digit , recall problems , compared with other high reactives . cooling of the fingertips is the result of sympathetic inervation of the arteriovenous anastomoses under the surface of the skin . these children were evaluated most recently when they were 11 years old . the 3-hour battery consisted of both behavioral and biological assessments . the behavioral data included the number of spontaneous comments and smiles displayed toward the examiner during the first 18 min of interaction , a reliable rating ( 4-point scale ) of the degree of uncertainty , tension , and anxiety displayed by the child in this setting , and a maternal qsort of 28 items describing the child 's behavior . four different , classes of biological variables , each under the potential influence of the amygdala , were also quantified . these biological variables were : ( i ) asymmetry in the magnitude of dcsynchronization of alpha frequencies in the eeg ; ( ii ) magnitude of the evoked potential from the inferior colliculus to a series of clicks ; ( iii ) sympathetic tone in the cardiovascular system ; and ( iv ) the magnitude of the wave form at 400 ms in the eventrelated potential to discrepant , visual scenes . most children and adults have less alpha power in the left than in the right , frontal area when at . rest , suggesting greater activation of cortical pyramidal neurons in the left , frontal lobe . further , individuals with this eeg profile report more sanguine moods and fewer signs of anxiety than the smaller proportion , who show greater activation on the right , side . the amygdala sends ipsilateral projections to the frontal lobe through the basal nucleus of meynert and it is likely that these projections contribute to the asymmetry in the alpha band of the eeg . a child who had greater activation in the right amygdala should show greater desynchronization of alpha frequencies in the right , hemisphere and would be classified as right hemisphere active . the brain stem auditory evoked response ( baer ) , elicited by a series of clicks delivered through earphones , was a relevant , measure because variation in the magnitude or latency of the fifth wave in the baer response - called wave 5 - differentiates between personality and clinical categories . in addition , adults with panic disorder show a larger wave 5 than do controls . the peak of the fifth wave is believed to represent , the termination of the lateral lemniscus on the inferior colliculus . the theoretical relevance of this fact is that , the amygdala projects to the inferior colliculus through both the central gray and the locus cereleus and , therefore , children with a more excitable amygdala should display a larger wave 5 than others . the rationale behind recording the event - related potential to discrepant , visual stimuli derived from the assumption that the amygdala reacts to discrepant or unexpected events and projects to cortical neurons that mediate the event - related potential . if high reactives possess a low threshold of reactivity in the amygdala and its projections , then they should show a larger eventrelated potential to discrepant events . the usual wave form to a discrepant , event , occurs between 1 50 and 800 ms with a peak between 300 and 400 ms . pradolescent children most often show a negative wave form that is called nc ( for negative component ) . each child was presented , through goggles , two series of pictures with 169 pictures in each series . in the first scries , 70% of the pictures were of the same item ( a fireworks display ) , 15% were of the same flower , the oddball stimulus , and the remaining 15% were each different , but ecologically valid ( a chair or kitchen utensil ) . the frequent picture presented 70% of the time was a yellow fire hydrant , the oddball stimulus was a different , flower , and the remaining fifteen percent of the pictures were each different , but ecologically invalid ( for example , a chair with three legs ) . these pictures were called novel invalid . finally , we recorded measures of cardiovascular activity as an index of reactivity in the sympathetic nervous system . the amygdala sends varied projections to the sympathetic system and , therefore , we assumed that high reactives would show signs of greater sympathetic reactivity than low reactives . the two major variables were the ratio of high- to lowfrequency power in the cardiac spectrum while the child was laying supine . a fast fourier transformation of the distribution of bcat - to - beat differences in the sample of resting heart rate usually reveals two peaks in the distribution . the higher frequency , around 0.2 hz , represents the parasympathetic influence of respiration on heart ratc - vagal tone . the lower frequency band , from 0.02 to 0.10 hz , represents both sympathetic and parasympathetic influences on heart , rate , due to cycles of change in blood pressure and body temperature . higher relative power in the low frequency band is usually correlated with a high resting heart rate and is indicative of higher sympathetic tone . my laboratory has been studying longitudinally a large group of healthy , caucasian children from middle - class families who have been followed from 4 months to 11 years : each infant was classified at 4 months of age into one of four temperamental groups based on their behavior to a standard battery of visual , auditory , and olfactory stimuli . infants who showed a combination of frequent , vigorous motor activity combined with frequent crying were classified as high reactive ( 22% of the sample ) . infants who showed the opposite profile of infrequent motor activity and minimal crying were classified as low reactive ( 40% ) . infants who showed infrequent , motor activity , but frequent , crying were classified as distressed ( 25% ) , and infants who showed frequent , motor activity , but minimal distress were classified as aroused ( 10% ) . it is assumed , but not yet proven , that the high- and lowreactive groups inherit different profiles of excitability in the amygdala and/or bed nucleus and their projections . the children from these temperamental groups were evaluated twice in the second year for their reaction to unfamiliar people , situations , and procedures . the 14and 21 -month - old children who had been categorized as high reactive as infants were more likely than the low reactives to display high levels of fear to unfamiliar people , rooms , and events . this relationship has been verified by fox and colleagues , who also found that 1-yearolds who had been classified as high - reactive infants at 4 months were more fearful than others when they encountered unfamiliar events . these children were observed when they were four and a half years old in a play session with two other unfamiliar children of the same sex and age , while the three mothers sat on a couch in the playroom . each child was classified reliably as inhibited , uninhibited , or neither , based on their behavior with the other children and their reactions to two unfamiliar events that occurred after the play session . they were quiet , spent long times close to their mother , and did not initiate social interaction with the other children . when the children were seven and a half years old , we evaluated the prevalence of anxious symptoms in 51 high ractives , 60 low reactives , and 53 children from the other two temperamental groups . the classification of anxious symptoms , which included extreme shyness , worry about , the future , fear of thunderstorms , animals , or loud noises , recurrent , nightmares , and occasional reluctance to go to school , was based on questionnaire and interview data with the mother and the child 's teacher . a total of 43 of the 164 children met criteria for possession of anxious symptoms . forty - five percent , of the children who had been high - reactive infants , compared with 15% of low ractives , had anxious symptoms ( chi - square=12.8 , p<0.01 ) high reactives who had anxious symptoms were more fearful in the second year and had higher sitting diastolic blood pressures and a greater magnitude of cooling of the temperature of the fingertips across a series of digit , recall problems , compared with other high reactives . cooling of the fingertips is the result of sympathetic inervation of the arteriovenous anastomoses under the surface of the skin . these children were evaluated most recently when they were 11 years old . the 3-hour battery consisted of both behavioral and biological assessments . the behavioral data included the number of spontaneous comments and smiles displayed toward the examiner during the first 18 min of interaction , a reliable rating ( 4-point scale ) of the degree of uncertainty , tension , and anxiety displayed by the child in this setting , and a maternal qsort of 28 items describing the child 's behavior . four different , classes of biological variables , each under the potential influence of the amygdala , were also quantified . these biological variables were : ( i ) asymmetry in the magnitude of dcsynchronization of alpha frequencies in the eeg ; ( ii ) magnitude of the evoked potential from the inferior colliculus to a series of clicks ; ( iii ) sympathetic tone in the cardiovascular system ; and ( iv ) the magnitude of the wave form at 400 ms in the eventrelated potential to discrepant , visual scenes . most children and adults have less alpha power in the left than in the right , frontal area when at . rest , suggesting greater activation of cortical pyramidal neurons in the left , frontal lobe . further , individuals with this eeg profile report more sanguine moods and fewer signs of anxiety than the smaller proportion , who show greater activation on the right , side . the amygdala sends ipsilateral projections to the frontal lobe through the basal nucleus of meynert and it is likely that these projections contribute to the asymmetry in the alpha band of the eeg . a child who had greater activation in the right amygdala should show greater desynchronization of alpha frequencies in the right , hemisphere and would be classified as right hemisphere active . the brain stem auditory evoked response ( baer ) , elicited by a series of clicks delivered through earphones , was a relevant , measure because variation in the magnitude or latency of the fifth wave in the baer response - called wave 5 - differentiates between personality and clinical categories . in addition , adults with panic disorder show a larger wave 5 than do controls . the peak of the fifth wave is believed to represent , the termination of the lateral lemniscus on the inferior colliculus . the theoretical relevance of this fact is that , the amygdala projects to the inferior colliculus through both the central gray and the locus cereleus and , therefore , children with a more excitable amygdala should display a larger wave 5 than others . the rationale behind recording the event - related potential to discrepant , visual stimuli derived from the assumption that the amygdala reacts to discrepant or unexpected events and projects to cortical neurons that mediate the event - related potential . if high reactives possess a low threshold of reactivity in the amygdala and its projections , then they should show a larger eventrelated potential to discrepant events . the usual wave form to a discrepant , event , occurs between 1 50 and 800 ms with a peak between 300 and 400 ms . pradolescent children most often show a negative wave form that is called nc ( for negative component ) . each child was presented , through goggles , two series of pictures with 169 pictures in each series . in the first scries , 70% of the pictures were of the same item ( a fireworks display ) , 15% were of the same flower , the oddball stimulus , and the remaining 15% were each different , but ecologically valid ( a chair or kitchen utensil ) . the frequent picture presented 70% of the time was a yellow fire hydrant , the oddball stimulus was a different , flower , and the remaining fifteen percent of the pictures were each different , but ecologically invalid ( for example , a chair with three legs ) . these pictures were called novel invalid . finally , we recorded measures of cardiovascular activity as an index of reactivity in the sympathetic nervous system . the amygdala sends varied projections to the sympathetic system and , therefore , we assumed that high reactives would show signs of greater sympathetic reactivity than low reactives . the two major variables were the ratio of high- to lowfrequency power in the cardiac spectrum while the child was laying supine . a fast fourier transformation of the distribution of bcat - to - beat differences in the sample of resting heart rate usually reveals two peaks in the distribution . the higher frequency , around 0.2 hz , represents the parasympathetic influence of respiration on heart ratc - vagal tone . the lower frequency band , from 0.02 to 0.10 hz , represents both sympathetic and parasympathetic influences on heart , rate , due to cycles of change in blood pressure and body temperature . higher relative power in the low frequency band is usually correlated with a high resting heart rate and is indicative of higher sympathetic tone . the high - reactive children were more subdued and anxious at the 11-year evaluation than the low ractives and were rated as more anxious and inhibited during the first 18 min of the interview ( table i ) . twice as many high as low reactives were rated as extremely inhibited ( rating of 4 ; awarded to children who made very few comments and smiles , displayed a great deal of motor tension , spoke in a soft voice , and showed other signs of concern ) . twice as many low as high ractives were rated as minimally anxious and uninhibited with a rating of 1 , which described a maximally relaxed and spontaneous child ( chi - square=11.8 , p<0.01 ) . further , more high than low reactives had values for both number of spontaneous comments and smiles in the lowest quartile of the two distributions ; more low ractives had values in the highest quartile for both measures ( chi - square=4.2,p<0.05 ) . thus , the infant temperamental profiles predicted , to a modest degree , spontaneity or a subdued style with the unfamiliar adult examiner . one half of the current group of high and low reactives had been seen when they were four and a half and seven and a half years of age . a similar rating of degree of anxiety / inhibition on a 4-point scale was assigned to each child based on 90 min of interaction with a different , but unfamiliar female examiner . seventy percent , of the low reactives , but only 13% of the high ractives were uninhibited at all three ages ; 38% of high reactives , but only 6% of low reactives were inhibited at all three ages ( chisquare=21.3 , p<0.001 ) . it was rare for a low - reactive infant to become a consistently inhibited child or for a high - reactive infant , to become a consistently uninhibited child . as expected , the uninhibited profile was better preserved because family and friends encourage sociability and discourage shyness and timidity . seven descriptive items on the maternal q - sort referred to shyness or sociability in the child . we computed the mean ranks the mother assigned to her child for the three shy and the four sociable items . high reactives were described as more shy and less sociable than the low ractives ( r(149)=3.91 , p<0.01 ) . figure 1 illustrates the mean standard scores for high and low reactives on the four biological variables at 11 years of age : right parietal activation in the eeg , wave 5 magnitudes , sympathetic tone ( a low ratio and a high resting heart rate ) , and the mean of the integrated voltages from 400 to 1000 ms for the event - related potential to the first oddball flower and the novel invalid scenes . the high ractives had greater eeg activation at the right parietal site ( r(152)=2.53 , p<0.05 ) . further , the high reactives who had been highly fearful in the second year showed greater activation in the right frontal area compared with low reactives who were equally fearful in the second year . the high reactives also had significantly larger wave 5 values ( r(125)=2.87 , p<0.05 ) , and this variable best separated the two temperamental groups . the high reactives also had greater sympathetic tone in the cardiovascular system ; 49% of high reactives , but only 32% of low reactives , combined a low ratio in the spectral analysis with a high resting heart rate ; 32% of the low ractives , but only 16% of high ractives , combined a high ratio with a low resting heart , rate ( chisquare=4.9 , p<0.05 ) . it is also of interest that high sympathetic tone was the best correlate of behavior . the children with high sympathetic tone , compared to those with high vagal tone , spoke less often , were rated as more anxious , and were described by their mother as shy . although the eeg , wave 5 , and event - related potential data also separated the two temperamental groups , these measures were less closely related to the child 's behavior . one explanation is that sympathetic activity is likely to influence the orbitofrontal cortex , which mediates a conscious awareness of feeling tone . a rise in heart , rate and blood pressure and a change in breathing results in information being sent to the brain through the medulla to provoke changes in the orbitofrontal cortex that can evoke an alteration in conscious feeling . a subdued mood and avoidance behavior can be consequences of this altered feeling tone . by contrast , activity in the inferior colliculus and the pyramidal neurons of the cortex are less likely to influence orbitofrontal neurons and , therefore , no change in feeling tone occurs and there should be a minimal relationship to behavior . it is important that among high and low reactives , who were equally subdued in their behavior in the laboratory , only the high ractives showed the biological features of right parietal asymmetry and a large wave 5 . that is why it is important , for investigators and clinicians to gather biological data to supplement , their behavioral observations and interviews . finally , the high reactives had significantly larger nc voltages to the first , oddball picture and the novel invalid pictures ( r(136)=2.00 , p<0.05 ) . further , the correlation between the voltages and these two classes of pictures across frontal and parietal sites were always positive and significant , for high - reactive children , but not for the low reactives . that is , only high reactives showed coherence in the magnitude of the nc across disparate cortical sites , implying that the discrepant scenes recruited neurons over a broader cortical area . there was an interesting asymmetry in the sensitivity of low compared with high values on the four biological measurements . low values better differentiated low from high reactives than did high values , suggesting that it is easier for low - reactive than for high - reactive children to attain a state of low cortical and autonomic arousal , even though the former can attain , temporarily , a state of higher arousal in a laboratory setting . the psychological advantages of low arousal are less obvious and apparently a smaller proportion of individuals are able to reach a state of relaxation . about 1 in 4 children who had been high reactive and 1 in 4 children who had been low reactive developed a behavioral and a biological profile at age 11 that was in accord with theoretical expectations , while only 1 of 20 children developed a profile of social behavior and biology that , violated their expected profile . this result , is of interest in light of the varied social experiences that these children have encountered over the prior 11 years . most children displayed behavioral and biological patterns that were characteristic of randomly selected children from middle - class , caucasian populations . thus , the prediction that a high - reactive infant will not be highly sociable and exuberant , and show low biological arousal at age 11 can be made with much greater confidence than the prediction that this category of child will be extremely subdued and anxious , and show signs of high arousal in cortical and autonomic targets . the suggestion that a temperamental bias constrains development more effectively than it determines particular outcomes applies to environmental conditions as well . if all one knows about a group of 1 00 children is that , they were born to economically secure , well - educated , nurturing parents and must predict the likely psychological adult , outcomes , the most accurate guesses will refer to the profiles that should not occur : criminality , school failure , psychosis , homelessness , drug addiction , and poverty . predictions concerning the more specific features that , will be part of the adult personality are less likely to be validated . the final fate of a neural crest cell in a 3-weekold embryo , whether sensory ganglion , melanocyte , or a muscle of the heart , is less certain than the fact that this cell will definitely not become connective tissue or part of the reproductive system . the high - reactive children were more subdued and anxious at the 11-year evaluation than the low ractives and were rated as more anxious and inhibited during the first 18 min of the interview ( table i ) . twice as many high as low reactives were rated as extremely inhibited ( rating of 4 ; awarded to children who made very few comments and smiles , displayed a great deal of motor tension , spoke in a soft voice , and showed other signs of concern ) . twice as many low as high ractives were rated as minimally anxious and uninhibited with a rating of 1 , which described a maximally relaxed and spontaneous child ( chi - square=11.8 , p<0.01 ) . further , more high than low reactives had values for both number of spontaneous comments and smiles in the lowest quartile of the two distributions ; more low ractives had values in the highest quartile for both measures ( chi - square=4.2,p<0.05 ) . thus , the infant temperamental profiles predicted , to a modest degree , spontaneity or a subdued style with the unfamiliar adult examiner . one half of the current group of high and low reactives had been seen when they were four and a half and seven and a half years of age . a similar rating of degree of anxiety / inhibition on a 4-point scale was assigned to each child based on 90 min of interaction with a different , but unfamiliar female examiner . seventy percent , of the low reactives , but only 13% of the high ractives were uninhibited at all three ages ; 38% of high reactives , but only 6% of low reactives were inhibited at all three ages ( chisquare=21.3 , p<0.001 ) . it was rare for a low - reactive infant to become a consistently inhibited child or for a high - reactive infant , to become a consistently uninhibited child . as expected , the uninhibited profile was better preserved because family and friends encourage sociability and discourage shyness and timidity . seven descriptive items on the maternal q - sort referred to shyness or sociability in the child . we computed the mean ranks the mother assigned to her child for the three shy and the four sociable items . high reactives were described as more shy and less sociable than the low ractives ( r(149)=3.91 , p<0.01 ) . figure 1 illustrates the mean standard scores for high and low reactives on the four biological variables at 11 years of age : right parietal activation in the eeg , wave 5 magnitudes , sympathetic tone ( a low ratio and a high resting heart rate ) , and the mean of the integrated voltages from 400 to 1000 ms for the event - related potential to the first oddball flower and the novel invalid scenes . the high ractives had greater eeg activation at the right parietal site ( r(152)=2.53 , p<0.05 ) . further , the high reactives who had been highly fearful in the second year showed greater activation in the right frontal area compared with low reactives who were equally fearful in the second year . the high reactives also had significantly larger wave 5 values ( r(125)=2.87 , p<0.05 ) , and this variable best separated the two temperamental groups . the high reactives also had greater sympathetic tone in the cardiovascular system ; 49% of high reactives , but only 32% of low reactives , combined a low ratio in the spectral analysis with a high resting heart rate ; 32% of the low ractives , but only 16% of high ractives , combined a high ratio with a low resting heart , rate ( chisquare=4.9 , p<0.05 ) . it is also of interest that high sympathetic tone was the best correlate of behavior . the children with high sympathetic tone , compared to those with high vagal tone , spoke less often , were rated as more anxious , and were described by their mother as shy . although the eeg , wave 5 , and event - related potential data also separated the two temperamental groups , these measures were less closely related to the child 's behavior . one explanation is that sympathetic activity is likely to influence the orbitofrontal cortex , which mediates a conscious awareness of feeling tone . a rise in heart , rate and blood pressure and a change in breathing results in information being sent to the brain through the medulla to provoke changes in the orbitofrontal cortex that can evoke an alteration in conscious feeling . a subdued mood and avoidance behavior can be consequences of this altered feeling tone . by contrast , activity in the inferior colliculus and the pyramidal neurons of the cortex are less likely to influence orbitofrontal neurons and , therefore , no change in feeling tone occurs and there should be a minimal relationship to behavior . it is important that among high and low reactives , who were equally subdued in their behavior in the laboratory , only the high ractives showed the biological features of right parietal asymmetry and a large wave 5 . that is why it is important , for investigators and clinicians to gather biological data to supplement , their behavioral observations and interviews . finally , the high reactives had significantly larger nc voltages to the first , oddball picture and the novel invalid pictures ( r(136)=2.00 , p<0.05 ) . further , the correlation between the voltages and these two classes of pictures across frontal and parietal sites were always positive and significant , for high - reactive children , but not for the low reactives . that is , only high reactives showed coherence in the magnitude of the nc across disparate cortical sites , implying that the discrepant scenes recruited neurons over a broader cortical area . there was an interesting asymmetry in the sensitivity of low compared with high values on the four biological measurements . low values better differentiated low from high reactives than did high values , suggesting that it is easier for low - reactive than for high - reactive children to attain a state of low cortical and autonomic arousal , even though the former can attain , temporarily , a state of higher arousal in a laboratory setting . the psychological advantages of low arousal are less obvious and apparently a smaller proportion of individuals are able to reach a state of relaxation . about 1 in 4 children who had been high reactive and 1 in 4 children who had been low reactive developed a behavioral and a biological profile at age 11 that was in accord with theoretical expectations , while only 1 of 20 children developed a profile of social behavior and biology that , violated their expected profile . this result , is of interest in light of the varied social experiences that these children have encountered over the prior 11 years . most children displayed behavioral and biological patterns that were characteristic of randomly selected children from middle - class , caucasian populations . thus , the prediction that a high - reactive infant will not be highly sociable and exuberant , and show low biological arousal at age 11 can be made with much greater confidence than the prediction that this category of child will be extremely subdued and anxious , and show signs of high arousal in cortical and autonomic targets . the suggestion that a temperamental bias constrains development more effectively than it determines particular outcomes applies to environmental conditions as well . if all one knows about a group of 1 00 children is that , they were born to economically secure , well - educated , nurturing parents and must predict the likely psychological adult , outcomes , the most accurate guesses will refer to the profiles that should not occur : criminality , school failure , psychosis , homelessness , drug addiction , and poverty . predictions concerning the more specific features that , will be part of the adult personality are less likely to be validated . the final fate of a neural crest cell in a 3-weekold embryo , whether sensory ganglion , melanocyte , or a muscle of the heart , is less certain than the fact that this cell will definitely not become connective tissue or part of the reproductive system . the evidence affirms the view that a temperamental bias for high reactivity in infancy , detectable early in development , is predictive of a personality profile marked by shyness , timidity , and anxiety to unfamiliar events and this behavioral phenotype is accompanied by a select biological pattern that implies amygdalar excitability . the question of greater relevance for clinicians is whether this category of child is at higher risk for any of the current psychiatric anxiety disorders . an independent group of 1 3-year - olds , who had been classified as inhibited or uninhibited in the second year , were interviewed by a psychiatrist who had no knowledge of their initial temperamental classification or later laboratory behavior . more of the adolescents who had been inhibited rather than uninhibited in the second year had symptoms of social anxiety ( 61% versus 27% ) . however , these inhibited children were not more likely to have developed specific target phobias or separation anxiety , implying that , inhibited children might be at special risk for the development of social phobia during the adolescent or adult years . the feared target of the social phobic is concern over the evaluations made by unfamiliar people in unfamiliar situations . by contrast , the feared target of the phobic patient is a very specific object that can harm or contaminate the agent . the feared target of the panic patient is anxiety over an unexpected autonomic surge . the targets and physiological profiles of these three groups are different and probably comprise different psychiatric categories . first , 20% of the children were high - reactive infants , but the prevalence of social phobia is less that this fact suggests that many high reactives find an adaptive niche in their society that allows them to titer unpredictable social encounters . the biography of t. s. eliot implies that he may have been a high - reactive infant , for he certainly was a shy child . his decision to become a poet permitted a degree of isolation that his temperament required . the second fact is that more females than males are diagnosed with social phobia , although there is no excess of girls over boys who are classified as high reactive during infancy . this fact suggests that cultural ideals and differential socialization of boys and girls contribute to the sex difference in social phobia . an excerpt from an essay written by one of the 11-year - old children , who was a high - reactive infant and a fearful toddler supports this claim . i have always been more of an anxious person than some other people ... it took me a very long time to realize how to cope with this heightened slate of nervousness ... i have also found that the manifestation of my anxiety can be overcome by using simple mind over matter techniques . a good example of this is when i was 8 , after learning about what asthma was , [ started , to feel like i was having trouble breathing . in a heightened slate of anxiety , this has happened many times . besides just general fears , it was a struggle to overcome this anxiety manifestation . because i now understand my predisposition towards anxiety , i can talk myself out of simple fears . it is also important to note that a high - reactive temperament protects the child from engaging in risky behavior - whether drugs , driving at high speeds , or temptations for delinquent , behavior . thus , the child with a high - reactive temperament has some advantages in our society and parents of such infants might decide not to change their child 's behavior when the next set of pharmacological advances permits them that choice .

development of the characteristics of social phobia often requires a diathesis in the form of a temperamental bias . a behavioral profile marked by vigorous motor activity and crying to unfamiliar stimuli at 4 months of age - called high reactivity- is characteristic of about 20% of healthy , caucasian infants . this pattern predicts shy behavior in preschool children and symptoms of social anxiety at age 7 , and , at age 11 , a subdued personality and biological features that are consonant with a hypothesis of amygdalar excitability . the biological variables that best characterize the children who had been high - reactive infants are right - hemisphere activity in the electroencephalogram ( eec ) , a larger evoked potential from the inferior colliculus , higher sympathetic tone in the cardiovascular system , and larger event - related potentials to discrepant stimuli . about a quarter of 11-year - olds who had been high reactives displayed behavioral and biological characteristics that are in theoretical accord with the hypothesis of amygdalar excitability , while only 1 of 20 displayed a profile characterized by features in opposition to their temperament . the evidence points to a modest temperamental contribution to the development of symptoms currently regarded as diagnostic of social phobia .

Study design Evaluation from age 4 months to 7 years Evaluation at age 11 years Results Behavior Biological variables Prediction of states of anxiety Conclusion

my laboratory has been studying longitudinally a large group of healthy , caucasian children from middle - class families who have been followed from 4 months to 11 years : each infant was classified at 4 months of age into one of four temperamental groups based on their behavior to a standard battery of visual , auditory , and olfactory stimuli . infants who showed a combination of frequent , vigorous motor activity combined with frequent crying were classified as high reactive ( 22% of the sample ) . infants who showed infrequent , motor activity , but frequent , crying were classified as distressed ( 25% ) , and infants who showed frequent , motor activity , but minimal distress were classified as aroused ( 10% ) . the children from these temperamental groups were evaluated twice in the second year for their reaction to unfamiliar people , situations , and procedures . the 14and 21 -month - old children who had been categorized as high reactive as infants were more likely than the low reactives to display high levels of fear to unfamiliar people , rooms , and events . this relationship has been verified by fox and colleagues , who also found that 1-yearolds who had been classified as high - reactive infants at 4 months were more fearful than others when they encountered unfamiliar events . these children were observed when they were four and a half years old in a play session with two other unfamiliar children of the same sex and age , while the three mothers sat on a couch in the playroom . they were quiet , spent long times close to their mother , and did not initiate social interaction with the other children . when the children were seven and a half years old , we evaluated the prevalence of anxious symptoms in 51 high ractives , 60 low reactives , and 53 children from the other two temperamental groups . forty - five percent , of the children who had been high - reactive infants , compared with 15% of low ractives , had anxious symptoms ( chi - square=12.8 , p<0.01 ) high reactives who had anxious symptoms were more fearful in the second year and had higher sitting diastolic blood pressures and a greater magnitude of cooling of the temperature of the fingertips across a series of digit , recall problems , compared with other high reactives . the behavioral data included the number of spontaneous comments and smiles displayed toward the examiner during the first 18 min of interaction , a reliable rating ( 4-point scale ) of the degree of uncertainty , tension , and anxiety displayed by the child in this setting , and a maternal qsort of 28 items describing the child 's behavior . these biological variables were : ( i ) asymmetry in the magnitude of dcsynchronization of alpha frequencies in the eeg ; ( ii ) magnitude of the evoked potential from the inferior colliculus to a series of clicks ; ( iii ) sympathetic tone in the cardiovascular system ; and ( iv ) the magnitude of the wave form at 400 ms in the eventrelated potential to discrepant , visual scenes . the amygdala sends ipsilateral projections to the frontal lobe through the basal nucleus of meynert and it is likely that these projections contribute to the asymmetry in the alpha band of the eeg . the brain stem auditory evoked response ( baer ) , elicited by a series of clicks delivered through earphones , was a relevant , measure because variation in the magnitude or latency of the fifth wave in the baer response - called wave 5 - differentiates between personality and clinical categories . the peak of the fifth wave is believed to represent , the termination of the lateral lemniscus on the inferior colliculus . the theoretical relevance of this fact is that , the amygdala projects to the inferior colliculus through both the central gray and the locus cereleus and , therefore , children with a more excitable amygdala should display a larger wave 5 than others . the rationale behind recording the event - related potential to discrepant , visual stimuli derived from the assumption that the amygdala reacts to discrepant or unexpected events and projects to cortical neurons that mediate the event - related potential . if high reactives possess a low threshold of reactivity in the amygdala and its projections , then they should show a larger eventrelated potential to discrepant events . in the first scries , 70% of the pictures were of the same item ( a fireworks display ) , 15% were of the same flower , the oddball stimulus , and the remaining 15% were each different , but ecologically valid ( a chair or kitchen utensil ) . the frequent picture presented 70% of the time was a yellow fire hydrant , the oddball stimulus was a different , flower , and the remaining fifteen percent of the pictures were each different , but ecologically invalid ( for example , a chair with three legs ) . the amygdala sends varied projections to the sympathetic system and , therefore , we assumed that high reactives would show signs of greater sympathetic reactivity than low reactives . higher relative power in the low frequency band is usually correlated with a high resting heart rate and is indicative of higher sympathetic tone . my laboratory has been studying longitudinally a large group of healthy , caucasian children from middle - class families who have been followed from 4 months to 11 years : each infant was classified at 4 months of age into one of four temperamental groups based on their behavior to a standard battery of visual , auditory , and olfactory stimuli . infants who showed a combination of frequent , vigorous motor activity combined with frequent crying were classified as high reactive ( 22% of the sample ) . infants who showed infrequent , motor activity , but frequent , crying were classified as distressed ( 25% ) , and infants who showed frequent , motor activity , but minimal distress were classified as aroused ( 10% ) . the children from these temperamental groups were evaluated twice in the second year for their reaction to unfamiliar people , situations , and procedures . the 14and 21 -month - old children who had been categorized as high reactive as infants were more likely than the low reactives to display high levels of fear to unfamiliar people , rooms , and events . this relationship has been verified by fox and colleagues , who also found that 1-yearolds who had been classified as high - reactive infants at 4 months were more fearful than others when they encountered unfamiliar events . these children were observed when they were four and a half years old in a play session with two other unfamiliar children of the same sex and age , while the three mothers sat on a couch in the playroom . they were quiet , spent long times close to their mother , and did not initiate social interaction with the other children . when the children were seven and a half years old , we evaluated the prevalence of anxious symptoms in 51 high ractives , 60 low reactives , and 53 children from the other two temperamental groups . forty - five percent , of the children who had been high - reactive infants , compared with 15% of low ractives , had anxious symptoms ( chi - square=12.8 , p<0.01 ) high reactives who had anxious symptoms were more fearful in the second year and had higher sitting diastolic blood pressures and a greater magnitude of cooling of the temperature of the fingertips across a series of digit , recall problems , compared with other high reactives . the behavioral data included the number of spontaneous comments and smiles displayed toward the examiner during the first 18 min of interaction , a reliable rating ( 4-point scale ) of the degree of uncertainty , tension , and anxiety displayed by the child in this setting , and a maternal qsort of 28 items describing the child 's behavior . these biological variables were : ( i ) asymmetry in the magnitude of dcsynchronization of alpha frequencies in the eeg ; ( ii ) magnitude of the evoked potential from the inferior colliculus to a series of clicks ; ( iii ) sympathetic tone in the cardiovascular system ; and ( iv ) the magnitude of the wave form at 400 ms in the eventrelated potential to discrepant , visual scenes . the amygdala sends ipsilateral projections to the frontal lobe through the basal nucleus of meynert and it is likely that these projections contribute to the asymmetry in the alpha band of the eeg . the brain stem auditory evoked response ( baer ) , elicited by a series of clicks delivered through earphones , was a relevant , measure because variation in the magnitude or latency of the fifth wave in the baer response - called wave 5 - differentiates between personality and clinical categories . the peak of the fifth wave is believed to represent , the termination of the lateral lemniscus on the inferior colliculus . the theoretical relevance of this fact is that , the amygdala projects to the inferior colliculus through both the central gray and the locus cereleus and , therefore , children with a more excitable amygdala should display a larger wave 5 than others . the rationale behind recording the event - related potential to discrepant , visual stimuli derived from the assumption that the amygdala reacts to discrepant or unexpected events and projects to cortical neurons that mediate the event - related potential . if high reactives possess a low threshold of reactivity in the amygdala and its projections , then they should show a larger eventrelated potential to discrepant events . in the first scries , 70% of the pictures were of the same item ( a fireworks display ) , 15% were of the same flower , the oddball stimulus , and the remaining 15% were each different , but ecologically valid ( a chair or kitchen utensil ) . the frequent picture presented 70% of the time was a yellow fire hydrant , the oddball stimulus was a different , flower , and the remaining fifteen percent of the pictures were each different , but ecologically invalid ( for example , a chair with three legs ) . the amygdala sends varied projections to the sympathetic system and , therefore , we assumed that high reactives would show signs of greater sympathetic reactivity than low reactives . higher relative power in the low frequency band is usually correlated with a high resting heart rate and is indicative of higher sympathetic tone . the high - reactive children were more subdued and anxious at the 11-year evaluation than the low ractives and were rated as more anxious and inhibited during the first 18 min of the interview ( table i ) . twice as many high as low reactives were rated as extremely inhibited ( rating of 4 ; awarded to children who made very few comments and smiles , displayed a great deal of motor tension , spoke in a soft voice , and showed other signs of concern ) . thus , the infant temperamental profiles predicted , to a modest degree , spontaneity or a subdued style with the unfamiliar adult examiner . one half of the current group of high and low reactives had been seen when they were four and a half and seven and a half years of age . figure 1 illustrates the mean standard scores for high and low reactives on the four biological variables at 11 years of age : right parietal activation in the eeg , wave 5 magnitudes , sympathetic tone ( a low ratio and a high resting heart rate ) , and the mean of the integrated voltages from 400 to 1000 ms for the event - related potential to the first oddball flower and the novel invalid scenes . further , the high reactives who had been highly fearful in the second year showed greater activation in the right frontal area compared with low reactives who were equally fearful in the second year . the high reactives also had significantly larger wave 5 values ( r(125)=2.87 , p<0.05 ) , and this variable best separated the two temperamental groups . the high reactives also had greater sympathetic tone in the cardiovascular system ; 49% of high reactives , but only 32% of low reactives , combined a low ratio in the spectral analysis with a high resting heart rate ; 32% of the low ractives , but only 16% of high ractives , combined a high ratio with a low resting heart , rate ( chisquare=4.9 , p<0.05 ) . the children with high sympathetic tone , compared to those with high vagal tone , spoke less often , were rated as more anxious , and were described by their mother as shy . although the eeg , wave 5 , and event - related potential data also separated the two temperamental groups , these measures were less closely related to the child 's behavior . by contrast , activity in the inferior colliculus and the pyramidal neurons of the cortex are less likely to influence orbitofrontal neurons and , therefore , no change in feeling tone occurs and there should be a minimal relationship to behavior . it is important that among high and low reactives , who were equally subdued in their behavior in the laboratory , only the high ractives showed the biological features of right parietal asymmetry and a large wave 5 . that is , only high reactives showed coherence in the magnitude of the nc across disparate cortical sites , implying that the discrepant scenes recruited neurons over a broader cortical area . low values better differentiated low from high reactives than did high values , suggesting that it is easier for low - reactive than for high - reactive children to attain a state of low cortical and autonomic arousal , even though the former can attain , temporarily , a state of higher arousal in a laboratory setting . about 1 in 4 children who had been high reactive and 1 in 4 children who had been low reactive developed a behavioral and a biological profile at age 11 that was in accord with theoretical expectations , while only 1 of 20 children developed a profile of social behavior and biology that , violated their expected profile . most children displayed behavioral and biological patterns that were characteristic of randomly selected children from middle - class , caucasian populations . thus , the prediction that a high - reactive infant will not be highly sociable and exuberant , and show low biological arousal at age 11 can be made with much greater confidence than the prediction that this category of child will be extremely subdued and anxious , and show signs of high arousal in cortical and autonomic targets . if all one knows about a group of 1 00 children is that , they were born to economically secure , well - educated , nurturing parents and must predict the likely psychological adult , outcomes , the most accurate guesses will refer to the profiles that should not occur : criminality , school failure , psychosis , homelessness , drug addiction , and poverty . the high - reactive children were more subdued and anxious at the 11-year evaluation than the low ractives and were rated as more anxious and inhibited during the first 18 min of the interview ( table i ) . twice as many high as low reactives were rated as extremely inhibited ( rating of 4 ; awarded to children who made very few comments and smiles , displayed a great deal of motor tension , spoke in a soft voice , and showed other signs of concern ) . thus , the infant temperamental profiles predicted , to a modest degree , spontaneity or a subdued style with the unfamiliar adult examiner . one half of the current group of high and low reactives had been seen when they were four and a half and seven and a half years of age . figure 1 illustrates the mean standard scores for high and low reactives on the four biological variables at 11 years of age : right parietal activation in the eeg , wave 5 magnitudes , sympathetic tone ( a low ratio and a high resting heart rate ) , and the mean of the integrated voltages from 400 to 1000 ms for the event - related potential to the first oddball flower and the novel invalid scenes . further , the high reactives who had been highly fearful in the second year showed greater activation in the right frontal area compared with low reactives who were equally fearful in the second year . the high reactives also had significantly larger wave 5 values ( r(125)=2.87 , p<0.05 ) , and this variable best separated the two temperamental groups . the high reactives also had greater sympathetic tone in the cardiovascular system ; 49% of high reactives , but only 32% of low reactives , combined a low ratio in the spectral analysis with a high resting heart rate ; 32% of the low ractives , but only 16% of high ractives , combined a high ratio with a low resting heart , rate ( chisquare=4.9 , p<0.05 ) . the children with high sympathetic tone , compared to those with high vagal tone , spoke less often , were rated as more anxious , and were described by their mother as shy . although the eeg , wave 5 , and event - related potential data also separated the two temperamental groups , these measures were less closely related to the child 's behavior . by contrast , activity in the inferior colliculus and the pyramidal neurons of the cortex are less likely to influence orbitofrontal neurons and , therefore , no change in feeling tone occurs and there should be a minimal relationship to behavior . it is important that among high and low reactives , who were equally subdued in their behavior in the laboratory , only the high ractives showed the biological features of right parietal asymmetry and a large wave 5 . that is , only high reactives showed coherence in the magnitude of the nc across disparate cortical sites , implying that the discrepant scenes recruited neurons over a broader cortical area . low values better differentiated low from high reactives than did high values , suggesting that it is easier for low - reactive than for high - reactive children to attain a state of low cortical and autonomic arousal , even though the former can attain , temporarily , a state of higher arousal in a laboratory setting . about 1 in 4 children who had been high reactive and 1 in 4 children who had been low reactive developed a behavioral and a biological profile at age 11 that was in accord with theoretical expectations , while only 1 of 20 children developed a profile of social behavior and biology that , violated their expected profile . most children displayed behavioral and biological patterns that were characteristic of randomly selected children from middle - class , caucasian populations . thus , the prediction that a high - reactive infant will not be highly sociable and exuberant , and show low biological arousal at age 11 can be made with much greater confidence than the prediction that this category of child will be extremely subdued and anxious , and show signs of high arousal in cortical and autonomic targets . if all one knows about a group of 1 00 children is that , they were born to economically secure , well - educated , nurturing parents and must predict the likely psychological adult , outcomes , the most accurate guesses will refer to the profiles that should not occur : criminality , school failure , psychosis , homelessness , drug addiction , and poverty . the evidence affirms the view that a temperamental bias for high reactivity in infancy , detectable early in development , is predictive of a personality profile marked by shyness , timidity , and anxiety to unfamiliar events and this behavioral phenotype is accompanied by a select biological pattern that implies amygdalar excitability . an independent group of 1 3-year - olds , who had been classified as inhibited or uninhibited in the second year , were interviewed by a psychiatrist who had no knowledge of their initial temperamental classification or later laboratory behavior . more of the adolescents who had been inhibited rather than uninhibited in the second year had symptoms of social anxiety ( 61% versus 27% ) . however , these inhibited children were not more likely to have developed specific target phobias or separation anxiety , implying that , inhibited children might be at special risk for the development of social phobia during the adolescent or adult years . first , 20% of the children were high - reactive infants , but the prevalence of social phobia is less that this fact suggests that many high reactives find an adaptive niche in their society that allows them to titer unpredictable social encounters . an excerpt from an essay written by one of the 11-year - old children , who was a high - reactive infant and a fearful toddler supports this claim . thus , the child with a high - reactive temperament has some advantages in our society and parents of such infants might decide not to change their child 's behavior when the next set of pharmacological advances permits them that choice .

cirrhosis is the end stage of every chronic liver disease , resulting in formation of fibrous tissue , disorganization of liver architecture , and nodule formation , which interferes with liver function and results in portal hypertension . portal hypertension is associated with development of a hyperdynamic circulation and complications such as ascites , hepatic encephalopathy , and oesophago - gastric varices . patients with cirrhosis and gastro - oesophageal varices have a hepatic venous pressure gradient during haemodynamic catheterization of at least 1012 mmhg . oesophageal varices are present at diagnosis in approximately 50% of cirrhotic patients , being more common in child - pugh class c patients compared to child - pugh class a patients ( 85% versus 40% ) [ 1 , 2 ] . de novo formation of varices occurs at a rate of 5% per year , with a higher incidence in patients continuing to consume alcohol or with worsening liver function . once varices form , they enlarge from small to large at a rate of 512% per year and bleed at a rate of 515% per year . the greatest bleeding risk is seen in large varices classified as being > 5 mm diameter and is also influenced by liver disease severity as assessed by child - pugh score , and by the presence of red wale markings on varices at endoscopy . therefore , these factors should also be taken into consideration to classify high - risk varices . reports from the 1940 's to the 1980 's demonstrate poor outcomes from variceal bleeding with mortality rates between 3060% [ 46 ] , but studies suggest that the outcomes have improved over the last few decades [ 79 ] . this is demonstrated in a study by carbonell et al . , who showed that between 19802000 , the inhospital mortality from variceal bleeding decreased from 42.6% to 14.5% and was associated with decreased rebleeding and rates of bacterial infection . although mortality from a bleeding episode has decreased with improved endoscopic and radiological techniques together with new pharmacologic therapies , a 2030% mortality means that bleeding from oesophageal varices remains of significant clinical importance . early diagnosis of varices before the first bleed is essential as studies of primary prophylaxis clearly show that the risk of variceal haemorrhage can be reduced by 50% to about 15% for large oesophageal varices . current guidelines , therefore , recommend that all cirrhotic patients should be screened for varices at diagnosis , with followup every 2 - 3 years for patients without varices ( depending upon liver disease severity ) and 1 - 2 years for patients with small varices , to assess for enlargement of varices and need for prophylactic treatment . upper gi endoscopy remains the gold standard for screening , but this test is not without its own limitations . there is conflicting evidence with regard to the interobserver agreement for endoscopic diagnosis of variceal presence , grade , or presence of red signs [ 1416 ] . . found in 100 cirrhotic patients that the interobserver agreement between four independent observers for the size of oesophageal varices and presence of red signs was good with kappa values of 0.59 and 0.60 , respectively . however , bendtsen et al . found considerable variation in the interobserver agreement on the diagnosis and grading of oesophageal varices between 22 endoscopists with a large variation in kappa values . the current guidelines cause a significant burden and cost to endoscopy units and necessitate patients having repeated unpleasant procedures even when up to 50% may still not have developed oesophageal varices 10 years after the initial diagnosis . if it were possible to predict oesophageal varices by noninvasive means this would restrict testing to the population deemed to be at most risk and reduce the number of endoscopies required . such a screening test should be simple , quick , reproducible , and cost effective . the utility of current noninvasive tests to predict oesophageal varices will be reviewed in this paper . several studies have examined the usefulness of different clinical and laboratory parameters as predictors of the presence or size of oesophageal varices . these are discussed below . a number of clinical signs and other laboratory markers have been identified either alone or in combination as factors predicting the presence of oesophageal varices . these include the presence of spider naevi , splenomegaly or ascites , child - pugh classification , serum albumin , and prothrombin time . spider naevi , a low - albumin and low - platelet count were shown to be independent risk factors for the presence of varices in a study by garcia - tsao et al . . in a further study by berzigotti et al . , spider naevi , alt , and albumin were found to predict oesophageal varices with the best cutoff giving a sensitivity of 93% , specificity of 37% , and correctly classifying 72% of patients . similarly , spider naevi have been found to be predictive of large oesophageal varices with a diagnostic accuracy of 72% when using the variables platelet count , prothrombin index , and spider naevi . found that splenomegaly detected on clinical examination was an independent risk factor for the presence of large varices . . demonstrated that cirrhotic patients in child - pugh classes b or c were almost 3 times as likely to have oesophageal varices or large oesophageal varices as compared to patients in child - pugh class a. the baveno iv international consensus workshop on methodology of diagnosis and treatment concluded that no study reached a high enough level of significance to warrant the widespread use of such noninvasive markers of oesophageal varices . chronic liver injury and inflammation leads to fibrosis and ultimately cirrhosis , through the deposition of extracellular matrix ( ecm ) complexes . the collagen fibrils of the complex undergo secondary processing , becoming cross - linked , which confers resistance to degradative enzymes and irreversibility . normally , deposition of the ecm is a dynamic , reversible process with removal of ecm mediated by several specific matrix metalloproteinases ( mmps ) , which in turn are regulated by soluble inhibitors termed timps ( tissue inhibitor of metalloproteinase ) . a number of serum markers for ecm deposition and removal have been evaluated as candidate markers for liver fibrosis , and a small number of studies have evaluated their usefulness in predicting oesophageal varices . potential markers examined to date include the glycoproteins , hyaluronic acid and laminin , and members of the collagen family including procollagen iii and type iv collagen . assessed the ability of serum hyaluronic acid to predict medium - to - large oesophageal varices and showed the sensitivity , specificity , positive and negative predictive values , and diagnostic accuracy at a cutoff of 207 g / l to be 94% , 77.8% , 88.7% , 87.5% , and 88.3% , respectively . krner et al . showed no association between concentrations of hyaluronic acid or laminin and grade of oesophageal varices , and a further study by bahr et al . confirmed the lack of association of serum laminin to size of oesophageal varices similar conflict is seen when examining the evidence with regard to the role of the collagens . in the first of only 2 studies in this area , the aminoterminal propeptide of type iii procollagen was shown to have a weak correlation to the degree of oesophageal varices . the second study by mamori et al . included 44 patients with alcoholic liver disease and demonstrated a significant difference in serum type iv collagen levels between patients with and without varices ( 712.3 versus 404.3 ng / ml , p < 0.001 ) , giving an auroc of 0.78 for predicting the presence of oesophageal varices . none of the aforementioned markers could currently be utilised to predict oesophageal varices in portal hypertension ; in view of this several different biomarkers have been combined with the aim of improving their diagnostic ability . fibrotest is a composite score generated by combining the results of five serum blood tests ( alpha-2-macroglobulin , apolipoprotein a1 , haptoglobin , -glutamyltranspeptidase , and bilirubin and alanine ) corrected for the age and gender of the patient . results have shown high predictive values for significant fibrosis in patients with chronic hepatitis c , chronic hepatitis b , fatty liver disease , and chronic alcoholic liver disease [ 2831 ] . a single study has assessed the predictive value of fibrotest in the diagnosis of large oesophageal varices in 99 cirrhotic patients . significant differences in fibrotest value ( 0.89 versus 0.82 ) , platelet count ( 110 versus 150 ) , and prothrombin time ( 50 versus 66% ) were seen between patients with and without large oesophageal varices . fibrotest had the highest discriminative power of all the variables with an auroc curve of 0.77 . using a cutoff of 0.80 , this gave a sensitivity of 92% , specificity 21% , ppv 33% , and npv 86% . a fibrotest score < 0.75 was found to be associated with the absence of large oesophageal varices with a npv of 100% . the limitations to the study are that it was a retrospective study with significant population bias and has not been reproduced in a prospective study of compensated cirrhotics . fibrotest is not readily available to most clinicians , which limit its utility as a screening test . thrombocytopenia may occur in portal hypertension - induced splenomegaly , in part due to platelet sequestration , and a large number of studies have been performed assessing the relationship between platelet count and oesophageal varices [ 17 , 1921 ] . a low - platelet count is regularly identified as predictive of oesophageal varices and large oesophageal varices , but there is a wide variation in the cut - off level of platelets used , ranging from 68,000 to 160,000 with sensitivities ranging from 7190% and specificities from 3673% . bias is likely to account for much of this variation , with the majority of studies being retrospective in nature , having heterogeneous cohorts of patients resulting in both selection and spectrum bias . a longitudinal study by qamar et al . of 213 patients , with compensated cirrhosis with portal hypertension but without varices , demonstrated that the median platelet count at the time of occurrence of varices was 91,000 . however , no platelet count could be identified that accurately predicted the presence of oesophageal varices ( auroc curve 0.63 ) , and they , therefore , concluded that platelet count is an inadequate noninvasive marker for prediction of the presence of oesophageal varices . in an attempt to improve the predictive value of the platelet count , it has been combined with other variables , and the results of these studies are discussed below . oesophageal collaterals develop as a consequence of portal hypertension , being formed by vascular remodelling and angiogenesis . key molecules thought to be involved in this include nitric oxide and vascular endothelial growth factor ( vegf ) . a single study of 85 cirrhotic patients examined the predictive capability of serum nitrate levels to detect oesophageal varices . significant differences in serum nitrate levels were found between patients with large oesophageal varices compared to patients without oesophageal varices ( p < 0.01 ) . mol / l , giving a sensitivity 86.5% , specificity 83.3% , ppv 95% , and npv 62.5% . animal studies suggest that the formation of oesophageal varices results not only from opening up of preexisting collateral vessels but also as a result of angiogenesis which may in part be mediated by vegf . use of vegf as a noninvasive biomarker has only been investigated in a single study , and no correlation between vegf levels and grade of oesophageal varices was detected . the development of portosystemic collaterals and the resultant shunting is responsible for the complication hepatic encephalopathy , in which ammonia plays a role . one study has examined the role of blood ammonia concentrations in the noninvasive detection of oesophageal varices . in this study of 153 cirrhotic patients , a significant correlation was demonstrated between oesophageal variceal grade and venous ammonia levels ( r = 0.43 , p < 0.001 ) . the auroc curve for predicting the presence of oesophageal varices was 0.78 , and using a cut - off of 42 therefore , variables associated with portal hypertension and hypersplenism are not accurate enough to be used as noninvasive markers of oesophageal varices . ( 1 ) platelet count / spleen diameter ratiothis ratio is calculated by dividing the platelet number / mm by the maximum spleen bipolar diameter in mm as estimated by abdominal ultrasound . the first by giannini et al . in 2003 , reported the platelet count / spleen diameter ratio to be the only independent variable associated with presence of ov on multivariate analysis and identified a cut - off value of 909 , giving a ppv of 96% and npv of 100% . the second part of the study confirmed the reproducibility of this cut - off level with a ppv of 74% and npv of 100% in compensated cirrhotic patients . the same group then followed up 68 patients without ov with repeat endoscopy and calculation of the platelet / spleen diameter ratio . at followup , patients with a platelet count / spleen diameter ratio < 909 had 100% npv and 84% ppv , and they concluded that the platelet count spleen diameter ratio was effective in ruling out the presence of ov when cirrhotic patients were followed longitudinally . subsequently , a multicentre , international validation study using the 909 ratio was performed in 218 patients . the test performed less well than in the original study with a ppv of 76.6% and a npv of 87.0% . this has been a consistent feature in all studies subsequently performed which vary from being retrospective or prospective in nature and utilise different cut off points [ 3943 ] . therefore , despite promising early results the platelet count / spleen diameter ratio is not a reliable tool to screen for oesophageal varices . this ratio is calculated by dividing the platelet number / mm by the maximum spleen bipolar diameter in mm as estimated by abdominal ultrasound . the first by giannini et al . in 2003 , reported the platelet count / spleen diameter ratio to be the only independent variable associated with presence of ov on multivariate analysis and identified a cut - off value of 909 , giving a ppv of 96% and npv of 100% . the second part of the study confirmed the reproducibility of this cut - off level with a ppv of 74% and npv of 100% in compensated cirrhotic patients . the same group then followed up 68 patients without ov with repeat endoscopy and calculation of the platelet / spleen diameter ratio . at followup , patients with a platelet count / spleen diameter ratio < 909 had 100% npv and 84% ppv , and they concluded that the platelet count spleen diameter ratio was effective in ruling out the presence of ov when cirrhotic patients were followed longitudinally . subsequently , a multicentre , international validation study using the 909 ratio was performed in 218 patients . the test performed less well than in the original study with a ppv of 76.6% and a npv of 87.0% . this has been a consistent feature in all studies subsequently performed which vary from being retrospective or prospective in nature and utilise different cut off points [ 3943 ] . therefore , despite promising early results the platelet count / spleen diameter ratio is not a reliable tool to screen for oesophageal varices . ( 2 ) platelet count and child - pugh classin 2007 , burton et al . published the validation of a model for predicting size and presence of varices based upon platelet count and child - pugh class . the first model aimed to detect large varices in child - pugh a patients with a platelet count < 80 and had a sensitivity of 58% , specificity 79% , ppv 30% , and npv 92% . the second model aimed to identifying any varices in child b / c patients with a platelet count < 90 and had a sensitivity of 60% , specificity of 59% , ppv 80% , and npv 34% . once again , the performance of these models would not reliably predict the presence of oesophageal varices . in 2007 , burton et al . published the validation of a model for predicting size and presence of varices based upon platelet count and child - pugh class . the first model aimed to detect large varices in child - pugh a patients with a platelet count < 80 and had a sensitivity of 58% , specificity 79% , ppv 30% , and npv 92% . the second model aimed to identifying any varices in child b / c patients with a platelet count < 90 and had a sensitivity of 60% , specificity of 59% , ppv 80% , and npv 34% . once again , the performance of these models would not reliably predict the presence of oesophageal varices . ( 3 ) ast / alt ratiothe ast / alt ratio has been used to predict cirrhosis , and by natural extension studies have been performed to assess its usefulness in predicting oesophageal varices . in a retrospective study , significantly higher ast / alt ratios were seen in patients with varices compared to those without ( ratio : 1.8 versus 1.0 , p < 0.0001 ) . a further prospective study found an ast / alt ratio > 1.12 to be significantly associated with the presence of varices at initial endoscopy ( or 3.9 , p = 0.02 95% ci 1.311.8 ) . this cutoff gave a sensitivity of 47.8% , specificity of 87% , ppv 42.3% , and npv 89.2% , and an auroc of 0.69 . a further study using a different cut - off of 1.0 demonstrated a sensitivity of 68% , specificity of 89% , ppv 77% , and npv 83% , with an auroc 0.83 ( 0.720.94 ) for predicting the presence of oesophageal varices . for the prediction of large oesophageal varices , this gave a sensitivity 68% , specificity 77% , ppv 41% , and npv 92% , and auroc 0.79 ( 0.640.94 ) . overall , the ast / alt ratio correctly classified 81% patients for the detection of varices and 76% of those with large varices . therefore these studies , which include patients with different aetiologies of liver disease and used different cutoffs for the ast / alt ratio can not confidently predict the presence of oesophageal varices in clinical practice to avoid screening all cirrhotic patients with endoscopy . the ast / alt ratio has been used to predict cirrhosis , and by natural extension studies have been performed to assess its usefulness in predicting oesophageal varices . in a retrospective study , significantly higher ast / alt ratios were seen in patients with varices compared to those without ( ratio : 1.8 versus 1.0 , p < 0.0001 ) . a further prospective study found an ast / alt ratio > 1.12 to be significantly associated with the presence of varices at initial endoscopy ( or 3.9 , p = 0.02 95% ci 1.311.8 ) . this cutoff gave a sensitivity of 47.8% , specificity of 87% , ppv 42.3% , and npv 89.2% , and an auroc of 0.69 . a further study using a different cut - off of 1.0 demonstrated a sensitivity of 68% , specificity of 89% , ppv 77% , and npv 83% , with an auroc 0.83 ( 0.720.94 ) for predicting the presence of oesophageal varices . for the prediction of large oesophageal varices , this gave a sensitivity 68% , specificity 77% , ppv 41% , and npv 92% , and auroc 0.79 ( 0.640.94 ) . overall , the ast / alt ratio correctly classified 81% patients for the detection of varices and 76% of those with large varices . therefore these studies , which include patients with different aetiologies of liver disease and used different cutoffs for the ast / alt ratio can not confidently predict the presence of oesophageal varices in clinical practice to avoid screening all cirrhotic patients with endoscopy . ( 4 ) right lobe liver albumin ratiothis ratio is calculated by dividing the right liver lobe diameter ( as assessed by abdominal ultrasound and measured in millimetres ) by the serum albumin concentration ( g / l ) . right liver lobe / albumin ratio correlated with presence and size of oesophageal varices ( r = 0.488 , p < 0.01 ; r = 0.481 , p < 0.01 , respectively ) . for a cut - off value of 4.425 this gave a sensitivity of 83.1% and specificity 73.9% and thus once again can not be used as a reliable screening test . this ratio is calculated by dividing the right liver lobe diameter ( as assessed by abdominal ultrasound and measured in millimetres ) by the serum albumin concentration ( g / l ) . right liver lobe / albumin ratio correlated with presence and size of oesophageal varices ( r = 0.488 , p < 0.01 ; r = 0.481 , p < 0.01 , respectively ) . for a cut - off value of 4.425 this gave a sensitivity of 83.1% and specificity 73.9% and thus once again can not be used as a reliable screening test . transient elastography ( te , fibroscan , echosens , france ) is a noninvasive technique developed to assess hepatic fibrosis in patients with chronic liver diseases . fibrosis causes an increase in liver stiffness , and measurement of this forms the basis of te , which is painless , rapid , and easy to perform . studies suggest that te is highly reproducible and reliable with very high interobserver and intraobserver agreement overall but that patient related and liver disease related factors may have a negative effect on the reproducibility of this technique . a wide range of liver stiffness values have been reported ranging from 2.5 to 75 kpa , being influenced by gender , body mass index , disease aetiology , and presence of necroinflammatory change [ 5053 ] . as a rough guide , normal te values are considered to be 3.88 kpa in men and 3.37.8 kpa in women , significant fibrosis ( metavir fibrosis stage 2 ) 7 - 8 kpa and cirrhosis 1317 kpa . a number of studies have been performed examining the relationship of liver stiffness to size and presence of oesophageal varices , and these results are summarised in table 1 [ 47 , 5457 ] . these studies demonstrate a significant correlation between liver stiffness measurements and the presence of oesophageal varices but are divided with regard to the relationship of liver stiffness to variceal size . for the diagnosis of variceal presence , auroc curves varied from 0.760.85 , with a sensitivities of 8495% , specificities of 4378% , ppv 5789% , and npv 6691% using cutoffs between 13.921.5 kpa . for the diagnosis of large oesophageal varices , auroc varied from 0.760.87 , with sensitivities of 7791% , specificities of 6085% , ppv 4856% and npv 94 - 95% using cut - offs between 1930.5 kpa . the other limitations of the study relate to inclusion of patients with liver disease of different aetiologies and of different severity , according to child - pugh class . all 70 patients were child - pugh class a and had cirrhosis secondary to hepatitis c. they demonstrated that lsm values increased with the grade of ov ( p < 0.001 ) . a cutoff of 21.5 kpa predicted the presence of ov with a sensitivity of 76% , specificity 78% , ppv 68% , and npv 84% and correctly classified 73% of patients . at a cutoff of 30.5 kpa , the presence of large ov was predicted with a sensitivity 77% , specificity 85% , ppv 56% , and npv 94% , and correctly classified 79% of patients . therefore , the predictive performance of liver stiffness measurement is poor for the diagnosis of ov with low specificity and ppv , particularly with regard to large ov . however , it may be useful as a screening test to identify patients in whom variceal screening is not required , but at present can not be advocated as a surrogate for gastroscopy . transient elastography has also been used to determine spleen stiffness , using the hypothesis that splenomegaly resulting from portal hypertension causes changes in the spleen 's density . in a study of 191 patients ( 135 cirrhotic ) recently published , it was demonstrated that spleen stiffness was significantly higher in cirrhotics than noncirrhotics and in patients with oesophageal varices compared to those without . 52.5 kpa was determined to be the best cutoff giving an auroc curve of 0.74 . they found a better diagnostic accuracy , of 89.95% , in predicting the presence but not the grade of oesophageal varices when liver and spleen stiffness were used together . mr elastographic spleen stiffness has also been assessed in a small study of 17 compensated cirrhotics . all of the 7 patients with oesophageal varices had a mean spleen stiffness of > 10.5 kpa . further larger studies are needed to investigate the diagnostic accuracy of mr elastographic spleen stiffness for noninvasive prediction of oesophageal varices . doppler ultrasonography ( us ) imaging provides a real - time , inexpensive , and repeatable examination of the portal system and allows estimation of both arterial and venous flow . portal vein diameter , portal blood velocity and congestion index , spleen size , flow pattern in the hepatic veins , and the presence of abdominal portosystemic collaterals are all us parameters previously thought to have with prognostic significance but all with poor sensitivity and specificity . greater than 13 mm , and platelet count < 100 10 as noninvasive predictive tools to discriminate cirrhotic patients with and without oesophageal varices ( ov ) . the roc curve constructed from all possible combinations of these dichotomous variables initially looked promising with an area under the curve ( auc ) value of 0.80 . to assess the validity of this tool , the investigators used a matched second cohort where the positive predictive value was found to be significantly reduced . a further validity study was repeated in another centre with a similarly poor sensitivity and specificity . thus , us has limited specificity and can not replace endoscopy as a screening tool for large oesophageal varices [ 61 , 63 ] . three recent studies suggest that multidetector ct is comparable to upper endoscopy in detecting small and large varices [ 6466 ] . only two of these studies were carried out prospectively [ 64 , 65 ] , and only one included a cost analysis . in one of the previous studies , virtual oesophagography could be carried out using the ct scans , but this procedure requires time - consuming and invasive intubation of the oesophagus with a catheter for air insufflation . ct was found to have approximately 90% sensitivity in the identification of oesophageal varices determined to be large on endoscopy , but only about 50% specificity . in addition , a significant number of gastric varices , perioesophageal varices and extraluminal pathology were identified by ct that were not identified by endoscopy . use of ct as the initial screening modality for the detection of varices was significantly cost effective compared to endoscopy irrespective of the prevalence of large varices . one of the major limitations identified in all studies was the differing rates of interobserver agreement in variceal size of both modalities , with only one study finding agreement between radiologists being higher than between endoscopists . there are also major concerns over the risk of cumulative radiation exposure in prolonged screening programmes . new capsule endoscopy devices have been developed , specifically for use in the oesophagus , acquiring images from both ends of the device . several studies have been performed , assessing the ability of these capsule endoscopy devices to detect any varices and identify large varices requiring primary prophylaxis [ 6873 ] . conventional ogd was used as the gold standard . with regards to the detection of varices , sensitivity varied between 68100% , and specificity 86100% [ 7072 , 74 ] . in the largest study performed to date , 288 patients were recruited in a multicentre trial . conventional ogd identified ov in 180 patients ( 62.5% ) and capsule endoscopy identified ov in 152 of these , giving a difference in diagnosing ov of 15.6% in favour of ogd . in 13 cases ( 14.5% ) , varices were identified by capsule but not confirmed by ogd . overall agreement for detection of varices was 85.8% , with a sensitivity of 84% , specificity 88% , positive likelihood ratio 7.0 , and negative likelihood ratio 0.18 . with regard to the grading of varices , there was complete agreement on the grade in 79% . in differentiating between varices requiring treatment or not , the sensitivity , specificity , ppv , and npv for capsule endoscopy were 78% , 96% , 87% , and 92% , respectively . overall agreement on treatment decisions based on variceal size was 91% ( kappa = 0.77 ) . other studies have correctly identified patients requiring primary prophylaxis in 74100% of patients [ 69 , 71 , 72 , 74 ] . 2 meta - analyses produced similar results with pooled sensitivities of 83% and 83.8% and pooled specificities of 85% and 80.5% , respectively for the diagnosis of oesophageal varices [ 75 , 76 ] . capsule endoscopy is reported to be feasible in 9499% of patients with the main reasons for failure being because patients were unable to swallow the capsule or due to technical problems with the recording or function of the capsule . adverse events have been reported in 01.4% of cases , including episodes of capsule retention necessitating removal . tolerability of the capsule is found to be better than conventional ogd , with better preprocedure perception and postprocedure satisfaction . 2683% patients prefer capsule endoscopy over conventional ogd in the studies performed to date [ 6870 , 7274 , 77 ] . with regard to cost - effectiveness , 2 studies have been performed , the first concluding that both screening methods are equivalent , the second that screening with capsule endoscopy followed by beta - blocker therapy may be cost - effective compared to ogd followed by beta - blocker therapy but is highly sensitive to local costs [ 78 , 79 ] . therefore in summary , capsule endoscopy is feasible in the majority of patients and with regard to patient preference , capsule endoscopy appears to be preferable to conventional endoscopy and may improve compliance with screening programmes , although this remains to be determined . the jury is still out with regard to cost but when it comes to performance , conventional ogd remains the gold standard . cirrhosis and portal hypertension are characterized by the development of a hyperdynamic circulation with elevated cardiac output and stroke volume and reduced systemic vascular resistance . these haemodynamic variables are independently associated with portal pressure and size of oesophageal varices [ 8184 ] . measurement is traditionally invasive , the thermodilution technique requiring introduction of a catheter into the pulmonary artery . the finometer ( finapres medical systems , amsterdam , the netherlands ) is a noninvasive device that allows continuous beat - to - beat blood pressure and haemodynamic monitoring over a number of hours . we have demonstrated the presence of the hyperdynamic circulation using this technique and shown significant differences in cardiac output and systemic vascular resistance according to the size of oesophageal varices . we have also shown significant correlation of these haemodynamic variables to the 1-year probability of variceal bleeding . data as yet unpublished examining the predictive ability of noninvasive parameters has shown promising initial results , with an auroc curve of 0.86 for cardiac output and 0.77 for peripheral vascular resistance for the diagnosis of large oesophageal varices . optimal cutoffs for these haemodynamic parameters remain to be defined . considering a cutoff of 7.06 l / min for cardiac output , this gave a sensitivity of 91% and a negative predictive value of 93% , maintaining a diagnostic accuracy of 86% . using a cutoff of 0.99 mu for peripheral vascular resistance gave a sensitivity of 91% and negative predictive value of 91% . proteomics is the large - scale study of proteins , particularly their structure and function and interactions in a biological system . proteomics does not require prior knowledge of the proteins present and , therefore , is ideal to screen for the best biomarkers of disease . promising results have been seen in patients with liver cirrhosis to search for markers of hepatic fibrosis [ 8688 ] and has been demonstrated to be more accurate than fibrotest . the optimal biomarker needs to be able to predict clinically significant endpoints as well as liver histology , and so further research is needed to know whether proteomics will ever be useful in the noninvasive diagnosis of oesophageal varices . bacterial products increase portal pressure by activating macrophages and releasing vasoconstrictive prostaglandins [ 9092 ] . a recent study demonstrated that scd163 is increased in cirrhosis , levels correlating with portal pressure , but that levels do not drop following reduction of portal pressure after transjugular intrahepatic portosystemic shunt . therefore , chronic activation of these cells may play a role in establishing and maintaining portal hypertension . further work is needed to assess their potential not only as a noninvasive marker of oesophageal varices but of varices with the highest bleeding risk . in conclusion , based on all the available evidence to date , upper gi endoscopy remains the gold standard for the diagnosis of oesophageal varices in cirrhotic patients despite its own limitations . clinical , biochemical , and radiological parameters currently are not accurate enough to avoid screening endoscopy , due to the risks associated with missing patients with large oesophageal varices . a screening test must be simple and inexpensive , and therefore current promising tools such as ct scanning or capsule endoscopy which are highly acceptable to patients may not prove to be cost - effective or suitable for repeated measurement . assessment of systemic haemodynamics and other serum markers may hold promise for the future , and more studies are needed to better understand and identify high risk groups , which may in time be facilitated by proteomic approaches .

current guidelines recommend that all cirrhotic patients should undergo screening endoscopy at diagnosis to identify patients with varices at high risk of bleeding who will benefit from primary prophylaxis . this approach places a heavy burden upon endoscopy units and the repeated testing over time may have a detrimental effect on patient compliance . noninvasive identification of patients at highest risk for oesophageal varices would limit investigation to those most likely to benefit . upper gi endoscopy is deemed to be the gold standard against which all other tests are compared , but is not without its limitations . multiple studies have been performed assessing clinical signs and variables relating to liver function , variables relating to liver fibrosis , and also to portal hypertension and hypersplenism . whilst some tests are clearly preferable to patients , none appear to be as accurate as upper gi endoscopy in the diagnosis of oesophageal varices . the search for noninvasive tests continues .

1. Introduction 2. Current Perspectives: Possible Approachesto Noninvasive Diagnosis of Oesophageal Varices 3. The Future Approach to Noninvasive Detection of Oesophageal Varices 4. Conclusions

cirrhosis is the end stage of every chronic liver disease , resulting in formation of fibrous tissue , disorganization of liver architecture , and nodule formation , which interferes with liver function and results in portal hypertension . portal hypertension is associated with development of a hyperdynamic circulation and complications such as ascites , hepatic encephalopathy , and oesophago - gastric varices . patients with cirrhosis and gastro - oesophageal varices have a hepatic venous pressure gradient during haemodynamic catheterization of at least 1012 mmhg . oesophageal varices are present at diagnosis in approximately 50% of cirrhotic patients , being more common in child - pugh class c patients compared to child - pugh class a patients ( 85% versus 40% ) [ 1 , 2 ] . the greatest bleeding risk is seen in large varices classified as being > 5 mm diameter and is also influenced by liver disease severity as assessed by child - pugh score , and by the presence of red wale markings on varices at endoscopy . early diagnosis of varices before the first bleed is essential as studies of primary prophylaxis clearly show that the risk of variceal haemorrhage can be reduced by 50% to about 15% for large oesophageal varices . current guidelines , therefore , recommend that all cirrhotic patients should be screened for varices at diagnosis , with followup every 2 - 3 years for patients without varices ( depending upon liver disease severity ) and 1 - 2 years for patients with small varices , to assess for enlargement of varices and need for prophylactic treatment . upper gi endoscopy remains the gold standard for screening , but this test is not without its own limitations . found in 100 cirrhotic patients that the interobserver agreement between four independent observers for the size of oesophageal varices and presence of red signs was good with kappa values of 0.59 and 0.60 , respectively . found considerable variation in the interobserver agreement on the diagnosis and grading of oesophageal varices between 22 endoscopists with a large variation in kappa values . the current guidelines cause a significant burden and cost to endoscopy units and necessitate patients having repeated unpleasant procedures even when up to 50% may still not have developed oesophageal varices 10 years after the initial diagnosis . if it were possible to predict oesophageal varices by noninvasive means this would restrict testing to the population deemed to be at most risk and reduce the number of endoscopies required . several studies have examined the usefulness of different clinical and laboratory parameters as predictors of the presence or size of oesophageal varices . a number of clinical signs and other laboratory markers have been identified either alone or in combination as factors predicting the presence of oesophageal varices . , spider naevi , alt , and albumin were found to predict oesophageal varices with the best cutoff giving a sensitivity of 93% , specificity of 37% , and correctly classifying 72% of patients . similarly , spider naevi have been found to be predictive of large oesophageal varices with a diagnostic accuracy of 72% when using the variables platelet count , prothrombin index , and spider naevi . demonstrated that cirrhotic patients in child - pugh classes b or c were almost 3 times as likely to have oesophageal varices or large oesophageal varices as compared to patients in child - pugh class a. the baveno iv international consensus workshop on methodology of diagnosis and treatment concluded that no study reached a high enough level of significance to warrant the widespread use of such noninvasive markers of oesophageal varices . a number of serum markers for ecm deposition and removal have been evaluated as candidate markers for liver fibrosis , and a small number of studies have evaluated their usefulness in predicting oesophageal varices . assessed the ability of serum hyaluronic acid to predict medium - to - large oesophageal varices and showed the sensitivity , specificity , positive and negative predictive values , and diagnostic accuracy at a cutoff of 207 g / l to be 94% , 77.8% , 88.7% , 87.5% , and 88.3% , respectively . showed no association between concentrations of hyaluronic acid or laminin and grade of oesophageal varices , and a further study by bahr et al . in the first of only 2 studies in this area , the aminoterminal propeptide of type iii procollagen was shown to have a weak correlation to the degree of oesophageal varices . included 44 patients with alcoholic liver disease and demonstrated a significant difference in serum type iv collagen levels between patients with and without varices ( 712.3 versus 404.3 ng / ml , p < 0.001 ) , giving an auroc of 0.78 for predicting the presence of oesophageal varices . none of the aforementioned markers could currently be utilised to predict oesophageal varices in portal hypertension ; in view of this several different biomarkers have been combined with the aim of improving their diagnostic ability . a single study has assessed the predictive value of fibrotest in the diagnosis of large oesophageal varices in 99 cirrhotic patients . significant differences in fibrotest value ( 0.89 versus 0.82 ) , platelet count ( 110 versus 150 ) , and prothrombin time ( 50 versus 66% ) were seen between patients with and without large oesophageal varices . thrombocytopenia may occur in portal hypertension - induced splenomegaly , in part due to platelet sequestration , and a large number of studies have been performed assessing the relationship between platelet count and oesophageal varices [ 17 , 1921 ] . a low - platelet count is regularly identified as predictive of oesophageal varices and large oesophageal varices , but there is a wide variation in the cut - off level of platelets used , ranging from 68,000 to 160,000 with sensitivities ranging from 7190% and specificities from 3673% . of 213 patients , with compensated cirrhosis with portal hypertension but without varices , demonstrated that the median platelet count at the time of occurrence of varices was 91,000 . however , no platelet count could be identified that accurately predicted the presence of oesophageal varices ( auroc curve 0.63 ) , and they , therefore , concluded that platelet count is an inadequate noninvasive marker for prediction of the presence of oesophageal varices . in an attempt to improve the predictive value of the platelet count , it has been combined with other variables , and the results of these studies are discussed below . a single study of 85 cirrhotic patients examined the predictive capability of serum nitrate levels to detect oesophageal varices . significant differences in serum nitrate levels were found between patients with large oesophageal varices compared to patients without oesophageal varices ( p < 0.01 ) . animal studies suggest that the formation of oesophageal varices results not only from opening up of preexisting collateral vessels but also as a result of angiogenesis which may in part be mediated by vegf . use of vegf as a noninvasive biomarker has only been investigated in a single study , and no correlation between vegf levels and grade of oesophageal varices was detected . one study has examined the role of blood ammonia concentrations in the noninvasive detection of oesophageal varices . the auroc curve for predicting the presence of oesophageal varices was 0.78 , and using a cut - off of 42 therefore , variables associated with portal hypertension and hypersplenism are not accurate enough to be used as noninvasive markers of oesophageal varices . in 2003 , reported the platelet count / spleen diameter ratio to be the only independent variable associated with presence of ov on multivariate analysis and identified a cut - off value of 909 , giving a ppv of 96% and npv of 100% . at followup , patients with a platelet count / spleen diameter ratio < 909 had 100% npv and 84% ppv , and they concluded that the platelet count spleen diameter ratio was effective in ruling out the presence of ov when cirrhotic patients were followed longitudinally . therefore , despite promising early results the platelet count / spleen diameter ratio is not a reliable tool to screen for oesophageal varices . in 2003 , reported the platelet count / spleen diameter ratio to be the only independent variable associated with presence of ov on multivariate analysis and identified a cut - off value of 909 , giving a ppv of 96% and npv of 100% . at followup , patients with a platelet count / spleen diameter ratio < 909 had 100% npv and 84% ppv , and they concluded that the platelet count spleen diameter ratio was effective in ruling out the presence of ov when cirrhotic patients were followed longitudinally . therefore , despite promising early results the platelet count / spleen diameter ratio is not a reliable tool to screen for oesophageal varices . the first model aimed to detect large varices in child - pugh a patients with a platelet count < 80 and had a sensitivity of 58% , specificity 79% , ppv 30% , and npv 92% . the second model aimed to identifying any varices in child b / c patients with a platelet count < 90 and had a sensitivity of 60% , specificity of 59% , ppv 80% , and npv 34% . once again , the performance of these models would not reliably predict the presence of oesophageal varices . the first model aimed to detect large varices in child - pugh a patients with a platelet count < 80 and had a sensitivity of 58% , specificity 79% , ppv 30% , and npv 92% . the second model aimed to identifying any varices in child b / c patients with a platelet count < 90 and had a sensitivity of 60% , specificity of 59% , ppv 80% , and npv 34% . ( 3 ) ast / alt ratiothe ast / alt ratio has been used to predict cirrhosis , and by natural extension studies have been performed to assess its usefulness in predicting oesophageal varices . in a retrospective study , significantly higher ast / alt ratios were seen in patients with varices compared to those without ( ratio : 1.8 versus 1.0 , p < 0.0001 ) . a further study using a different cut - off of 1.0 demonstrated a sensitivity of 68% , specificity of 89% , ppv 77% , and npv 83% , with an auroc 0.83 ( 0.720.94 ) for predicting the presence of oesophageal varices . for the prediction of large oesophageal varices , this gave a sensitivity 68% , specificity 77% , ppv 41% , and npv 92% , and auroc 0.79 ( 0.640.94 ) . therefore these studies , which include patients with different aetiologies of liver disease and used different cutoffs for the ast / alt ratio can not confidently predict the presence of oesophageal varices in clinical practice to avoid screening all cirrhotic patients with endoscopy . the ast / alt ratio has been used to predict cirrhosis , and by natural extension studies have been performed to assess its usefulness in predicting oesophageal varices . in a retrospective study , significantly higher ast / alt ratios were seen in patients with varices compared to those without ( ratio : 1.8 versus 1.0 , p < 0.0001 ) . a further prospective study found an ast / alt ratio > 1.12 to be significantly associated with the presence of varices at initial endoscopy ( or 3.9 , p = 0.02 95% ci 1.311.8 ) . a further study using a different cut - off of 1.0 demonstrated a sensitivity of 68% , specificity of 89% , ppv 77% , and npv 83% , with an auroc 0.83 ( 0.720.94 ) for predicting the presence of oesophageal varices . for the prediction of large oesophageal varices , this gave a sensitivity 68% , specificity 77% , ppv 41% , and npv 92% , and auroc 0.79 ( 0.640.94 ) . therefore these studies , which include patients with different aetiologies of liver disease and used different cutoffs for the ast / alt ratio can not confidently predict the presence of oesophageal varices in clinical practice to avoid screening all cirrhotic patients with endoscopy . right liver lobe / albumin ratio correlated with presence and size of oesophageal varices ( r = 0.488 , p < 0.01 ; r = 0.481 , p < 0.01 , respectively ) . studies suggest that te is highly reproducible and reliable with very high interobserver and intraobserver agreement overall but that patient related and liver disease related factors may have a negative effect on the reproducibility of this technique . a number of studies have been performed examining the relationship of liver stiffness to size and presence of oesophageal varices , and these results are summarised in table 1 [ 47 , 5457 ] . these studies demonstrate a significant correlation between liver stiffness measurements and the presence of oesophageal varices but are divided with regard to the relationship of liver stiffness to variceal size . for the diagnosis of variceal presence , auroc curves varied from 0.760.85 , with a sensitivities of 8495% , specificities of 4378% , ppv 5789% , and npv 6691% using cutoffs between 13.921.5 kpa . for the diagnosis of large oesophageal varices , auroc varied from 0.760.87 , with sensitivities of 7791% , specificities of 6085% , ppv 4856% and npv 94 - 95% using cut - offs between 1930.5 kpa . the other limitations of the study relate to inclusion of patients with liver disease of different aetiologies and of different severity , according to child - pugh class . however , it may be useful as a screening test to identify patients in whom variceal screening is not required , but at present can not be advocated as a surrogate for gastroscopy . in a study of 191 patients ( 135 cirrhotic ) recently published , it was demonstrated that spleen stiffness was significantly higher in cirrhotics than noncirrhotics and in patients with oesophageal varices compared to those without . they found a better diagnostic accuracy , of 89.95% , in predicting the presence but not the grade of oesophageal varices when liver and spleen stiffness were used together . all of the 7 patients with oesophageal varices had a mean spleen stiffness of > 10.5 kpa . further larger studies are needed to investigate the diagnostic accuracy of mr elastographic spleen stiffness for noninvasive prediction of oesophageal varices . portal vein diameter , portal blood velocity and congestion index , spleen size , flow pattern in the hepatic veins , and the presence of abdominal portosystemic collaterals are all us parameters previously thought to have with prognostic significance but all with poor sensitivity and specificity . greater than 13 mm , and platelet count < 100 10 as noninvasive predictive tools to discriminate cirrhotic patients with and without oesophageal varices ( ov ) . ct was found to have approximately 90% sensitivity in the identification of oesophageal varices determined to be large on endoscopy , but only about 50% specificity . new capsule endoscopy devices have been developed , specifically for use in the oesophagus , acquiring images from both ends of the device . several studies have been performed , assessing the ability of these capsule endoscopy devices to detect any varices and identify large varices requiring primary prophylaxis [ 6873 ] . other studies have correctly identified patients requiring primary prophylaxis in 74100% of patients [ 69 , 71 , 72 , 74 ] . 2 meta - analyses produced similar results with pooled sensitivities of 83% and 83.8% and pooled specificities of 85% and 80.5% , respectively for the diagnosis of oesophageal varices [ 75 , 76 ] . capsule endoscopy is reported to be feasible in 9499% of patients with the main reasons for failure being because patients were unable to swallow the capsule or due to technical problems with the recording or function of the capsule . with regard to cost - effectiveness , 2 studies have been performed , the first concluding that both screening methods are equivalent , the second that screening with capsule endoscopy followed by beta - blocker therapy may be cost - effective compared to ogd followed by beta - blocker therapy but is highly sensitive to local costs [ 78 , 79 ] . therefore in summary , capsule endoscopy is feasible in the majority of patients and with regard to patient preference , capsule endoscopy appears to be preferable to conventional endoscopy and may improve compliance with screening programmes , although this remains to be determined . the jury is still out with regard to cost but when it comes to performance , conventional ogd remains the gold standard . these haemodynamic variables are independently associated with portal pressure and size of oesophageal varices [ 8184 ] . we have demonstrated the presence of the hyperdynamic circulation using this technique and shown significant differences in cardiac output and systemic vascular resistance according to the size of oesophageal varices . data as yet unpublished examining the predictive ability of noninvasive parameters has shown promising initial results , with an auroc curve of 0.86 for cardiac output and 0.77 for peripheral vascular resistance for the diagnosis of large oesophageal varices . promising results have been seen in patients with liver cirrhosis to search for markers of hepatic fibrosis [ 8688 ] and has been demonstrated to be more accurate than fibrotest . the optimal biomarker needs to be able to predict clinically significant endpoints as well as liver histology , and so further research is needed to know whether proteomics will ever be useful in the noninvasive diagnosis of oesophageal varices . in conclusion , based on all the available evidence to date , upper gi endoscopy remains the gold standard for the diagnosis of oesophageal varices in cirrhotic patients despite its own limitations . clinical , biochemical , and radiological parameters currently are not accurate enough to avoid screening endoscopy , due to the risks associated with missing patients with large oesophageal varices . a screening test must be simple and inexpensive , and therefore current promising tools such as ct scanning or capsule endoscopy which are highly acceptable to patients may not prove to be cost - effective or suitable for repeated measurement .

recent studies have shown that linear leak currents play an important role in the generation of network oscillations ( cymbalyuk et al . , 2002 ; blethyn et al . , 2006 ; koizumi and smith , 2008 ; pang et al . , 2009 ) and regulating excitability ( rekling et al . , 2000 ; brickley et al . , 2007 ) . neuromodulatory substances are known to modify neuronal excitability by activating metabotropic receptors that can regulate ionic currents and pumps ( bayliss et al . , 1992 ; cantrell and catterall , 2001 ; lebeau et al . , 2005 ; tobin and calabrese , 2005 ) . in particular , the negative regulation of leak currents by several neurotransmitters has been proposed to play an important role in the regulation of neuronal excitability ( bayliss et al . , 1992 ; lee and boden , 1997 ; aller et al . , 2005 ; kim , 2005 ; pratt and aizenman , 2007 ; weber et al . , 2008 ) , oscillations ( xu et al . , 2007 ) and sleep - wake transitions ( goldstein et al . , 2001 nevertheless , few studies have directly tested the dependence of pacemaker and network oscillatory activity on leak currents and the role of neuromodulators in this process . in this study we examine the role of linear conductances on the generation of oscillations by the pyloric network of the crab cancer borealis . the pyloric network , located in the stomatogastric ganglion ( stg ) , generates a rhythmic pattern of bursting activity driven by a pacemaker group of one anterior burster ( ab ) and two pyloric dilator ( pd ) neurons . the pyloric oscillations are dependent on and regulated by a variety of neuromodulators that are released hormonally or by descending projection neurons ( nusbaum and beenhakker , 2002 ) . when neuromodulators are removed , e.g. , by decentralization , the bursting oscillations are usually disrupted and the pyloric neurons either spike tonically or become quiescent ( marder and bucher , 2006 ) . using the dynamic clamp technique , we show that in a decentralized preparation , a reduction in the level of linear ( leak ) conductance in a single pyloric pacemaker neuron recovers oscillatory activity in that neuron , and interestingly , brings back the entire network oscillation . many of peptidergic and other modulators converge to unmask a voltage - dependent inward current ( imi ) characterized by an inverted bell - shaped current - voltage relationship ( golowasch and marder , 1992b ; swensen and marder , 2000 ) . an inverted bell - shaped i v curve has traditionally been thought to confer a region of negative - slope conductance to the overall i v curve of a neuron , enhancing their excitability and their tendency to produce regenerative membrane potential changes , including slow oscillations . v curve could be approximated by two linear segments , one with negative and one with positive slope . the positive - conductance component can depolarize the cell but reduces excitability whereas the negative - conductance component both increases excitability and reduces the stability of the membrane potential near the resting potential . our dynamic clamp results indicate that regulation of a linear conductance with a reversal potential near the pacemaker neuron 's resting potential , controls the ability of the pyloric pacemaker neuron to generate oscillations . furthermore , we show that the only essential element of the non - linear imi that contributes to the enhancement of membrane excitability and promoting membrane potential oscillations is the negative - conductance region spanning a voltage range that is aligned with the resting potential of the pacemaker neuron . experiments were carried on adult male crabs ( cancer borealis ) purchased from local distributors ( newark , nj , usa ) . the animals were maintained in artificial seawater tanks at 1215c until they were used , when they were first anesthetized by cooling on ice for 1530 min prior to each dissection . the stomatogastric nervous system ( including the stomatogastric ganglion , stg , the esophageal ganglion , og , and the commissural ganglia , cogs , figure 1a ) was removed using standard methods ( selverston et al . , 1976 ; harris - warrick et al . , 1992 ) and each preparation was pinned down in a sylgard - coated petri dish . the stg was desheathed to expose the cell bodies for microelectrode impalement , and to allow effective superfusion . preparations were superfused using normal saline containing ( in mm ) : 11 kcl , 440 nacl , 13 cacl2 , 26 mgcl2 , 11.2 trizma base , 5.1 maleic acid , ph 7.47.5 , and kept at 12c . effect of decentralization on the pyloric network oscillations . the medial and lateral ventral motor nerves ( mvn , lvn ) contain axons from motor neurons located in the stomatogastric ganglion ( stg ) . the stg receives descending projections from neurons with somata located in the oesophageal ganglion ( og ) and the paired commissural ganglion ( cog , one such projection neuron is labeled in red ) . a petroleum jelly well ( yellow ) around the desheathed stomatogastric nerve ( stn ) is filled with 0.75-m sucrose plus 1-m ttx to block action potential transmission of descending projections to the stg ( decentralization ) . the pacemaker neuron ab is strongly electrically coupled to the two pd neurons and , together , they make a synchronously bursting pacemaker group . ( c ) extracellular recordings from lvn and mvn under control ( left ) and decentralized ( right ) conditions , demonstrating that removal of input from descending projections results in the cessation of pyloric oscillations . neurons pd , lp and py are labeled on the lvn recording and neurons ic and vd are labeled on the mvn recording . the axon of the pacemaker ab neuron runs along stn ( recording not shown ) . intracellular recordings of pd , ab and lp neurons are shown in the bottom three traces . microelectrodes were pulled using a flaming - brown micropipette puller ( p97 , sutter instruments ) and filled with 0.6 m k2so4 + 0.02 m kcl ( resistance of 1525 m ) . extracellular recordings were made with two stainless steel pins , one placed in the bath and the other within a petroleum jelly well built around the nerve of interest . neuronal identification was accomplished by matching the intracellular action potential recordings to their corresponding extracellular nerve recordings ( selverston et al . , 1976 ; harris - warrick et al . , 1992 ) . intracellular recordings were made from the soma of the neurons using axoclamp 2b amplifiers ( molecular devices ) and extracellular recordings were amplified using a differential ac amplifier model 1700 ( a m systems ) . we used the dynamic clamp technique ( sharp et al . , 1992 ) to introduce ionic conductances into , or subtract them from , the biological neurons . a ni pci-6070-e board ( national instruments , austin , tx , usa ) was used for current injection in dynamic clamp experiments . data acquisition was performed using the digidata 1332a data acquisition board and pclamp 9.2 software ( molecular devices ) . the dynamic clamp software was developed in our laboratories ( available for download at http://stg.rutgers.edu/software ) in the labwindows / cvi software environment ( national instruments ) on a windows platform . in dynamic clamp , ionic currents are calculated using hodgkin - huxley - type equations as described below and continuously updated by recording the membrane potential ( v ) of the neurons in real time . imi was described by : where emi = 10 mv and m = 4 ms . the standard leak current is described by il = gl(vel ) where el = 68 mv is the leak current reversal potential and gl is the leak conductance . when the value of leak conductance was set to be negative in the dynamic clamp software it resulted in a reduction of the total neuronal conductance . we describe this current as the voltage - dependent current imi was divided into regions approximating a linear i v curve with positive linear conductance ( mi pl ) or negative linear conductance ( mi nl ) . the linear conductance regions were restricted in their voltage range so that they represented the negative and positive - conductance regions of imi : where = 4 ms . although imi is non - inactivating , to build imi nl , an inactivation variable hn with very fast kinetics is used in dynamic clamp as a method of restricting the voltage range of activation below the upper limit of 46 mv ( see figure 6a , red trace ) . a similar use of the activation variable mp was made to produce imi pl ( figure 6a , gray trace ) . it should be noted that dynamic clamp conductance manipulations introduce artificial ionic currents into the point of penetration , in this case the soma . the ionic currents in biological neurons are distributed over the entire structure of the cell and it is possible therefore that the size of the currents injected by dynamic clamp does not properly represent the equivalent biological ionic current . in particular , the currents injected in our dynamic clamp experiments may be much larger than what is actually needed for the pacemaker neurons to produce oscillations . we measured neuronal input conductance before and after injecting negative conductances with dynamic clamp to avoid reducing the total conductance of the neuron to less than zero . when simultaneous dynamic clamp current and constant current injections were required , a brownlee precision amplifier ( brownlee precision ) was used to add the currents before input to the axoclamp amplifier the acquired data were saved as binary files and were analyzed with the software readscope ( http://stg.rutgers.edu/software ) and clampfit 9.2 ( molecular devices ) . for experiments performed to compare the effect of neuromodulatory input , the stg was isolated from anterior neuromodulatory inputs ( i.e. decentralized ) by replacing the saline solution within a petroleum jelly well built around the single stomatogastric nerve ( stn , figure 1a ) with 0.75-m sucrose and 1-m ttx ( biotium ) to block action potential transmission . in experiments to measure the input resistance of neurons 1-m ttx was used to superfuse the ganglion . sas ( sas institute ) , sigmastat 2.3 ( systat software ) and origin 7 ( originlab ) software packages were used for statistical and graphical analysis and all final figures were made in coreldraw 12 ( corel corp . ) . reported statistical significance indicated a significance level p 0.05 . in all statistical tests all error bars shown and error values reported are standard deviations of the mean ( sd ) . the pyloric network is a central pattern generator located in the stomatogastric ganglion , one of the four ganglia in the stomatogastric nervous system , an extension of the crab cns ( figure 1a ) . the pyloric network is an extremely well - studied network and consists , primarily , of six types of neurons that are connected with inhibitory synapses as well as gap junctions ( figure 1b ) ( nusbaum and beenhakker , 2002 ; marder and bucher , 2006 ) . oscillations in the pyloric network are relatively stereotypical and consist of three phases with a cycle frequency of 1 hz ( figure 1c ) . all pyloric neurons take part in the network oscillation by producing bursts of action potentials on top of slow wave oscillations of their membrane potential . these oscillations are generated by a pacemaker group consisting of the ab and two pd neurons which are strongly coupled through gap junctions and produce synchronous bursting activity . pyloric oscillations are conditional upon the presence of neuromodulators that are released hormonally and in paracrine fashion from descending neurons that project to the stg via the stomatogastric nerve ( stn ; figure 1a ) . blocking action potential conduction in the stn ( decentralization ) in vitro results in the disruption of the pyloric oscillations ( figure 1c ) . neuromodulatory inputs have been shown to target ionic currents in all pyloric neurons and modify the strengths of chemical synapses and gap junctions in the network ( johnson et al . although distinct modulators diverge in their target neurons , many modulators of the pyloric network , particularly neuropeptides and muscarinic agonists , have been shown to converge to activate a single voltage - gated inward current ( imi ) ( golowasch and marder , 1992b ; swensen and marder , 2000 ) . it should be noted that not all modulatory actions converge on this current and a variety of other ionic currents are also regulated by neuromodulators , particularly neuroamines ( harris - warrick et al . , 1995 ; johnson et al . , 2003 ) . the intact pyloric network is presumed to be subject to modulation by a variety of substances , each targeting a subset of neurons and synapses , the combined actions of which result in the observed triphasic pyloric oscillations . here we examine the hypothesis that the primary action of neuromodulators that results in the triphasic pyloric oscillation is a simple modification of the leakiness of the pacemaker neurons . during pyloric oscillations the pacemaker neurons ab and pd produce synchronous oscillations and follower neurons such as the lp neuron burst out of phase with the pacemakers ( figures 1c and 2a ) . when the preparation was decentralized , bursting oscillations in all the neurons ceased and the slow wave oscillation of their membrane potentials were suppressed and replaced with a almost completely quiescent membrane potential of 60 to 50 mv ( figure 2b ) . using the dynamic clamp technique , we reduced the magnitude of the leak current ( i.e. increased the input resistance rin ) in individual pyloric neurons in a way that the resting potential was not significantly changed . 2 ) with a reversal potential near the resting potential of the neuron . a small value of gnl in any of the neurons did not affect the output of a decentralized preparation . when a sufficiently large gnl value was added to the ab neuron ( n = 3 ) , bursting activity in the ab neuron resumed and other pyloric neurons also resumed their oscillations with a triphasic pyloric activity pattern ( figure 2c ) . all oscillatory activity ceased immediately as soon as the dynamic clamp current was removed ( not shown ) . similarly , if a sufficiently large gnl was added to either one of the two pd neurons , it resulted in the recovery of the triphasic pyloric activity ( figure 2d ; n = 8) . in contrast , addition of an inl to the lp neuron never resulted in the recovery of oscillations in either the lp neuron or the network no matter what conductance value gnl was used ( figure 2e ; n = 3 ) . simultaneous recordings of lvn and mvn ( extracellular ) , and of pd , ab and lp neurons ( intracellular ) are shown in all panels . bottom traces show the current injected with dynamic clamp ( idc ) in the respective neuron . ( c , d ) rhythmic activity that strongly resembles the control pyloric activity is observed when negative leak current inl ( gnl = 80 ns ) is injected into the ab neuron ( c ) as well as into one of the two pd neurons ( d ) . ( e ) no rhythmic activity can be induced when negative leak current inl ( gnl = 80 ns ) is injected into the follower lp neuron . arrowheads point to 60 mv and 0 na . these results indicated that reducing the amount of leak current in a single pacemaker neuron , but not a follower neuron , was sufficient to recover the oscillations of the entire pyloric network . there appeared to be no difference in the ability to recover the oscillations by the dynamic clamp application of inl in either the ab or pd neurons . thus , from this point on we focused our experiments on the pd neurons because the single small ab neuron is often difficult to find and harder to record from than the pd neurons . we examined the effect of the conductance of the inl on the recovered pyloric oscillation . although in different preparations different amounts of gnl led to the recovery of pyloric oscillations , increasingly negative gnl in a recovered rhythm always led to a faster oscillation frequency ( figure 3a ) . on average , a gnl value of 70 to 80 ns was required to bring back the oscillation frequency to the average control value observed before decentralization ( horizontal line ) . we also examined the effect of inl on the phase of activity of the individual pyloric neurons . the phase of individual neurons in the pyloric oscillation is defined as the fraction of each cycle duration ( t ) during which that neuron is active ( figure 3b ) . as seen in figure 3c , the inl not only recovered network oscillations but produced activity phases ( open boxes ) that were statistically similar to those in the control pyloric oscillation recorded before decentralization ( filled boxes ; two - way rm anova ; all pair - wise p values > 0.1 ; n = 6 ) . this was observed in all preparation in which inl led to a recovery of rhythmic pyloric activity . ( a ) negative leak conductance was injected into one pd neuron and the effect on rhythm frequency is plotted against the maximal conductance gnl . ( b ) sample extracellular recordings of lvn and mvn used to determine the phase diagram shown in ( c ) . vertical dashed lines indicate mark the beginning and end of the cycle divided by t. ( c ) phase diagram of pyloric activity . each neuron 's spiking time is represented as a fraction of t ( sd ) . pd neuron firing is used as reference and thus begins at phase = 0 . filled bars are control conditions ; open bars are in response to negative leak current injected with dynamic clamp ( gnl = 80 ns ) . ( n = 6 ; n = 2 for the ic neuron which is not active in all preparations ) . the fact that pyloric oscillations can be recovered in a decentralized preparation by reducing the leak conductance in the pacemaker neurons , leads to the hypothesis that modulatory inputs result in a conductance decrease in these neurons . we examined this hypothesis by measuring the leak conductance in the pd neurons in control and in a decentralized preparation in which action potential transmission along the stn was blocked with only 0.75-m sucrose to allow reversal of the block . these experiments were done in two - electrode voltage clamp in which the pd membrane potential was ramped from a holding value of 60 mv to a final value of 80 mv in 500 ms and the resulting i v curve was fit with a linear function to measure the slope ( figure 4a ) . the current that is blocked by decentralization can then be measured as a difference current ( figure 4b ) . the average reversal potential for the difference current was 63.6 9.5 mv . ( a ) current vs. voltage relationship ( i v ) of a pd neuron obtained by ramping the voltage in two - electrode voltage clamp between 60 and 80 mv in 500 ms . recovery indicates reversal of decentralization after several washes with normal saline of the sucrose well around the stn . dashed blue lines indicate linear fits ( i = av + b ; control : a = 0.071 , b = 3.67 ; decentralized : a = 0.149 , b = 8.14 ; recovery : a = 0.127 , b = 7.16 ) . ( c ) conductance was measured as the slope of the best linear fit to the i v curves in ( a ) ( a values of the fits multiplied by 1000 ) . the input conductance of the decentralized pd neurons was significantly higher than either the control or the recovered cells ( one - way anova , n = 6 , p = 0.04 ) . to prevent any possible contamination of the leak conductance measurement by activation of voltage - gated currents the neuron was kept in two - electrode voltage clamp while the preparation was decentralized ( see materials and methods ) and while it recovered from decentralization after sucrose was washed from around the stn . these experiments showed that the leak conductance in the pd neuron was significantly increased when the preparation was decentralized , compared to control and recovery ( figure 4c ; n = 6 ; p = 0.04 ) , supporting the hypothesis that the conductance in the decentralized system is reduced when neuromodulators are restored . as mentioned above , several neuromodulators of the pyloric network are known to activate a single voltage - gated inward current imi in pyloric neurons ( swensen and marder , 2000 ) . imi was first characterized through actions of proctolin ( golowasch and marder , 1992b ) , an endogenously released neuromodulator that is known to activate the pyloric oscillations in a decentralized preparation ( nusbaum and marder , 1989 ) . if neuromodulators could affect the rhythm by affecting the pacemaker 's leak current or by targeting imi it is important to know if these effects are similar or related . we therefore compared the effect of inl with adding a dynamic clamp version of imi in the pd neuron and with bath application of 10 m proctolin in decentralized preparations . we found that the dynamic clamp injections of inl or imi and bath application of proctolin all had very similar effects in recovering the pyloric oscillation . top traces correspond to the membrane potential of a pd neuron and the bottom traces to the injected dynamic clamp current in ( a ) control , ( b ) after decentralization , ( c ) during injection of inl ( eq . 2 ) with gnl = 80 ns , ( d ) during injection of imi ( eq . 1 ) with gmi = 80 ns , and ( e ) during bath application of 1-m proctolin . this result led us to the idea that inl and imi act on the pd neuron through similar mechanisms . how could the actions of a non - linear inward current and a linear current with negative conductance be similar ? v curve ( black trace ) can be divided into two regions , one with negative - slope conductance ( red trace ) and the other with positive slope conductance ( gray trace ) . the section of the i v curve with negative - slope conductance can be argued to be well approximated with inl ( dashed blue curve in figure 6a ) . we denoted the negative ( positive ) slope conductance section of imi as imi nl ( imi pl ) and compared the effect of dynamic clamp injection of inl , imi , imi nl and imi pl . these currents were modeled in dynamic clamp with the appropriate parameters so that imi was well approximated by imi nl + imi pl ( eq . 3 ) and inl matched the negative slope of imi nl in the appropriate voltage range as shown in figure 6a ( see materials and methods ) . when imi nl , inl or imi were added to the pd neuron using dynamic clamp the result was always the recovery of oscillatory activity ( figures 6d , f , g ) . in contrast , adding imi pl had little effect on the pd neuron and never resulted in oscillatory activity ( figure 6e ) . these results indicated that the actions of imi on the pd neuron were mainly through the limited region of negative - slope conductance which effectively resulted in a reduction of the leak current in the pd neuron . full imi is the black trace , inl is the dashed blue line that extends across the entire voltage range , imi nl is the red trace , and imi pl is the gray trace . ( d ) activity with imi nl injected ( red trace in ( a ) , gnl = 80 ns ) . ( e ) activity with imi pl injected ( gray trace in ( a ) , gpl = 80 ns ) . ( f ) activity with inl injected ( dashed blue trace in ( a ) , gnl = 80 ns ) . ( g ) activity with the full imi injected ( black trace in ( a ) , gmi = 80 ns ) . all panels in ( b g ) show the membrane potential of the same pd neuron ( top , arrowheads point to 70 mv ) and the dynamic clamp - injected current ( bottom , arrowheads point to 0 na ) . regulation of leak currents has been proposed to play an important role in the control of neuronal excitability , oscillatory activity and sleep - wake transitions ( bayliss et al . , 1992 ; lee and boden , 1997 ; goldstein et al . , 2001 ; aller et al . , 2005 ; kim , 2005 ; pratt and aizenman , 2007 ; xu et al . , 2007 ; weber et al . , 2008 ) . a number of neuromodulators acting on metabotropic receptors have been shown to negatively regulate leak currents by decreasing their conductance ; these include glutamate ( blethyn et al . , 2006 ; brickley et al . , 2007 ) , thyrotropin - releasing hormone ( trh ) ( bayliss et al . , 1992 ) and serotonin ( weber et al . , 2008 ) . we show that the action of the neuromodulatory inputs in producing pyloric oscillations can be mimicked by a simple reduction in the leak current of the pyloric pacemaker neurons . when a leak current with negative conductance is injected , using the dynamic clamp technique , in any one of the neurons that comprise the pyloric pacemaker group , the rhythmic pattern of the entire network is recovered . this recovered rhythm is essentially identical to the ongoing pyloric rhythm prior to decentralization and to the rhythm produced by bath application of a modulatory neuropeptide in a decentralized preparation . in contrast , injecting the negative - conductance leak current into non - pacemaker neurons in the pyloric network fails to activate the pyloric rhythm or any rhythmic activity in that neuron . how does the recovery of the pyloric rhythm by reducing the leak conductance in the pacemaker neurons relate to the known effects of neuromodulators in this network ? much of the neuromodulatory actions on individual pyloric neurons converge , through unknown second messenger mechanisms , to activate a voltage - gated inward current imi ( swensen and marder , 2000 ) . we show that the i v curve of this non - linear inward current can basically be decomposed into two linear components , one with positive - conductance imi pl and one with negative conductance imi nl . when we introduced one or the other of these two components into the pd neuron using dynamic clamp , we found that imi nl led to the recovery of oscillations whereas imi pl did not . the recovered oscillation was identical to oscillations produced with the full i v range of imi or that produced by bath application of the neuropeptide proctolin . the red and gray traces shown in figure 6 suggest that the sum of these two linear components , restricted in their voltage range , very closely match the total current imi over the full voltage range . however , we observed that the linear negative - conductance component ( imi nl or inl ) effectively activates the pyloric rhythm only when its reversal potential was negative relative to the membrane potentials trajectory of the pacemaker neurons slow wave . reversal potential values more positive than that led to currents that were outward for most of this trajectory and thus did not enhance their excitability ( not shown ) . conversely , with its positive reversal potential , the positive - conductance linear component ( imi pl ) only led to a slight depolarization of the baseline voltage but decreased the input resistance of the cells and did not enhance their excitability . electrical loading of a neuron can affect its membrane potential amplitude changes and temporal properties such as spiking and bursting activity . such electrical loading can occur via two mechanisms : electrical coupling to other cells or changes in the passive membrane conductance . in the pyloric network , electrical coupling of the intrinsically oscillatory ab neuron to non - oscillatory neurons , such as the pd neuron , affects the oscillatory activity of the whole ensemble ( hooper and marder , 1987 ; kepler et al . , 1990 ; if the load produced by the non - oscillatory neurons is large , it would kill the oscillations . a negative conductance in any of the coupled neurons effectively reduces the electric load on the oscillatory cell , leading to an enhanced excitability . any reduction in leak current has this effect as long as the membrane potential of the cell is more positive than the current 's reversal potential ( e.g. sirois et al . , 2002 ; thus , the reduction of the leak current in the pd neuron may lead to the recovery of oscillatory activity because of the strong electrical coupling of the pd neurons to the ab neuron . however , our experiments do not exclude the possibility that the pd neuron itself becomes an intrinsic oscillator under these conditions . previous studies of the pyloric network have shown that blocking outward currents can result in a recovery of oscillations in the ab and pd neurons , although a recovery of the full network activity was not reported ( harris - warrick and johnson , 1987 ) . additionally , modulatory actions by dopamine , that do not target imi , have also been reported to result in a significant increase of the input resistance in the ab neuron and a simultaneous decrease in the pd neuron ( johnson et al . , 1993 ) and dopamine has been shown to induce oscillatory activity in the isolated ab neuron in lobsters ( johnson et al . , 1992 ) . it is therefore possible that recovery of oscillations may occur through mechanisms that include , among other effects , a decrease in leak conductance , but that are independent of imi . changes in leak conductance have been shown to be the likely mechanism underlying the excitatory action of several substances acting on metabotropic receptors ( bayliss et al . , 1992 ; sirois et al . , 2002 ; blethyn et al . , 2006 ; brickley et al . , 2007 ; koizumi and smith , 2008 in many if not all these cases , it appears that the consequence of a reduced leak current is to move the voltage of the cell to the activation range of voltage - dependent pacemaker currents . of course , excitability can not result from a simple reduction in the endogenous leak current ; it is necessary to produce a negative - slope conductance in the i v curve that creates a regenerative mechanism for membrane depolarization . in the case of the pacemaker pyloric network neurons , we suggest that the negative - conductance component of imi is itself the pacemaker current since no other current has been reported to be active in the voltage range of this conductance component ( graubard and hartline , 1991 ; golowasch and marder , 1992a ; soto - trevino et al . , although this hypothesis needs to be tested experimentally , it effectively implies that the fundamental pacemaker properties in this network result from the actions of a linear portion of a non - linear current that is activated by neuromodulatory inputs . in other words , it appears that pacemaker activity in this network depends on a linear current , whose essential properties are implemented by the modulator - activated inward current imi . 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 .

linear leak currents have been implicated in the regulation of neuronal excitability , generation of neuronal and network oscillations , and network state transitions . yet , few studies have directly tested the dependence of network oscillations on leak currents or explored the role of leak currents on network activity . in the oscillatory pyloric network of decapod crustaceans neuromodulatory inputs are necessary for pacemaker activity . a large subset of neuromodulators is known to activate a single voltage - gated inward current imi , which has been shown to regulate the rhythmic activity of the network and its pacemaker neurons . using the dynamic clamp technique , we show that the crucial component of imi for the generation of oscillatory activity is only a close - to - linear portion of the current - voltage relationship . the nature of this conductance is such that the presence or the absence of neuromodulators effectively regulates the amount of leak current and the input resistance in the pacemaker neurons . when deprived of neuromodulatory inputs , pyloric oscillations are disrupted ; yet , a linear reduction of the total conductance in a single neuron within the pacemaker group recovers not only the pacemaker activity in that neuron , but also leads to a recovery of oscillations in the entire pyloric network . the recovered activity produces proper frequency and phasing that is similar to that induced by neuromodulators . these results show that the passive properties of pacemaker neurons can significantly affect their capacity to generate and regulate the oscillatory activity of an entire network , and that this feature is exploited by neuromodulatory inputs .

Introduction Materials and Methods Results Discussion Conflict of Interest Statement

recent studies have shown that linear leak currents play an important role in the generation of network oscillations ( cymbalyuk et al . in particular , the negative regulation of leak currents by several neurotransmitters has been proposed to play an important role in the regulation of neuronal excitability ( bayliss et al . , 2001 nevertheless , few studies have directly tested the dependence of pacemaker and network oscillatory activity on leak currents and the role of neuromodulators in this process . in this study we examine the role of linear conductances on the generation of oscillations by the pyloric network of the crab cancer borealis . the pyloric network , located in the stomatogastric ganglion ( stg ) , generates a rhythmic pattern of bursting activity driven by a pacemaker group of one anterior burster ( ab ) and two pyloric dilator ( pd ) neurons . the pyloric oscillations are dependent on and regulated by a variety of neuromodulators that are released hormonally or by descending projection neurons ( nusbaum and beenhakker , 2002 ) . using the dynamic clamp technique , we show that in a decentralized preparation , a reduction in the level of linear ( leak ) conductance in a single pyloric pacemaker neuron recovers oscillatory activity in that neuron , and interestingly , brings back the entire network oscillation . many of peptidergic and other modulators converge to unmask a voltage - dependent inward current ( imi ) characterized by an inverted bell - shaped current - voltage relationship ( golowasch and marder , 1992b ; swensen and marder , 2000 ) . our dynamic clamp results indicate that regulation of a linear conductance with a reversal potential near the pacemaker neuron 's resting potential , controls the ability of the pyloric pacemaker neuron to generate oscillations . furthermore , we show that the only essential element of the non - linear imi that contributes to the enhancement of membrane excitability and promoting membrane potential oscillations is the negative - conductance region spanning a voltage range that is aligned with the resting potential of the pacemaker neuron . the stomatogastric nervous system ( including the stomatogastric ganglion , stg , the esophageal ganglion , og , and the commissural ganglia , cogs , figure 1a ) was removed using standard methods ( selverston et al . the pacemaker neuron ab is strongly electrically coupled to the two pd neurons and , together , they make a synchronously bursting pacemaker group . ( c ) extracellular recordings from lvn and mvn under control ( left ) and decentralized ( right ) conditions , demonstrating that removal of input from descending projections results in the cessation of pyloric oscillations . the axon of the pacemaker ab neuron runs along stn ( recording not shown ) . intracellular recordings were made from the soma of the neurons using axoclamp 2b amplifiers ( molecular devices ) and extracellular recordings were amplified using a differential ac amplifier model 1700 ( a m systems ) . we used the dynamic clamp technique ( sharp et al . the dynamic clamp software was developed in our laboratories ( available for download at http://stg.rutgers.edu/software ) in the labwindows / cvi software environment ( national instruments ) on a windows platform . in dynamic clamp , ionic currents are calculated using hodgkin - huxley - type equations as described below and continuously updated by recording the membrane potential ( v ) of the neurons in real time . when the value of leak conductance was set to be negative in the dynamic clamp software it resulted in a reduction of the total neuronal conductance . we describe this current as the voltage - dependent current imi was divided into regions approximating a linear i v curve with positive linear conductance ( mi pl ) or negative linear conductance ( mi nl ) . the ionic currents in biological neurons are distributed over the entire structure of the cell and it is possible therefore that the size of the currents injected by dynamic clamp does not properly represent the equivalent biological ionic current . in particular , the currents injected in our dynamic clamp experiments may be much larger than what is actually needed for the pacemaker neurons to produce oscillations . we measured neuronal input conductance before and after injecting negative conductances with dynamic clamp to avoid reducing the total conductance of the neuron to less than zero . when simultaneous dynamic clamp current and constant current injections were required , a brownlee precision amplifier ( brownlee precision ) was used to add the currents before input to the axoclamp amplifier the acquired data were saved as binary files and were analyzed with the software readscope ( http://stg.rutgers.edu/software ) and clampfit 9.2 ( molecular devices ) . the pyloric network is a central pattern generator located in the stomatogastric ganglion , one of the four ganglia in the stomatogastric nervous system , an extension of the crab cns ( figure 1a ) . the pyloric network is an extremely well - studied network and consists , primarily , of six types of neurons that are connected with inhibitory synapses as well as gap junctions ( figure 1b ) ( nusbaum and beenhakker , 2002 ; marder and bucher , 2006 ) . oscillations in the pyloric network are relatively stereotypical and consist of three phases with a cycle frequency of 1 hz ( figure 1c ) . all pyloric neurons take part in the network oscillation by producing bursts of action potentials on top of slow wave oscillations of their membrane potential . these oscillations are generated by a pacemaker group consisting of the ab and two pd neurons which are strongly coupled through gap junctions and produce synchronous bursting activity . pyloric oscillations are conditional upon the presence of neuromodulators that are released hormonally and in paracrine fashion from descending neurons that project to the stg via the stomatogastric nerve ( stn ; figure 1a ) . blocking action potential conduction in the stn ( decentralization ) in vitro results in the disruption of the pyloric oscillations ( figure 1c ) . neuromodulatory inputs have been shown to target ionic currents in all pyloric neurons and modify the strengths of chemical synapses and gap junctions in the network ( johnson et al . although distinct modulators diverge in their target neurons , many modulators of the pyloric network , particularly neuropeptides and muscarinic agonists , have been shown to converge to activate a single voltage - gated inward current ( imi ) ( golowasch and marder , 1992b ; swensen and marder , 2000 ) . the intact pyloric network is presumed to be subject to modulation by a variety of substances , each targeting a subset of neurons and synapses , the combined actions of which result in the observed triphasic pyloric oscillations . here we examine the hypothesis that the primary action of neuromodulators that results in the triphasic pyloric oscillation is a simple modification of the leakiness of the pacemaker neurons . during pyloric oscillations the pacemaker neurons ab and pd produce synchronous oscillations and follower neurons such as the lp neuron burst out of phase with the pacemakers ( figures 1c and 2a ) . when the preparation was decentralized , bursting oscillations in all the neurons ceased and the slow wave oscillation of their membrane potentials were suppressed and replaced with a almost completely quiescent membrane potential of 60 to 50 mv ( figure 2b ) . using the dynamic clamp technique , we reduced the magnitude of the leak current ( i.e. increased the input resistance rin ) in individual pyloric neurons in a way that the resting potential was not significantly changed . all oscillatory activity ceased immediately as soon as the dynamic clamp current was removed ( not shown ) . similarly , if a sufficiently large gnl was added to either one of the two pd neurons , it resulted in the recovery of the triphasic pyloric activity ( figure 2d ; n = 8) . in contrast , addition of an inl to the lp neuron never resulted in the recovery of oscillations in either the lp neuron or the network no matter what conductance value gnl was used ( figure 2e ; n = 3 ) . bottom traces show the current injected with dynamic clamp ( idc ) in the respective neuron . ( c , d ) rhythmic activity that strongly resembles the control pyloric activity is observed when negative leak current inl ( gnl = 80 ns ) is injected into the ab neuron ( c ) as well as into one of the two pd neurons ( d ) . these results indicated that reducing the amount of leak current in a single pacemaker neuron , but not a follower neuron , was sufficient to recover the oscillations of the entire pyloric network . there appeared to be no difference in the ability to recover the oscillations by the dynamic clamp application of inl in either the ab or pd neurons . although in different preparations different amounts of gnl led to the recovery of pyloric oscillations , increasingly negative gnl in a recovered rhythm always led to a faster oscillation frequency ( figure 3a ) . we also examined the effect of inl on the phase of activity of the individual pyloric neurons . the phase of individual neurons in the pyloric oscillation is defined as the fraction of each cycle duration ( t ) during which that neuron is active ( figure 3b ) . as seen in figure 3c , the inl not only recovered network oscillations but produced activity phases ( open boxes ) that were statistically similar to those in the control pyloric oscillation recorded before decentralization ( filled boxes ; two - way rm anova ; all pair - wise p values > 0.1 ; n = 6 ) . this was observed in all preparation in which inl led to a recovery of rhythmic pyloric activity . the fact that pyloric oscillations can be recovered in a decentralized preparation by reducing the leak conductance in the pacemaker neurons , leads to the hypothesis that modulatory inputs result in a conductance decrease in these neurons . we examined this hypothesis by measuring the leak conductance in the pd neurons in control and in a decentralized preparation in which action potential transmission along the stn was blocked with only 0.75-m sucrose to allow reversal of the block . these experiments were done in two - electrode voltage clamp in which the pd membrane potential was ramped from a holding value of 60 mv to a final value of 80 mv in 500 ms and the resulting i v curve was fit with a linear function to measure the slope ( figure 4a ) . the input conductance of the decentralized pd neurons was significantly higher than either the control or the recovered cells ( one - way anova , n = 6 , p = 0.04 ) . to prevent any possible contamination of the leak conductance measurement by activation of voltage - gated currents the neuron was kept in two - electrode voltage clamp while the preparation was decentralized ( see materials and methods ) and while it recovered from decentralization after sucrose was washed from around the stn . these experiments showed that the leak conductance in the pd neuron was significantly increased when the preparation was decentralized , compared to control and recovery ( figure 4c ; n = 6 ; p = 0.04 ) , supporting the hypothesis that the conductance in the decentralized system is reduced when neuromodulators are restored . as mentioned above , several neuromodulators of the pyloric network are known to activate a single voltage - gated inward current imi in pyloric neurons ( swensen and marder , 2000 ) . imi was first characterized through actions of proctolin ( golowasch and marder , 1992b ) , an endogenously released neuromodulator that is known to activate the pyloric oscillations in a decentralized preparation ( nusbaum and marder , 1989 ) . we therefore compared the effect of inl with adding a dynamic clamp version of imi in the pd neuron and with bath application of 10 m proctolin in decentralized preparations . we found that the dynamic clamp injections of inl or imi and bath application of proctolin all had very similar effects in recovering the pyloric oscillation . top traces correspond to the membrane potential of a pd neuron and the bottom traces to the injected dynamic clamp current in ( a ) control , ( b ) after decentralization , ( c ) during injection of inl ( eq . how could the actions of a non - linear inward current and a linear current with negative conductance be similar ? we denoted the negative ( positive ) slope conductance section of imi as imi nl ( imi pl ) and compared the effect of dynamic clamp injection of inl , imi , imi nl and imi pl . 3 ) and inl matched the negative slope of imi nl in the appropriate voltage range as shown in figure 6a ( see materials and methods ) . when imi nl , inl or imi were added to the pd neuron using dynamic clamp the result was always the recovery of oscillatory activity ( figures 6d , f , g ) . these results indicated that the actions of imi on the pd neuron were mainly through the limited region of negative - slope conductance which effectively resulted in a reduction of the leak current in the pd neuron . all panels in ( b g ) show the membrane potential of the same pd neuron ( top , arrowheads point to 70 mv ) and the dynamic clamp - injected current ( bottom , arrowheads point to 0 na ) . regulation of leak currents has been proposed to play an important role in the control of neuronal excitability , oscillatory activity and sleep - wake transitions ( bayliss et al . a number of neuromodulators acting on metabotropic receptors have been shown to negatively regulate leak currents by decreasing their conductance ; these include glutamate ( blethyn et al . we show that the action of the neuromodulatory inputs in producing pyloric oscillations can be mimicked by a simple reduction in the leak current of the pyloric pacemaker neurons . when a leak current with negative conductance is injected , using the dynamic clamp technique , in any one of the neurons that comprise the pyloric pacemaker group , the rhythmic pattern of the entire network is recovered . in contrast , injecting the negative - conductance leak current into non - pacemaker neurons in the pyloric network fails to activate the pyloric rhythm or any rhythmic activity in that neuron . how does the recovery of the pyloric rhythm by reducing the leak conductance in the pacemaker neurons relate to the known effects of neuromodulators in this network ? much of the neuromodulatory actions on individual pyloric neurons converge , through unknown second messenger mechanisms , to activate a voltage - gated inward current imi ( swensen and marder , 2000 ) . we show that the i v curve of this non - linear inward current can basically be decomposed into two linear components , one with positive - conductance imi pl and one with negative conductance imi nl . when we introduced one or the other of these two components into the pd neuron using dynamic clamp , we found that imi nl led to the recovery of oscillations whereas imi pl did not . the recovered oscillation was identical to oscillations produced with the full i v range of imi or that produced by bath application of the neuropeptide proctolin . the red and gray traces shown in figure 6 suggest that the sum of these two linear components , restricted in their voltage range , very closely match the total current imi over the full voltage range . however , we observed that the linear negative - conductance component ( imi nl or inl ) effectively activates the pyloric rhythm only when its reversal potential was negative relative to the membrane potentials trajectory of the pacemaker neurons slow wave . conversely , with its positive reversal potential , the positive - conductance linear component ( imi pl ) only led to a slight depolarization of the baseline voltage but decreased the input resistance of the cells and did not enhance their excitability . such electrical loading can occur via two mechanisms : electrical coupling to other cells or changes in the passive membrane conductance . in the pyloric network , electrical coupling of the intrinsically oscillatory ab neuron to non - oscillatory neurons , such as the pd neuron , affects the oscillatory activity of the whole ensemble ( hooper and marder , 1987 ; kepler et al . a negative conductance in any of the coupled neurons effectively reduces the electric load on the oscillatory cell , leading to an enhanced excitability . any reduction in leak current has this effect as long as the membrane potential of the cell is more positive than the current 's reversal potential ( e.g. , 2002 ; thus , the reduction of the leak current in the pd neuron may lead to the recovery of oscillatory activity because of the strong electrical coupling of the pd neurons to the ab neuron . previous studies of the pyloric network have shown that blocking outward currents can result in a recovery of oscillations in the ab and pd neurons , although a recovery of the full network activity was not reported ( harris - warrick and johnson , 1987 ) . additionally , modulatory actions by dopamine , that do not target imi , have also been reported to result in a significant increase of the input resistance in the ab neuron and a simultaneous decrease in the pd neuron ( johnson et al . , 1993 ) and dopamine has been shown to induce oscillatory activity in the isolated ab neuron in lobsters ( johnson et al . it is therefore possible that recovery of oscillations may occur through mechanisms that include , among other effects , a decrease in leak conductance , but that are independent of imi . changes in leak conductance have been shown to be the likely mechanism underlying the excitatory action of several substances acting on metabotropic receptors ( bayliss et al . , 2007 ; koizumi and smith , 2008 in many if not all these cases , it appears that the consequence of a reduced leak current is to move the voltage of the cell to the activation range of voltage - dependent pacemaker currents . of course , excitability can not result from a simple reduction in the endogenous leak current ; it is necessary to produce a negative - slope conductance in the i v curve that creates a regenerative mechanism for membrane depolarization . in the case of the pacemaker pyloric network neurons , we suggest that the negative - conductance component of imi is itself the pacemaker current since no other current has been reported to be active in the voltage range of this conductance component ( graubard and hartline , 1991 ; golowasch and marder , 1992a ; soto - trevino et al . , although this hypothesis needs to be tested experimentally , it effectively implies that the fundamental pacemaker properties in this network result from the actions of a linear portion of a non - linear current that is activated by neuromodulatory inputs . in other words , it appears that pacemaker activity in this network depends on a linear current , whose essential properties are implemented by the modulator - activated inward current imi . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the international diabetes federation ( idf ) has reported that there were 382 million people living with diabetes worldwide in 2013 , and this number is expected to rise to 592 million by 2035 . most people with diabetes live in low- and middle - income countries , where rapid changes in lifestyle have increased the prevalence of diabetes , cardiovascular diseases , and cancer , and these countries are expected to experience the greatest increase in cases of diabetes in the next 20 years . in 2010 , the chinese center for disease control and prevention ( cdc ) and the endocrinology branch of the chinese medical association conducted a cross - sectional survey in a nationally representative sample of 98,658 chinese adults with diabetes . the prevalence of diabetes in chinese adults was estimated to be 9.7% using the 1999 world health organization criteria . however , when glycosylated hemoglobin ( hba1c ) 6.5% was included in the diagnostic criteria , the prevalence of diabetes in china was estimated to be approximately 11.6% , of which only one - quarter ( 25.8% ) received treatment for diabetes , and only roughly one - third of those receiving treatment ( 39.7% ) achieved adequate glycemic control . xinjiang is located in the middle of the eurasian continent occupying one sixth of the mainland . it is a multi - ethnic region where the uyghur nationality comprises about 46% of the total population . in 2012 , yang et al . screened an uyghur population of 4620 adults aged over 35 years and found that 6.23% had type 2 diabetes ( t2d ) . only 35% of these participants were aware of their diabetes , 7.3% were taking hypoglycemic agents , and 3.13% achieved blood glucose control . these data demonstrate an urgent need to develop effective strategies to improve prevention , detection , and treatment of diabetes in the uyghur population . development of t2d can be attributed to the combined effect of genetic and environmental factors . establishment of single - nucleotide polymorphism ( snp ) databases , development and improvement of cost - effective high - throughput genotyping technology , and multi - center consortium large - scale genome - wide association studies ( gwas ) is an effective method to investigate genetic susceptibility to t2d . by searching susceptibility variants across the entire genome in an unbiased , hypothesis - free manner , gwas have successfully identified genetic loci robustly associated with t2d , the number of which has risen from just three in 2006 to approximately 70 today . the first gwas analysis for t2d the study was initially conducted in 600 t2d patients and 600 control patients , and was followed by a related study of nearly 3,000 t2d patients and 3000 controls from an independent european cohort . this study identified 3 novel susceptibility genes associated with t2d including tcf7l2 , hhex / ide and slc30a8 . subsequently , a large number of t2d associated susceptibility genes have been characterized by gwas , including the cyclin - dependent kinase inhibitor 2a/2b ( cdkn2a/2b ) gene , the cdk5 regulatory subunit associated protein 1-like 1 ( cdkal1 ) gene , and the fat mass and obesity - associated ( fto ) gene . the hapmap project has provided data for genome - wide snps in diverse populations such as han chinese and european residents . nevertheless , the association between cdkn2a/2b , cdkal1 and fto and t2d has not been verified in an uyghur population . this study aims to investigate the genotype distribution of snps and to assess the association between gene polymorphisms and t2d in uyghur adults . furthermore , using multifactor dimensionality reduction ( mdr ) , a nonparametric and genetic model - free method for detecting and characterizing nonlinear interactions between discrete genetic and environmental attributes , the capacity of gene - gene and gene - environment interactions to predict t2d will be investigated . mdr has been successfully applied in studies investigating the etiology of diseases such as multiple sclerosis and t2d . proposed a modified generalized mdr ( gmdr ) method applicable to both dichotomous and continuous variables in population - based study designs , which improves predictive ability . we will apply gmdr to provide information about the molecular pathogenesis of t2d in the uyghur population . a case - controlled study of uyghur patients with t2d and non - diabetic controls was conducted at the first affiliated hospital of xinjiang medical university between march 2012 and september 2013 . written informed consent was obtained from each participant prior to the study , and the protocol was approved by the ethics committee of the first affiliated hospital of xinjiang medical university . patients with t2d were recruited according to the following inclusion criteria : newly diagnosed t2d according to who criteria , fasting plasma glucose ( fpg ) 7.0 mmol / l , and/or oral glucose tolerance test 2 hour sample ( ogtt-2h ) 11.0 mmol / l , or previously diagnosed t2d treated with hypoglycemic drugs where fpg and ogtt-2h levels were below the diagnostic levels . the exclusion criteria for the patient group included : patients with type 1 diabetes , or any wasting diseases such as viral infections , tuberculosis , cancer , mental illness , epilepsy , or acute inflammation . non - diabetic control patients were enrolled during health examination at the hospital during according to the following inclusion criteria : fasting plasma glucose < 7.0 mmol / l , and/or ogtt-2h < 11.1 mmol / l , and without a history of treatment with hypoglycemic agents . the exclusion criteria included : any wasting diseases such as viral infections , tuberculosis , cancer , mental illness , epilepsy , and acute inflammation . any previous diagnoses , current clinical characteristics and family medical history of all diabetic and non - diabetic control patients were recorded . clinical characteristics included height , weight , body mass index ( bmi ) , waist circumference at the midpoint between the lateral iliac crest and the lower edge of the ribs in the midaxillary line , and systolic and diastolic blood pressures . peripheral blood samples were drawn from all subjects after fasting for 8 hours , with 4 ml of blood mixed with or without anticoagulants . serum creatinine ( scr ) , uric acid ( ua ) , fasting plasma glucose ( fpg ) , triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl ) , low density lipoproteins ( ldl ) , aspartate aminotransferase ( ast ) , and alanine aminotransferase ( alt ) were measured with qualified controls using a hitachi 7600 automatic biochemical analyzer in the clinical laboratory of the first affiliated hospital of xinjiang medical university . hypertension was diagnosed according to the 2010 guidelines for prevention and treatment of hypertension in china . coronary heart disease ( chd ) was diagnosed by a cardiology specialist according to the 1979 who diagnostic criteria , or according to a previous medical history of angina or myocardial infarction previously confirmed as chd . non - alcoholic fatty liver disease ( nafld ) was diagnosed according to the nonalcoholic fatty liver disease diagnostic criteria established in 2010 by the chinese society of hepatology . dyslipidemia was diagnosed according to the guidelines on prevention and treatment of blood lipid abnormality in chinese adults established in 2007 , or previous diagnosis of dyslipidemia and treatment with a lipid - lowering drug . stroke was diagnosed according to the criteria established by the fourth national conference on cerebrovascular diseases in 1995 . anticoagulated blood samples were stored at 80c for dna extraction using the iautomag dna extraction system ( bioteke corp . , samples containing greater than 50 ng/l dna , an a260/a280 ratio ( od ) between 1.82.0 , and a clear band in gel electrophoresis were used for further analysis and transferred to 96-well plates and stored at 20c for later use . eight hotspot snps within the cdkal1 , cdkn2a/2b , and fto genes were selected , including loci rs10946398 , rs7754840 ( cdkal1 ) , rs10811661 , rs3088440 , rs11515 ( cdkn2a/2b ) , rs7195539 , rs8050136 , and rs9939609 ( fto ) . snps were detected by capitalbio corporation ( beijing , china ) using the sequenom massarraysnp technology with a genotyping success rate and accuracy > 98% . pcr reactions were carried out in 5 l per well of a 384-well plate using 4 l of pcr master mix , 2050 ng template dna , 0.5 u of hotstar taq , 0.5 pmol of each primer , and 0.1 l of 25 mm dntps incubated at 94c for 4 min ; 94c for 20 sec ; 56c for 30 sec ; 72c for 1 min ; for 45 cycles and 72c for 3 min . pcr products were treated with shrimp alkaline phosphatase ( sap ) to remove free dntps and pcr was performed with 2 l of sap mix ( sap 0.5 u , buffer 0.17 l ) and 5 l of pcr product at 37c for 40 min and 85c for 5 min . after sap treatment , a single - base extension reaction was carried out with 7 l of sap - treated pcr product and 2 l of extend mix ( primer mix 0.94 l , iplex enzyme 0.041 l , and extending mixture 0.2 l ) at 94c for 30 sec ; 94c for 5 sec ; 52c for 5 sec ; 80c for 5 sec ; goto iii , 4 more times ; goto ii , 39 more times ; and 72c for 3 min . purified extension products were loaded onto the 384 spectrochip ( sequenom ) chip using a massarray nanodispenser rs1000 spotter . the spotted spectrochip chips were analyzed using matrix - assisted laser desorption / ionization - time of flight ( maldi - tof ) mass spectrometry . all statistical analyses were performed using ibm spss 21.0 software ( ibm corp . , armonk the hardy - weinberg equilibrium ( hwe ) of the control group was calculated to evaluate whether our samples were representative of a population . all normally distributed qualitative data are expressed as mean standard deviation , and comparisons between two groups were carried out using the t test . abnormally distributed data are presented as median and quartiles , and group - group comparisons were conducted using the wilcoxon rank test . differences in qualitative data , snp genotype or allele frequency were analyzed using the test . logistic regression analysis was applied to evaluate risk factors associated with t2d , using co - dominant , dominant , recessive , additive , and over - dominant models for each snp . for gene - environment interaction analysis , clinical characteristics were categorized accordingly . according to the guidelines for prevention and control of overweight and obesity in chinese adults established in 2004 , patients were categorized as underweight if bmi < 18.5 kg / m ( value=0 ) ; normal weight if 18.5 kg / m bmi < 24 kg / m ( value=1 ) ; overweight if 24 kg / m bmi < 28 kg / m ( value=2 ) ; or obese if bmi 28 kg / m ( value=3 ) . the waistline ( wl ) values were categorized as 0 where male wl < 85 cm or female wl < 80 cm , and 1 where male wl 85 cm or female wl 80 cm . according the guidelines on prevention and treatment of blood lipid abnormality in chinese adults established in 2007 , the scores associated with tg , tc , hdl , and ldl levels were categorized as 0 where tg < 1.7 mmol / l , tc < 5.18 mmol / l , hdl 1.04 mmol / l , and ldl < 3.37 mmol / l , and 1 where tg 1.7 mmol / l , tc 5.18 mmol / l , hdl < 1.04 mmol / l , and ldl 3.37 mmol / l . gene - gene interactions within each snp were analyzed using generalized mdr ( gmdr ) 0.9 software ( http://www.ssg.uab.edu/gmdr/ ) using the best model with a relatively high cross - validation count and balanced testing accuracy . a case - controlled study of uyghur patients with t2d and non - diabetic controls was conducted at the first affiliated hospital of xinjiang medical university between march 2012 and september 2013 . written informed consent was obtained from each participant prior to the study , and the protocol was approved by the ethics committee of the first affiliated hospital of xinjiang medical university . patients with t2d were recruited according to the following inclusion criteria : newly diagnosed t2d according to who criteria , fasting plasma glucose ( fpg ) 7.0 mmol / l , and/or oral glucose tolerance test 2 hour sample ( ogtt-2h ) 11.0 mmol / l , or previously diagnosed t2d treated with hypoglycemic drugs where fpg and ogtt-2h levels were below the diagnostic levels . the exclusion criteria for the patient group included : patients with type 1 diabetes , or any wasting diseases such as viral infections , tuberculosis , cancer , mental illness , epilepsy , or acute inflammation . non - diabetic control patients were enrolled during health examination at the hospital during according to the following inclusion criteria : fasting plasma glucose < 7.0 mmol / l , and/or ogtt-2h < 11.1 mmol / l , and without a history of treatment with hypoglycemic agents . the exclusion criteria included : any wasting diseases such as viral infections , tuberculosis , cancer , mental illness , epilepsy , and acute inflammation . any previous diagnoses , current clinical characteristics and family medical history of all diabetic and non - diabetic control patients were recorded . clinical characteristics included height , weight , body mass index ( bmi ) , waist circumference at the midpoint between the lateral iliac crest and the lower edge of the ribs in the midaxillary line , and systolic and diastolic blood pressures . peripheral blood samples were drawn from all subjects after fasting for 8 hours , with 4 ml of blood mixed with or without anticoagulants . serum creatinine ( scr ) , uric acid ( ua ) , fasting plasma glucose ( fpg ) , triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl ) , low density lipoproteins ( ldl ) , aspartate aminotransferase ( ast ) , and alanine aminotransferase ( alt ) were measured with qualified controls using a hitachi 7600 automatic biochemical analyzer in the clinical laboratory of the first affiliated hospital of xinjiang medical university . hypertension was diagnosed according to the 2010 guidelines for prevention and treatment of hypertension in china . coronary heart disease ( chd ) was diagnosed by a cardiology specialist according to the 1979 who diagnostic criteria , or according to a previous medical history of angina or myocardial infarction previously confirmed as chd . non - alcoholic fatty liver disease ( nafld ) was diagnosed according to the nonalcoholic fatty liver disease diagnostic criteria established in 2010 by the chinese society of hepatology . dyslipidemia was diagnosed according to the guidelines on prevention and treatment of blood lipid abnormality in chinese adults established in 2007 , or previous diagnosis of dyslipidemia and treatment with a lipid - lowering drug . stroke was diagnosed according to the criteria established by the fourth national conference on cerebrovascular diseases in 1995 . anticoagulated blood samples were stored at 80c for dna extraction using the iautomag dna extraction system ( bioteke corp . , samples containing greater than 50 ng/l dna , an a260/a280 ratio ( od ) between 1.82.0 , and a clear band in gel electrophoresis were used for further analysis and transferred to 96-well plates and stored at 20c for later use . eight hotspot snps within the cdkal1 , cdkn2a/2b , and fto genes were selected , including loci rs10946398 , rs7754840 ( cdkal1 ) , rs10811661 , rs3088440 , rs11515 ( cdkn2a/2b ) , rs7195539 , rs8050136 , and rs9939609 ( fto ) . snps were detected by capitalbio corporation ( beijing , china ) using the sequenom massarraysnp technology with a genotyping success rate and accuracy > 98% . pcr reactions were carried out in 5 l per well of a 384-well plate using 4 l of pcr master mix , 2050 ng template dna , 0.5 u of hotstar taq , 0.5 pmol of each primer , and 0.1 l of 25 mm dntps incubated at 94c for 4 min ; 94c for 20 sec ; 56c for 30 sec ; 72c for 1 min ; for 45 cycles and 72c for 3 min . pcr products were treated with shrimp alkaline phosphatase ( sap ) to remove free dntps and pcr was performed with 2 l of sap mix ( sap 0.5 u , buffer 0.17 l ) and 5 l of pcr product at 37c for 40 min and 85c for 5 min . after sap treatment , a single - base extension reaction was carried out with 7 l of sap - treated pcr product and 2 l of extend mix ( primer mix 0.94 l , iplex enzyme 0.041 l , and extending mixture 0.2 l ) at 94c for 30 sec ; 94c for 5 sec ; 52c for 5 sec ; 80c for 5 sec ; goto iii , 4 more times ; goto ii , 39 more times ; and 72c for 3 min . purified extension products were loaded onto the 384 spectrochip ( sequenom ) chip using a massarray nanodispenser rs1000 spotter . the spotted spectrochip chips were analyzed using matrix - assisted laser desorption / ionization - time of flight ( maldi - tof ) mass spectrometry . all statistical analyses were performed using ibm spss 21.0 software ( ibm corp . , armonk the hardy - weinberg equilibrium ( hwe ) of the control group was calculated to evaluate whether our samples were representative of a population . all normally distributed qualitative data are expressed as mean standard deviation , and comparisons between two groups were carried out using the t test . abnormally distributed data are presented as median and quartiles , and group - group comparisons were conducted using the wilcoxon rank test . differences in qualitative data , snp genotype or allele frequency were analyzed using the test . logistic regression analysis was applied to evaluate risk factors associated with t2d , using co - dominant , dominant , recessive , additive , and over - dominant models for each snp . for gene - environment interaction analysis , clinical characteristics were categorized accordingly . according to the guidelines for prevention and control of overweight and obesity in chinese adults established in 2004 , patients were categorized as underweight if bmi < 18.5 kg / m ( value=0 ) ; normal weight if 18.5 kg / m bmi < 24 kg / m ( value=1 ) ; overweight if 24 kg / m bmi < 28 kg / m ( value=2 ) ; or obese if bmi 28 kg / m ( value=3 ) . the waistline ( wl ) values were categorized as 0 where male wl < 85 cm or female wl < 80 cm , and 1 where male wl 85 cm or female wl 80 cm . according the guidelines on prevention and treatment of blood lipid abnormality in chinese adults established in 2007 , the scores associated with tg , tc , hdl , and ldl levels were categorized as 0 where tg < 1.7 mmol / l , tc < 5.18 mmol / l , hdl 1.04 mmol / l , and ldl < 3.37 mmol / l , tc 5.18 mmol / l , hdl < 1.04 mmol / l , and ldl 3.37 mmol / l . gene - gene interactions within each snp were analyzed using generalized mdr ( gmdr ) 0.9 software ( http://www.ssg.uab.edu/gmdr/ ) using the best model with a relatively high cross - validation count and balanced testing accuracy . in total 879 uyghur patients with t2d hospitalized in the first affiliated hospital of xinjiang medical university were enrolled between march 2012 and september 2013 , including 543 males and 336 females aged 2183 years . 895 non - diabetic , unrelated uyghur adults were included as controls , including 571 males and 324 females aged 2179 years . there were no significant differences in age or sex between the diabetes and control groups ( p=0.302 , and 0.378 , respectively ; table 1 ) . the levels of bmi , wl , sbp , fpg , and alt in the diabetes group were higher than those in the control group ( p<0.05 ) , whereas the ua , scr , hdl , and ldl levels were lower in diabetic patients than in controls ( p<0.05 ) . the proportion of patients with a family history of diabetes , or hypertension , chd , nafld , dyslipidemia , stroke , smoking , alcohol abuse or high pressure work environment was higher in the diabetes group than in the control group ( p<0.05 ) . however , the levels of dbp , tg , tc , and ast did not differ significantly between these groups ( p>0.05 ) ( table 1 ) . univariate logistic regression analyses were conducted to analyze the variables associated with t2d , and multivariate logistic regression analyses were conducted to analyze the variables showing statistically significant differences in the univariate logistic regression analyses ( table 2 ) . hypertension , chd , family history of diabetes , fpg , tc , and hdl were independent risk factors for t2d ( p<0.001 ) . the hardy - weinberg equilibrium ( hwe ) test was performed to assess the genotype distributions of snps in the control group . the genotype distributions of 7 of the 8 sites in the control group were within hwe ( p>0.05 ) , indicating that these samples were representative of the uyghur nationality . however , the genotype distribution of rs11515 did not fit the hwe in the control group ( p=0.041 ) , and was thus excluded from further analysis . logistic regression analyses of genetic models of snp genotype and allele frequency revealed statistically significant differences in the genotype distributions and allele frequencies between the diabetes and control groups within loci rs10811661 ( cdkn2a/2b ) , rs7195539 ( fto ) , rs8050136 ( fto ) , and rs9939609 ( fto ) ( p<0.05 ) , but not in loci rs10946398 ( cdkal1 ) , rs7754840 ( cdkal1 ) , or rs3088440 ( cdkn2a/2b ) , ( p>0.05 ; table 3 ) . the allele distributions in loci rs8050136 ( fto ) and rs9939609 ( fto ) exhibited significant divergence between the diabetes and control groups ( p<0.05 ) , and allele a was a common risk allele for both loci . the odds ratio ( or ) values were 1.198 ( 95% confidence interval [ ci ] : 1.0371.384 ) for rs8050136 ( fto ) and 1.238 ( 95% ci : 1.07514.427 ) for rs9939609 ( fto ) . the genotype distribution of rs10811661 ( cdkn2a/2b ) differed between the diabetes and control groups in co - dominant , additive and recessive models , with p values of 0.045 , 0.007 , and 0.011 , respectively , and or values of 1.484 ( 95% ci : 1.0102.183 ) , 1.230 ( 95% ci : 1.0571.431 ) , and 1.276 ( 95% ci : 1.0571.541 ) , respectively . similarly , the rs8050136 ( fto ) genotype distributions differed significantly between the two groups in co - dominant , additive and recessive models with p values of 0.027 , 0.015 , and 0.037 , respectively , and or values of 1.472 ( 95% ci : 1.0452.072 ) , 1.196 ( 95% ci : 1.0351.382 ) , and 1.220 ( 95% ci : 1.0121.470 ) , respectively . in addition , the genotype distributions of rs9939609 ( fto ) in co - dominant , additive , dominant , and recessive models differed significantly between the two groups ( p=0.046 , 0.010 , 0.004 , 0.010 , and 0.036 , respectively ) , with or values of 1.224 ( 95% ci : 1.0041.492 ) , 1.513 ( 95% ci : 1.1062.070 ) , 1.228 ( 95% ci : 1.0691.411 ) , 1.279 ( 95% ci : 1.0611.542 ) , and 1.377 ( 95% ci : 1.0211.858 ) , respectively . however , all the differences discussed above were eliminated after adjusting for sex , age , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl ( p>0.05 ) . nonetheless , the variation in rs7195539 ( fto ) genotype distribution between the diabetes and control groups remained significant in co - dominant and dominant models after adjusting for covariates , with p values of 0.009 , 0.012 , and 0.031 , respectively , and or values of 0.013 ( 95%ci : 0.0010.334 ) , 0.017 ( 95% ci : 0.0010.404 ) , and 0.026 ( 95% ci : 0.0010.713 ) . the snp loci were assigned according to co - dominant markers and gene - gene interactions were evaluated by gmdr software ( table 4 ) . after adjusting for covariates including age , sex , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , the two - locus interaction model rs10811661rs7195539 appeared to be the best model , with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.5483 , and a p value of 0.011 , which increased to only 0.014 after 1000 permutation tests . candidate variables for gene - environment interaction analyses were set as each snp , family history of diabetes , smoking history , alcohol history and work pressure , whereas covariates were set as age , gender , fpg , hypertension , and chd ( table 5 , figure 2 ) . the three - locus interaction model tg - hdl - family history of diabetes appeared to be the best model with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.7072 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . the second - best model was the four - locus interaction model of rs7195539-tg - hdl - family history of diabetes , with a cross - validation consistency of 8/10 , the highest balanced testing accuracy of 0.7002 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . in total 879 uyghur patients with t2d hospitalized in the first affiliated hospital of xinjiang medical university were enrolled between march 2012 and september 2013 , including 543 males and 336 females aged 2183 years . 895 non - diabetic , unrelated uyghur adults were included as controls , including 571 males and 324 females aged 2179 years . there were no significant differences in age or sex between the diabetes and control groups ( p=0.302 , and 0.378 , respectively ; table 1 ) . the levels of bmi , wl , sbp , fpg , and alt in the diabetes group were higher than those in the control group ( p<0.05 ) , whereas the ua , scr , hdl , and ldl levels were lower in diabetic patients than in controls ( p<0.05 ) . the proportion of patients with a family history of diabetes , or hypertension , chd , nafld , dyslipidemia , stroke , smoking , alcohol abuse or high pressure work environment was higher in the diabetes group than in the control group ( p<0.05 ) . however , the levels of dbp , tg , tc , and ast did not differ significantly between these groups ( p>0.05 ) ( table 1 ) . univariate logistic regression analyses were conducted to analyze the variables associated with t2d , and multivariate logistic regression analyses were conducted to analyze the variables showing statistically significant differences in the univariate logistic regression analyses ( table 2 ) . hypertension , chd , family history of diabetes , fpg , tc , and hdl were independent risk factors for t2d ( p<0.001 ) . the hardy - weinberg equilibrium ( hwe ) test was performed to assess the genotype distributions of snps in the control group . the genotype distributions of 7 of the 8 sites in the control group were within hwe ( p>0.05 ) , indicating that these samples were representative of the uyghur nationality . however , the genotype distribution of rs11515 did not fit the hwe in the control group ( p=0.041 ) , and was thus excluded from further analysis . logistic regression analyses of genetic models of snp genotype and allele frequency revealed statistically significant differences in the genotype distributions and allele frequencies between the diabetes and control groups within loci rs10811661 ( cdkn2a/2b ) , rs7195539 ( fto ) , rs8050136 ( fto ) , and rs9939609 ( fto ) ( p<0.05 ) , but not in loci rs10946398 ( cdkal1 ) , rs7754840 ( cdkal1 ) , or rs3088440 ( cdkn2a/2b ) , ( p>0.05 ; table 3 ) . the allele distributions in loci rs8050136 ( fto ) and rs9939609 ( fto ) exhibited significant divergence between the diabetes and control groups ( p<0.05 ) , and allele a was a common risk allele for both loci . the odds ratio ( or ) values were 1.198 ( 95% confidence interval [ ci ] : 1.0371.384 ) for rs8050136 ( fto ) and 1.238 ( 95% ci : 1.07514.427 ) for rs9939609 ( fto ) . the genotype distribution of rs10811661 ( cdkn2a/2b ) differed between the diabetes and control groups in co - dominant , additive and recessive models , with p values of 0.045 , 0.007 , and 0.011 , respectively , and or values of 1.484 ( 95% ci : 1.0102.183 ) , 1.230 ( 95% ci : 1.0571.431 ) , and 1.276 ( 95% ci : 1.0571.541 ) , respectively . similarly , the rs8050136 ( fto ) genotype distributions differed significantly between the two groups in co - dominant , additive and recessive models with p values of 0.027 , 0.015 , and 0.037 , respectively , and or values of 1.472 ( 95% ci : 1.0452.072 ) , 1.196 ( 95% ci : 1.0351.382 ) , and 1.220 ( 95% ci : 1.0121.470 ) , respectively . in addition , the genotype distributions of rs9939609 ( fto ) in co - dominant , additive , dominant , and recessive models differed significantly between the two groups ( p=0.046 , 0.010 , 0.004 , 0.010 , and 0.036 , respectively ) , with or values of 1.224 ( 95% ci : 1.0041.492 ) , 1.513 ( 95% ci : 1.1062.070 ) , 1.228 ( 95% ci : 1.0691.411 ) , 1.279 ( 95% ci : 1.0611.542 ) , and 1.377 ( 95% ci : 1.0211.858 ) , respectively . however , all the differences discussed above were eliminated after adjusting for sex , age , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl ( p>0.05 ) . nonetheless , the variation in rs7195539 ( fto ) genotype distribution between the diabetes and control groups remained significant in co - dominant and dominant models after adjusting for covariates , with p values of 0.009 , 0.012 , and 0.031 , respectively , and or values of 0.013 ( 95%ci : 0.0010.334 ) , 0.017 ( 95% ci : 0.0010.404 ) , and 0.026 ( 95% ci : 0.0010.713 ) . the snp loci were assigned according to co - dominant markers and gene - gene interactions were evaluated by gmdr software ( table 4 ) . after adjusting for covariates including age , sex , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , the two - locus interaction model rs10811661rs7195539 appeared to be the best model , with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.5483 , and a p value of 0.011 , which increased to only 0.014 after 1000 permutation tests . candidate variables for gene - environment interaction analyses were set as each snp , family history of diabetes , smoking history , alcohol history and work pressure , whereas covariates were set as age , gender , fpg , hypertension , and chd ( table 5 , figure 2 ) . the three - locus interaction model tg - hdl - family history of diabetes appeared to be the best model with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.7072 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . the second - best model was the four - locus interaction model of rs7195539-tg - hdl - family history of diabetes , with a cross - validation consistency of 8/10 , the highest balanced testing accuracy of 0.7002 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . identifying individuals at risk for t2d is extremely important , as this allows the implementation of appropriate strategies to potentially prevent the occurrence of t2d and its systemic complications , including atherosclerotic cardiovascular diseases . it is notable that prediabetes and a family history of diabetes are both associated with impairments of the cardiovascular system and increased cardiovascular risk in the absence of overt t2d , highlighting the importance of identifying susceptible individuals before the disease develops . it has been suggested that systemic inflammation and elevated blood glucose levels in prediabetes lead to microvascular and macrovascular disease that manifest as increased intima - media thickness of the common carotid artery , reduced flow - mediated dilatation , increased arterial stiffness and impaired coronary endothelial function . the insulin resistance associated with a family history of diabetes is though to cause endothelial dysfunction through activation of the ras / raf / mapk pathway , up - regulation of endothelin and its receptors , and reduction of no levels , as well as increased intima - media thickness of the common carotid artery . although several risk factors for t2d have already been determined , the discovery of new genetic markers will improve the identification of individuals predisposed to the development of this disease . this might help to reduce the burden of t2d on individuals and society through the use of screening and appropriate interventions in those at risk of developing the t2d . novel susceptibility genes that have been reported to be associated with t2d include tcf7l2 , hhex / ide and slc30a8 , cdkn2a/2b , cdkal1 and fto . this study aimed to analyze the association of cdkn2a/2b , cdkal1 and fto with t2d in a uyghur population . in a case - control study of 879 uyghur patients with t2d and 895 age- and sex - matched healthy controls , we found that the genotype distributions of the snps rs10811661 ( cdkn2a/2b ) and rs7195539 , rs8050136 and rs9939609 ( fto ) differed significantly between the t2d and control groups . the allele frequencies of rs8050136 and rs9939609 also differed significantly between the two groups , and allele a appeared to be a risk allele for both snps . genotype distributions of rs10811661 , rs7195539 , rs8050136 , and rs9939609 differed significantly between genetic models , but rs10811661 , rs8050136 , and rs9939609 were eliminated after adjusting for covariates , and only rs7195539 differed significantly in both co - dominant and dominant models . the amino acid sequence of cdkal1 is similar to that of cdk5-associated protein 1 , which inhibits cdk5 by interacting with its p35 and p39 subunits . due to the regulatory effects of cdk5 on insulin secretion , currently , five non - coding snps within the cdkal1 gene ( rs4712523 , rs10946398 , rs7754840 , rs7756992 , and rs9465871 ) have been found to associate with t2d ; rs7754840 and rs10946398 exhibited the strongest association with t2d , with complete linkage disequilibrium ( ld ) between the two loci ( r=1.0 ) . european carriers of the gc and cc genotypes of rs7754840 had lower first - phase insulin release than carriers of the gg genotype . on the other hand , a study of finnish men demonstrated that rs7754840 was significantly associated with t2d ; hence cdkal1 is thought to increase the risk of t2d by impairing insulin secretion . cdkal1 locus rs10946398 was also found to be associated with t2d in a british population , and rs10946398 was associated with t2d in a han population in china . nevertheless , we did not detect a significant difference in these genotype and allele frequencies between t2d and control groups in a uyghur population ( p>0.05 ) , indicating that the common susceptibility loci rs10946398 and rs7754840 in cdkal1 were not associated with t2d in uyghurs . are highly expressed in islet and fat cells , and cdkn2a is also expressed in brain cells . cdkn2a and b are tumor suppressor genes involved in cell proliferation , apoptosis and tumorigenesis , and encode cdk inhibitors p16 and p15 , respectively , which inhibit cdk4 and cdk6 . in a murine model , cdk4 influenced proliferation and quality of cells , yet its activity was reduced by overexpression of cdkn2a , which in turn caused islet hypoplasia and diabetes . cdkn2a/2b has been reported to be associated with the risk of t2d in a gwas . however , additional studies in europe and asia have both confirmed [ 26,2936 ] and failed to detect [ 32,3739 ] an association between the rs10811661 polymorphism upstream of the cdkn2a/2b gene and t2d . in our study , the genotype distribution of rs10811661 differed significantly between patients in the diabetes group and those in the control group , suggesting that rs1088661 polymorphism may be associated with t2d risk in uyghurs . the distribution of the rs1088661 genotype also differed significantly in co - dominant , additive and recessive models , indicating an increased risk of t2d in tt genotype carriers . nevertheless , after adjusting for covariates , such as age , sex , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , these differences were eliminated suggesting that the locus polymorphism might act via one of the covariates to impact the pathogenesis of t2d in uyghurs . several studies have found a correlation between the rs3088440 polymorphism in the cdkn2a/2b gene and the pathogenesis of malignant tumors , including elevated risk of salivary gland carcinoma and melanoma , but not the onset of cervical cancer or differentiated thyroid cancer . we found no significant difference between the genotype distributions and allele frequencies of locus rs3088440 between diabetic patients and controls in uyghurs , suggesting that in this population rs3088440 is not associated with t2d . the nucleic acid demethylation activity of fto was hypothesized to regulate the expression of genes involved in metabolism , and dysregulation of this process may cause obesity ; however , the precise molecular mechanism responsible for this activity remains unclear . fto mrna was found to be abundantly expressed in the brain tissue of wild - type mice , particularly in hypothalamic nuclei governing energy balance . thus , it is conceivable that the fto gene may regulate the pathogenesis and development of obesity primarily through its impact on the energy center in the hypothalamus . loci rs8050136 and rs9939609 have been reported to associate with t2d in multiple studies , yet there is some inconsistency . this association has been reported to be both mediated by bmi [ 25,44,4749 ] and independent of bmi [ 5053 ] . in this study , the genotype distributions of loci rs7195539 , rs8050136 , and rs9939609 in the fto gene differed significantly between the diabetes and control groups , implying that these polymorphisms are associated with t2d in uyghurs . in addition , the allele frequencies of loci rs8050136 and rs9939609 differed significantly between the two groups with a common allele a. this indicates that uyghurs with loci rs8050136 and rs9939609 containing allele a have an escalated risk of t2d . the rs8050136 genotype distributions in co - dominant , additive and recessive models also differed significantly , and the genotype distributions of rs9939609 in co - dominant , additive , dominant , and recessive models differed significantly between the two groups . these results suggest that the risk of t2d is higher in uyghurs carrying ca or aa genotypes at rs8050136 and/or ta or aa genotypes at rs9939609 than in those carrying the cc or tt genotype , respectively . nevertheless , these variations were eliminated after adjusting for sex , age , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , indicating that these snps are likely to influence the risk of t2d in uyghurs by affecting bmi . to date , few gmdr analyses of gene - gene and gene - environment interactions between the cdkal1 , cdkn2a/2b , and fto genes and t2d have been reported . we found that , after adjusting for demographic and environmental factors , the success of the two - locus model rs10811661rs7195539 indicated a potential association between these two loci and t2d in uyghurs . the three - locus interaction model tg - hdl - family history of diabetes appeared to be the best model for analyzing gene - environment interactions after assigning snps , biochemical characteristics , family history of diabetes , smoking and alcohol history and work pressure as candidate variables , and age , gender , fpg , hypertension , and chd as covariates . the next best model was the four - locus interaction model of rs7195539-tg - hdl - family history of diabetes , suggesting that interactions among these factors may be associated with t2d in uyghurs . our conclusions are somewhat limited by the relatively small sample size included in this study . we observed that rs11515 did not fit the hwe , which may indicate sampling error , and suggests that the control sample was not large enough to adequately represent the genetic diversity of the uyghur population . we also did not employ a 1:1 matching strategy , so the t2d and control group were not precisely matched . nevertheless , we observed that cdkal1 gene polymorphism is unlikely to be associated with a risk of t2d in the uyghur population . in contrast , cdkn2a/2b ( rs10811661 ) and fto ( rs7195539 , rs8050136 , rs9939609 ) polymorphisms may be associated with t2d in this population . rs8050136 allele a and rs9939609 allele a were risk alleles for t2d in the uyghur population . furthermore , our analysis suggests that a cdkn2a/2b ( rs10811661)-fto ( rs7195539 ) interaction , or an fto ( rs7195539)-tg - hdl - family history of diabetes interaction is relevant in determining t2d risk .

backgroundwe investigated the association between 8 single - nucleotide polymorphisms ( snps ) at 3 genetic loci ( cdkal1 , cdkn2a/2b and fto ) with type 2 diabetes ( t2d ) in a uyghur population.material/methodsa case - control study of 879 uyghur patients with t2d and 895 non - diabetic uyghur controls was conducted at the hospital of xinjiang medical university between 2010 and 2013 . eight snps in cdkal1 , cdkn2a/2b and fto were analyzed using sequenom massarraysnp genotyping . factors associated with t2d were assessed by logistic regression analyses . gene - gene and gene - environment interactions were analyzed by generalized multifactor dimensionality reduction.resultsgenotype distributions of rs10811661 ( cdkn2a/2b ) , rs7195539 , rs8050136 , and rs9939609 ( fto ) and allele frequencies of rs8050136 and rs9939609 differed significantly between diabetes and control groups ( all p<0.05 ) . while rs10811661 , rs8050136 , and rs9939609 were eliminated after adjusting for covariates ( p>0.05 ) , rs7195539 distribution differed significantly in co - dominant and dominant models ( p<0.05 ) . in gene - gene interaction analysis , after adjusting for covariates the two - locus rs10811661-rs7195539 interaction model had a cross - validation consistency of 10/10 and the highest balanced accuracy of 0.5483 ( p=0.014 ) . in gene - environment interaction analysis , the 3-locus interaction model tg - hdl - family history of diabetes had a cross - validation consistency of 10/10 and the highest balanced accuracy of 0.7072 ( p<0.001 ) . the 4-locus interaction model , rs7195539-tg - hdl - family history of diabetes had a cross - validation consistency of 8/10 ( p<0.001).conclusionspolymorphisms in cdkn2a/2b and fto , but not cdkal1 , may be associated with t2d , and alleles rs8050136 and rs9939609 are likely risk alleles for t2d in this population . there were potential interactions among cdkn2a/2b ( rs10811661 ) fto ( rs7195539 ) or fto ( rs7195539)-tg - hdl - family history of diabetes in the pathogenesis of t2d in a uyghur population .

Background Material and Methods Study design Subject clinical characteristics Detection of SNPs within CDKAL1, CDKN2A/2B, and FTO Sequenom MassARRAYSNP detection Statistical analysis Results General characteristics of the study population Analysis of risk factors associated with type 2 diabetes in the Uyghur population Distribution of SNP genotypes in the Uyghur population Gene-gene and gene-environment interactions between SNPs and environmental factors Discussion Conclusions

subsequently , a large number of t2d associated susceptibility genes have been characterized by gwas , including the cyclin - dependent kinase inhibitor 2a/2b ( cdkn2a/2b ) gene , the cdk5 regulatory subunit associated protein 1-like 1 ( cdkal1 ) gene , and the fat mass and obesity - associated ( fto ) gene . furthermore , using multifactor dimensionality reduction ( mdr ) , a nonparametric and genetic model - free method for detecting and characterizing nonlinear interactions between discrete genetic and environmental attributes , the capacity of gene - gene and gene - environment interactions to predict t2d will be investigated . a case - controlled study of uyghur patients with t2d and non - diabetic controls was conducted at the first affiliated hospital of xinjiang medical university between march 2012 and september 2013 . eight hotspot snps within the cdkal1 , cdkn2a/2b , and fto genes were selected , including loci rs10946398 , rs7754840 ( cdkal1 ) , rs10811661 , rs3088440 , rs11515 ( cdkn2a/2b ) , rs7195539 , rs8050136 , and rs9939609 ( fto ) . logistic regression analysis was applied to evaluate risk factors associated with t2d , using co - dominant , dominant , recessive , additive , and over - dominant models for each snp . a case - controlled study of uyghur patients with t2d and non - diabetic controls was conducted at the first affiliated hospital of xinjiang medical university between march 2012 and september 2013 . serum creatinine ( scr ) , uric acid ( ua ) , fasting plasma glucose ( fpg ) , triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl ) , low density lipoproteins ( ldl ) , aspartate aminotransferase ( ast ) , and alanine aminotransferase ( alt ) were measured with qualified controls using a hitachi 7600 automatic biochemical analyzer in the clinical laboratory of the first affiliated hospital of xinjiang medical university . eight hotspot snps within the cdkal1 , cdkn2a/2b , and fto genes were selected , including loci rs10946398 , rs7754840 ( cdkal1 ) , rs10811661 , rs3088440 , rs11515 ( cdkn2a/2b ) , rs7195539 , rs8050136 , and rs9939609 ( fto ) . logistic regression analysis was applied to evaluate risk factors associated with t2d , using co - dominant , dominant , recessive , additive , and over - dominant models for each snp . in total 879 uyghur patients with t2d hospitalized in the first affiliated hospital of xinjiang medical university were enrolled between march 2012 and september 2013 , including 543 males and 336 females aged 2183 years . the proportion of patients with a family history of diabetes , or hypertension , chd , nafld , dyslipidemia , stroke , smoking , alcohol abuse or high pressure work environment was higher in the diabetes group than in the control group ( p<0.05 ) . hypertension , chd , family history of diabetes , fpg , tc , and hdl were independent risk factors for t2d ( p<0.001 ) . logistic regression analyses of genetic models of snp genotype and allele frequency revealed statistically significant differences in the genotype distributions and allele frequencies between the diabetes and control groups within loci rs10811661 ( cdkn2a/2b ) , rs7195539 ( fto ) , rs8050136 ( fto ) , and rs9939609 ( fto ) ( p<0.05 ) , but not in loci rs10946398 ( cdkal1 ) , rs7754840 ( cdkal1 ) , or rs3088440 ( cdkn2a/2b ) , ( p>0.05 ; table 3 ) . the allele distributions in loci rs8050136 ( fto ) and rs9939609 ( fto ) exhibited significant divergence between the diabetes and control groups ( p<0.05 ) , and allele a was a common risk allele for both loci . the genotype distribution of rs10811661 ( cdkn2a/2b ) differed between the diabetes and control groups in co - dominant , additive and recessive models , with p values of 0.045 , 0.007 , and 0.011 , respectively , and or values of 1.484 ( 95% ci : 1.0102.183 ) , 1.230 ( 95% ci : 1.0571.431 ) , and 1.276 ( 95% ci : 1.0571.541 ) , respectively . similarly , the rs8050136 ( fto ) genotype distributions differed significantly between the two groups in co - dominant , additive and recessive models with p values of 0.027 , 0.015 , and 0.037 , respectively , and or values of 1.472 ( 95% ci : 1.0452.072 ) , 1.196 ( 95% ci : 1.0351.382 ) , and 1.220 ( 95% ci : 1.0121.470 ) , respectively . in addition , the genotype distributions of rs9939609 ( fto ) in co - dominant , additive , dominant , and recessive models differed significantly between the two groups ( p=0.046 , 0.010 , 0.004 , 0.010 , and 0.036 , respectively ) , with or values of 1.224 ( 95% ci : 1.0041.492 ) , 1.513 ( 95% ci : 1.1062.070 ) , 1.228 ( 95% ci : 1.0691.411 ) , 1.279 ( 95% ci : 1.0611.542 ) , and 1.377 ( 95% ci : 1.0211.858 ) , respectively . however , all the differences discussed above were eliminated after adjusting for sex , age , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl ( p>0.05 ) . nonetheless , the variation in rs7195539 ( fto ) genotype distribution between the diabetes and control groups remained significant in co - dominant and dominant models after adjusting for covariates , with p values of 0.009 , 0.012 , and 0.031 , respectively , and or values of 0.013 ( 95%ci : 0.0010.334 ) , 0.017 ( 95% ci : 0.0010.404 ) , and 0.026 ( 95% ci : 0.0010.713 ) . after adjusting for covariates including age , sex , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , the two - locus interaction model rs10811661rs7195539 appeared to be the best model , with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.5483 , and a p value of 0.011 , which increased to only 0.014 after 1000 permutation tests . the three - locus interaction model tg - hdl - family history of diabetes appeared to be the best model with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.7072 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . the second - best model was the four - locus interaction model of rs7195539-tg - hdl - family history of diabetes , with a cross - validation consistency of 8/10 , the highest balanced testing accuracy of 0.7002 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . in total 879 uyghur patients with t2d hospitalized in the first affiliated hospital of xinjiang medical university were enrolled between march 2012 and september 2013 , including 543 males and 336 females aged 2183 years . there were no significant differences in age or sex between the diabetes and control groups ( p=0.302 , and 0.378 , respectively ; table 1 ) . the proportion of patients with a family history of diabetes , or hypertension , chd , nafld , dyslipidemia , stroke , smoking , alcohol abuse or high pressure work environment was higher in the diabetes group than in the control group ( p<0.05 ) . however , the levels of dbp , tg , tc , and ast did not differ significantly between these groups ( p>0.05 ) ( table 1 ) . univariate logistic regression analyses were conducted to analyze the variables associated with t2d , and multivariate logistic regression analyses were conducted to analyze the variables showing statistically significant differences in the univariate logistic regression analyses ( table 2 ) . hypertension , chd , family history of diabetes , fpg , tc , and hdl were independent risk factors for t2d ( p<0.001 ) . logistic regression analyses of genetic models of snp genotype and allele frequency revealed statistically significant differences in the genotype distributions and allele frequencies between the diabetes and control groups within loci rs10811661 ( cdkn2a/2b ) , rs7195539 ( fto ) , rs8050136 ( fto ) , and rs9939609 ( fto ) ( p<0.05 ) , but not in loci rs10946398 ( cdkal1 ) , rs7754840 ( cdkal1 ) , or rs3088440 ( cdkn2a/2b ) , ( p>0.05 ; table 3 ) . the allele distributions in loci rs8050136 ( fto ) and rs9939609 ( fto ) exhibited significant divergence between the diabetes and control groups ( p<0.05 ) , and allele a was a common risk allele for both loci . the genotype distribution of rs10811661 ( cdkn2a/2b ) differed between the diabetes and control groups in co - dominant , additive and recessive models , with p values of 0.045 , 0.007 , and 0.011 , respectively , and or values of 1.484 ( 95% ci : 1.0102.183 ) , 1.230 ( 95% ci : 1.0571.431 ) , and 1.276 ( 95% ci : 1.0571.541 ) , respectively . similarly , the rs8050136 ( fto ) genotype distributions differed significantly between the two groups in co - dominant , additive and recessive models with p values of 0.027 , 0.015 , and 0.037 , respectively , and or values of 1.472 ( 95% ci : 1.0452.072 ) , 1.196 ( 95% ci : 1.0351.382 ) , and 1.220 ( 95% ci : 1.0121.470 ) , respectively . in addition , the genotype distributions of rs9939609 ( fto ) in co - dominant , additive , dominant , and recessive models differed significantly between the two groups ( p=0.046 , 0.010 , 0.004 , 0.010 , and 0.036 , respectively ) , with or values of 1.224 ( 95% ci : 1.0041.492 ) , 1.513 ( 95% ci : 1.1062.070 ) , 1.228 ( 95% ci : 1.0691.411 ) , 1.279 ( 95% ci : 1.0611.542 ) , and 1.377 ( 95% ci : 1.0211.858 ) , respectively . however , all the differences discussed above were eliminated after adjusting for sex , age , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl ( p>0.05 ) . nonetheless , the variation in rs7195539 ( fto ) genotype distribution between the diabetes and control groups remained significant in co - dominant and dominant models after adjusting for covariates , with p values of 0.009 , 0.012 , and 0.031 , respectively , and or values of 0.013 ( 95%ci : 0.0010.334 ) , 0.017 ( 95% ci : 0.0010.404 ) , and 0.026 ( 95% ci : 0.0010.713 ) . after adjusting for covariates including age , sex , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , the two - locus interaction model rs10811661rs7195539 appeared to be the best model , with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.5483 , and a p value of 0.011 , which increased to only 0.014 after 1000 permutation tests . candidate variables for gene - environment interaction analyses were set as each snp , family history of diabetes , smoking history , alcohol history and work pressure , whereas covariates were set as age , gender , fpg , hypertension , and chd ( table 5 , figure 2 ) . the three - locus interaction model tg - hdl - family history of diabetes appeared to be the best model with a cross - validation consistency of 10/10 , the highest balanced testing accuracy of 0.7072 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . the second - best model was the four - locus interaction model of rs7195539-tg - hdl - family history of diabetes , with a cross - validation consistency of 8/10 , the highest balanced testing accuracy of 0.7002 , and a p value of 0.001 , which was reduced to < 0.001 after 1000 permutation tests . this study aimed to analyze the association of cdkn2a/2b , cdkal1 and fto with t2d in a uyghur population . in a case - control study of 879 uyghur patients with t2d and 895 age- and sex - matched healthy controls , we found that the genotype distributions of the snps rs10811661 ( cdkn2a/2b ) and rs7195539 , rs8050136 and rs9939609 ( fto ) differed significantly between the t2d and control groups . the allele frequencies of rs8050136 and rs9939609 also differed significantly between the two groups , and allele a appeared to be a risk allele for both snps . genotype distributions of rs10811661 , rs7195539 , rs8050136 , and rs9939609 differed significantly between genetic models , but rs10811661 , rs8050136 , and rs9939609 were eliminated after adjusting for covariates , and only rs7195539 differed significantly in both co - dominant and dominant models . nevertheless , we did not detect a significant difference in these genotype and allele frequencies between t2d and control groups in a uyghur population ( p>0.05 ) , indicating that the common susceptibility loci rs10946398 and rs7754840 in cdkal1 were not associated with t2d in uyghurs . in our study , the genotype distribution of rs10811661 differed significantly between patients in the diabetes group and those in the control group , suggesting that rs1088661 polymorphism may be associated with t2d risk in uyghurs . the distribution of the rs1088661 genotype also differed significantly in co - dominant , additive and recessive models , indicating an increased risk of t2d in tt genotype carriers . nevertheless , after adjusting for covariates , such as age , sex , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , these differences were eliminated suggesting that the locus polymorphism might act via one of the covariates to impact the pathogenesis of t2d in uyghurs . several studies have found a correlation between the rs3088440 polymorphism in the cdkn2a/2b gene and the pathogenesis of malignant tumors , including elevated risk of salivary gland carcinoma and melanoma , but not the onset of cervical cancer or differentiated thyroid cancer . in this study , the genotype distributions of loci rs7195539 , rs8050136 , and rs9939609 in the fto gene differed significantly between the diabetes and control groups , implying that these polymorphisms are associated with t2d in uyghurs . in addition , the allele frequencies of loci rs8050136 and rs9939609 differed significantly between the two groups with a common allele a. this indicates that uyghurs with loci rs8050136 and rs9939609 containing allele a have an escalated risk of t2d . the rs8050136 genotype distributions in co - dominant , additive and recessive models also differed significantly , and the genotype distributions of rs9939609 in co - dominant , additive , dominant , and recessive models differed significantly between the two groups . nevertheless , these variations were eliminated after adjusting for sex , age , bmi , hypertension , chd , family history of diabetes , fpg , tc , and hdl , indicating that these snps are likely to influence the risk of t2d in uyghurs by affecting bmi . to date , few gmdr analyses of gene - gene and gene - environment interactions between the cdkal1 , cdkn2a/2b , and fto genes and t2d have been reported . we found that , after adjusting for demographic and environmental factors , the success of the two - locus model rs10811661rs7195539 indicated a potential association between these two loci and t2d in uyghurs . the three - locus interaction model tg - hdl - family history of diabetes appeared to be the best model for analyzing gene - environment interactions after assigning snps , biochemical characteristics , family history of diabetes , smoking and alcohol history and work pressure as candidate variables , and age , gender , fpg , hypertension , and chd as covariates . the next best model was the four - locus interaction model of rs7195539-tg - hdl - family history of diabetes , suggesting that interactions among these factors may be associated with t2d in uyghurs . in contrast , cdkn2a/2b ( rs10811661 ) and fto ( rs7195539 , rs8050136 , rs9939609 ) polymorphisms may be associated with t2d in this population . rs8050136 allele a and rs9939609 allele a were risk alleles for t2d in the uyghur population . furthermore , our analysis suggests that a cdkn2a/2b ( rs10811661)-fto ( rs7195539 ) interaction , or an fto ( rs7195539)-tg - hdl - family history of diabetes interaction is relevant in determining t2d risk .

in malawi , a country of 15.9 million people in southern africa , an estimated 10.3% ( 9.6%10.8% ) of adults are hiv positive , and an estimated 170,000 children younger than 15 years are living with hiv , most of whom were infected through vertical transmission . since july 2011 , the malawi ministry of health has embarked on a program to prevent mother - to - child transmission ( pmtct ) of hiv known as option b plus malawi became the first country to implement a test - and - treat approach in a national pmtct program : option b+ offers all hiv - infected pregnant and breastfeeding women lifelong antiretroviral therapy ( art ) , regardless of cd4 count or clinical stage . the overall goal of the program was to improve art uptake and retention , and thus the outcomes of hiv - infected pregnant women , their uninfected partners , and their infants within the continuum of hiv testing and treatment services . the option b+ strategy offers an attractive rapid art scale - up and was adopted in malawi ahead of the recommendations from the world health organization . although it has the potential to profoundly impact maternal and infant outcomes , operational challenges throughout the cascade of pmtct services may affect uptake and adherence to art by pregnant women , the follow - up of hiv - exposed infants , and the long - term retention of this population . from the onset , potential threats to the implementation and scale - up of option b+ were identified and concerns were raised in relation to initiation into care , retention in care , adherence to art , and the lack of psycho - social support in the context of lifelong retention of a relatively asymptomatic population group . during its first year of implementation in malawi , option b+ rapidly expanded uptake and access to pmtct services , with a 7-fold increase in the number of women starting art for pmtct . one of the main challenges encountered to date is the retention of women once initiated on art . in the first year of implementation , a large proportion of women ( 17% ) seemed to be lost to follow - up ( ltfu ) 6 months after art initiation , with most losses occurring in the first 3 months of therapy . many women recorded as initiated on treatment and who accepted their first month of art never come back , raising additional questions on whether they initiated treatment . early assessments into the reasons associated with stopping art among women who initiated treatment during option b+ pointed at difficulties linked to traveling to health facilities and lack of transport money , not understanding initial treatment guidance , and being too weak , sick , or experiencing treatment side effects . in light of these potential challenges to the implementation of option b+ in malawi , a cluster randomized control trial , pure ( pmtct uptake and retention in malawi ) , was designed to evaluate and compare strategies to strengthen uptake and retention of mothers and families in pmtct care . the trial compares facility - based peer support and community - based peer support to standard of care across the south east , south west , and central west regions . interventions include the introduction of trained expert patients ( mentor mothers and expert mothers ) , who are working within and outside health facilities to support women involved in option b+ and their families . in this article , we describe the results from a qualitative formative study designed to refine the pure trial intervention arms . the aim of this study was to explore the experience of patients and health care workers ( hcws ) in relation to the implementation of option b+ . the specific objectives were to elicit local perceptions of option b+ among hcws and women who have recently been enrolled in this program and to document the nature of the support provided to these women . this was descriptive , cross - sectional formative research , drawing on a qualitative approach to collect data through semi - structured interviews and focus group discussions with both option b+ patients and hcws . the study was conducted in 6 of 21 facilities within the pure trial sites across 3 health zones ( central west , south east , and south west ) . among the 21 study sites included in the pure trial , in addition , in each health zone , we selected 2 sites that were different according to their location ( rural vs urban settings ) , number of pregnant and/or breastfeeding women attending services in the past quarter ( large vs smaller facilities ) , and type of ownership of the health facility ( ministry of health vs christian health association malawi ) . the health facilities identified as study sites for the study are shown in figure 1 . geographic distribution of the 6 pure formative sites in the south east , south west , and central west zones of the country ( mulanje district hospital and muloza health centre in the south east zone ; trinity mission hospital and makhetha health centre in the south west zone ; and nsipe health centre and lobi rural hospital in the central east / central west zone ) . study participants were recruited through a purposive sampling approach at each study site until the sample of respondents was achieved . in each site , the research team identified 4 women newly initiating art services and enrolled in option b+ , and all available hcws involved in providing services as part of option b+ . our intention was to interview a small sample of individuals until thematic saturation is achieved . based on previous studies conducted in the same setting in relation to pmtct services , we expected that saturation would be achieved at 2030 interviews ; we therefore selected 24 interviews ( 4 per site ) as a suitable number of women to be interviewed . hcws recruited for the study included nurses , medical assistants , and community health workers ( health surveillance assistants ) who routinely provided pmtct care in the selected health facilities . patients involved in the study included hiv - infected pregnant or breastfeeding women seeking antenatal care ( anc ) or initiating art services at 1 of the 6 study sites . in total , 48 hcws and 24 women receiving care as part of option b+ all data were collected at the health facilities at a time identified as convenient by research participants themselves . semi - structured interviews were conducted with 4 patients in each study site ( n = 24 ) . one focus group discussion was conducted with 8 hcws per facility ( n = 6 groups of 8 individuals ) . ( a follow - up longitudinal study is collecting additional data alongside the pure trial implementation from 2014 to 2016 ) . a team of 4 data collectors was recruited and trained in recruitment of study participants , data collection , and appropriate conduct during fieldwork , including ethical considerations . all data collectors had previous experience in collecting qualitative data and conducting interviews and received additional training specific to this study . recordings of interviews and focus group discussions were transcribed in chichewa and later translated verbatim to english . the quality of the transcription was checked by a bilingual researcher who used the original transcripts and extracts from the audio files to check the final translation . data were anonymized and pseudonyms were used to protect the identity of research participants . based on a sample of 10 transcripts from interviews and focus groups , the research team identified themes primarily through a deductive approach . using a framework analysis approach , themes were developed to capture common issues , such as perception and acceptability of current care , adherence and loss to follow - up , care relationships , disclosure , and task - shifting involving community hcws . a coding structure reflecting each of the themes and subthemes was applied to the data using nvivo 7 . framework and thematic analysis of the retrieved data were followed by a narrative account of the findings . data were collected with respect for international ethical guidelines , aiming at protecting confidentiality and anonymity , ensuring informed consent , giving ample time and information about the study to respondents , and interviewing study participants in location allowing for audio privacy . study participants did not receive an incentive to be part of the study but they received a transport reimbursement ( equivalent usd 1.40 ) for taking part in the interviews and focus group discussions . the study protocol was approved by the national health sciences research committee in malawi , the university of north carolina institutional review board , the university of toronto research ethics board , and the world health organization ethics review committee . this was descriptive , cross - sectional formative research , drawing on a qualitative approach to collect data through semi - structured interviews and focus group discussions with both option b+ patients and hcws . the study was conducted in 6 of 21 facilities within the pure trial sites across 3 health zones ( central west , south east , and south west ) . among the 21 study sites included in the pure trial , in addition , in each health zone , we selected 2 sites that were different according to their location ( rural vs urban settings ) , number of pregnant and/or breastfeeding women attending services in the past quarter ( large vs smaller facilities ) , and type of ownership of the health facility ( ministry of health vs christian health association malawi ) . the health facilities identified as study sites for the study are shown in figure 1 . geographic distribution of the 6 pure formative sites in the south east , south west , and central west zones of the country ( mulanje district hospital and muloza health centre in the south east zone ; trinity mission hospital and makhetha health centre in the south west zone ; and nsipe health centre and lobi rural hospital in the central east / central west zone ) . study participants were recruited through a purposive sampling approach at each study site until the sample of respondents was achieved . in each site , the research team identified 4 women newly initiating art services and enrolled in option b+ , and all available hcws involved in providing services as part of option b+ . our intention was to interview a small sample of individuals until thematic saturation is achieved . based on previous studies conducted in the same setting in relation to pmtct services , we expected that saturation would be achieved at 2030 interviews ; we therefore selected 24 interviews ( 4 per site ) as a suitable number of women to be interviewed . hcws recruited for the study included nurses , medical assistants , and community health workers ( health surveillance assistants ) who routinely provided pmtct care in the selected health facilities . patients involved in the study included hiv - infected pregnant or breastfeeding women seeking antenatal care ( anc ) or initiating art services at 1 of the 6 study sites . in total , 48 hcws and 24 women receiving care as part of option b+ all data were collected at the health facilities at a time identified as convenient by research participants themselves . semi - structured interviews were conducted with 4 patients in each study site ( n = 24 ) . one focus group discussion was conducted with 8 hcws per facility ( n = 6 groups of 8 individuals ) . ( a follow - up longitudinal study is collecting additional data alongside the pure trial implementation from 2014 to 2016 ) . a team of 4 data collectors was recruited and trained in recruitment of study participants , data collection , and appropriate conduct during fieldwork , including ethical considerations . all data collectors had previous experience in collecting qualitative data and conducting interviews and received additional training specific to this study . recordings of interviews and focus group discussions were transcribed in chichewa and later translated verbatim to english . the quality of the transcription was checked by a bilingual researcher who used the original transcripts and extracts from the audio files to check the final translation . data were anonymized and pseudonyms were used to protect the identity of research participants . based on a sample of 10 transcripts from interviews and focus groups , the research team identified themes primarily through a deductive approach . using a framework analysis approach , themes were developed to capture common issues , such as perception and acceptability of current care , adherence and loss to follow - up , care relationships , disclosure , and task - shifting involving community hcws . a coding structure reflecting each of the themes and subthemes was applied to the data using nvivo 7 . framework and thematic analysis of the retrieved data were followed by a narrative account of the findings . data were collected with respect for international ethical guidelines , aiming at protecting confidentiality and anonymity , ensuring informed consent , giving ample time and information about the study to respondents , and interviewing study participants in location allowing for audio privacy . study participants did not receive an incentive to be part of the study but they received a transport reimbursement ( equivalent usd 1.40 ) for taking part in the interviews and focus group discussions . the study protocol was approved by the national health sciences research committee in malawi , the university of north carolina institutional review board , the university of toronto research ethics board , and the world health organization ethics review committee . women interviewed were between 19 and 40 years old ( mean age : 28 years ) . all but 2 were married ( one was not married and the other was divorced ) , and all but 3 had 1 child ( 3 were pregnant ) . most had reached junior primary school , and 8 of 24 had completed the first or second years of senior primary school . for those who had an economic activity , this ranged from farming ( 6 of 24 ) , small business ( 7 of 24 ) , and small labor or seasonal work in tea plantations ( 4 of 24 ) . results are presented across 3 main themes : the perception of option b+ by patients and hcws ; experiences of adherence and loss to follow - up in the context of option b+ ; and patients ' and hcws ' perspectives on improving option b+ . several of the women interviewed were tested for hiv during pregnancy and talked about how they accepted their diagnoses and sought treatment . some respondents also narrated their desire to have more children in the future , which was sometimes linked to the perspective of getting better on art . distance , privacy , and confidentiality remain problematic issues for women , especially for those living in rural areas ; they expressed feeling stigmatized in relation to living with hiv , within their social network and during their visits to the health centers . in most cases , women were reluctant to share their serostatus beyond a small circle of people because they feared discrimination . several of the women interviewed had not disclosed their serostatus to more than one person : no one knows because i have never explained this to anyone as it is only me and my husband that know about this . [ nsipe ] i have just told my husband , but i have not told my children since they are with my mother . [ makhetha ] no one knows because i have never explained this to anyone as it is only me and my husband that know about this . [ nsipe ] i have just told my husband , but i have not told my children since they are with my mother . [ makhetha ] across several health centers , women criticized the lack of privacy in hiv and art services , with confidentiality around their serostatus compromised each time they go to the clinic : if you meet a person who knows you and is seeing you enter the room where we get arvs [ antiretrovirals ] , they tend to tell people that so and so are on arvs . [ lobi ] if you meet a person who knows you and is seeing you enter the room where we get arvs [ antiretrovirals ] , they tend to tell people that so and so are on arvs . [ lobi ] in each health facility , the women interviewed expressed how much stigma is still part of their lives and that they still face discrimination when they choose to disclose their serostatus to others : disclosure of hiv in my community is difficult because when people know that you are hiv positive , they start telling each other about your status . [ trinity ] disclosure of hiv in my community is difficult because when people know that you are hiv positive , they start telling each other about your status . , i am open only to my family and close friends . [ trinity ] some of their close family members avoided coming to their houses and , in 2 cases , the husbands decided to leave after their partner tested positive : since i was found hiv positive [ ] my husband ran away from me . [ nsipe ] since i was found hiv positive [ ] my husband ran away from me . [ nsipe ] the lack of inclusion of male partners , and their reluctance to be tested for hiv , was cited as an ongoing problem by women ; some feared for the stability of their relationship if their partner did not test . many of the mothers who expressed being supported by their partner to get an hiv test and start treatment also reported that male partners refused to get tested or to start art themselves : my husband is refusing to go for testing , therefore i do n't know if my family will continue to live in this situation . [ nsipe ] my husband is refusing to go for testing , therefore i do n't know if my family will continue to live in this situation . [ nsipe ] from the hcws ' perspective , option b+ was positively received as it encouraged deliveries at health facilities to get babies on nevirapine within 72 hours . they also expressed positive views about the intensified testing of newborns and that option b+ would encourage continuous breastfeeding until 24 months . across all sites , hcws complained about the lack of infrastructure and the recurrent shortage of hiv test kits . some hcws expressed challenges to identify the address and exact name of patients for follow - up . importantly , hcws reported that some mothers experience difficulties in understanding why they are initiated on art immediately after testing . hcws were concerned that the test - and - treat approach is too fast and too much information is given to women at once , with no time to digest and communicate a new positive hiv diagnosis : we test her [ a patient ] today and she is found hiv positive , we immediately initiate her onto art whilst she is still thinking about how she contracted the virus and she hasn't yet accepted the reality about her serostatus , and we give her the drugs for her unborn child at the same time . so it 's like she is getting too much information at once before she makes the rightful decision [ mulanje ] . we test her [ a patient ] today and she is found hiv positive , we immediately initiate her onto art whilst she is still thinking about how she contracted the virus and she hasn't yet accepted the reality about her serostatus , and we give her the drugs for her unborn child at the same time . so it 's like she is getting too much information at once before she makes the rightful decision [ mulanje ] . another challenge experienced by hcws is that , because of staff shortages , some mothers are asked to come back another day to be tested . similarly to patients , hcws highlighted the lack of privacy and confidentiality when patients came for testing or to pick up art : the space where vct [ voluntary counseling and testing ] and art clinics are conducted is small , so on art day the space is overcrowded . [ nsipe ] the space where vct [ voluntary counseling and testing ] and art clinics are conducted is small , [ nsipe ] most women interviewed self - reported that they were adhering to treatment by taking art as prescribed and understood that they should take treatment for life : they explained to me that i would be taking these drugs daily for the rest of my life . [ nsipe ] they explained to me that i would be taking these drugs daily for the rest of my life . [ nsipe ] despite declaring that they adhered to treatment , and generally understanding well how to take art , nearly all mothers and hcws interviewed reported problems linked to retention in care and adherence to art ; some patients reported missing doses of art while they continued to collect treatment regularly from the clinic . hcws reported that several mothers did not come back after delivery or after their first visit to the health facility . hcws expressed that patients feel discouraged when they see people they know queuing for art . hcws reported that the ltfu may be explained by the fact that women are expected to start treatment immediately , sometimes while they are still in the labor ward : once we diagnose them [ patients ] positive , we immediately initiate them on art . so you find that most of them do n't come for subsequent visits because they were introduced to arvs while in pain . this is a common problem and most of the defaulters belong to this category of ladies initiated at labour ward . [ trinity ] once we diagnose them [ patients ] positive , we immediately initiate them on art . so you find that most of them do n't come for subsequent visits because they were introduced to arvs while in pain . this is a common problem and most of the defaulters belong to this category of ladies initiated at labour ward . [ trinity ] a key concern expressed by hcws was that they do not always know how to follow - up with the mothers who may be defaulting . some hcws noted that the loss to follow - up was also because of the lack of involvement of male partners in pmtct care , for instance , because men did not accompany women to anc or because they were not routinely tested for hiv and followed - up within pmtct services : because men do n't accompany their spouses to antenatal clinic , it happens that once the lady delivers and she is not suffering from any disease , she does not see the reasons for coming to the clinic to continue art treatment . [ trinity ] because men do n't accompany their spouses to antenatal clinic , it happens that once the lady delivers and she is not suffering from any disease , she does not see the reasons for coming to the clinic to continue art treatment . [ trinity ] when asked how one can improve the current pmtct care in malawi , the main suggestion from women and hcws was to encourage more men to come forward for hiv testing and to start treatment when required , for example , by improving couple counseling and testing initiatives . life skills advice ( for example , skills to be empowered to improve their day - to - day situation ) to be included in the care package that they received at health facilities . family planning and contraception as well as nutrition and income - generating activities were cited as important aspects of pmtct services to be considered . another suggestion from patients to improve pmtct care was to address the issues around privacy and confidentiality . some suggested having a specific day for art visits to get drugs as a group:[in order to improve confidentiality ] we should have a special day for art visits to get drugs as a group . [ nsipe ] [ in order to improve confidentiality ] we should have a special day for art visits to get drugs as a group . [ nsipe ] from the hcws ' perspective , more training was necessary to help patients enrolled in option b+ . distance , especially among rural health facilities , was an obstacle to trace patients ltfu . hcws suggested that task sharing with health surveillance assistants ( hsas ) was important to link patients back to care and that bikes and motorbikes could help them to address this issue : we have many clients who come from far and we do not know if their reason for defaulting might be that they are dead or not , so if we can have another motorbike and make the effective follow - ups we can have a successful art . [ lobi ] we have many clients who come from far and we do not know if their reason for defaulting might be that they are dead or not , so if we can have another motorbike and make the effective follow - ups we can have a successful art . [ lobi ] several of the women interviewed were tested for hiv during pregnancy and talked about how they accepted their diagnoses and sought treatment . some respondents also narrated their desire to have more children in the future , which was sometimes linked to the perspective of getting better on art . distance , privacy , and confidentiality remain problematic issues for women , especially for those living in rural areas ; they expressed feeling stigmatized in relation to living with hiv , within their social network and during their visits to the health centers . in most cases , women were reluctant to share their serostatus beyond a small circle of people because they feared discrimination . several of the women interviewed had not disclosed their serostatus to more than one person : no one knows because i have never explained this to anyone as it is only me and my husband that know about this . [ nsipe ] i have just told my husband , but i have not told my children since they are with my mother . [ makhetha ] no one knows because i have never explained this to anyone as it is only me and my husband that know about this . [ nsipe ] i have just told my husband , but i have not told my children since they are with my mother . [ makhetha ] across several health centers , women criticized the lack of privacy in hiv and art services , with confidentiality around their serostatus compromised each time they go to the clinic : if you meet a person who knows you and is seeing you enter the room where we get arvs [ antiretrovirals ] , they tend to tell people that so and so are on arvs . [ lobi ] if you meet a person who knows you and is seeing you enter the room where we get arvs [ antiretrovirals ] , they tend to tell people that so and so are on arvs . [ lobi ] in each health facility , the women interviewed expressed how much stigma is still part of their lives and that they still face discrimination when they choose to disclose their serostatus to others : disclosure of hiv in my community is difficult because when people know that you are hiv positive , they start telling each other about your status . [ trinity ] disclosure of hiv in my community is difficult because when people know that you are hiv positive , they start telling each other about your status . [ trinity ] some of their close family members avoided coming to their houses and , in 2 cases , the husbands decided to leave after their partner tested positive : since i was found hiv positive [ ] my husband ran away from me . [ nsipe ] since i was found hiv positive [ ] my husband ran away from me . [ nsipe ] the lack of inclusion of male partners , and their reluctance to be tested for hiv , was cited as an ongoing problem by women ; some feared for the stability of their relationship if their partner did not test . many of the mothers who expressed being supported by their partner to get an hiv test and start treatment also reported that male partners refused to get tested or to start art themselves : my husband is refusing to go for testing , therefore i do n't know if my family will continue to live in this situation . [ nsipe ] my husband is refusing to go for testing , therefore i do n't know if my family will continue to live in this situation . [ nsipe ] from the hcws ' perspective , option b+ was positively received as it encouraged deliveries at health facilities to get babies on nevirapine within 72 hours . they also expressed positive views about the intensified testing of newborns and that option b+ would encourage continuous breastfeeding until 24 months . across all sites , hcws complained about the lack of infrastructure and the recurrent shortage of hiv test kits . some hcws expressed challenges to identify the address and exact name of patients for follow - up . importantly , hcws reported that some mothers experience difficulties in understanding why they are initiated on art immediately after testing . hcws were concerned that the test - and - treat approach is too fast and too much information is given to women at once , with no time to digest and communicate a new positive hiv diagnosis : we test her [ a patient ] today and she is found hiv positive , we immediately initiate her onto art whilst she is still thinking about how she contracted the virus and she hasn't yet accepted the reality about her serostatus , and we give her the drugs for her unborn child at the same time . so it 's like she is getting too much information at once before she makes the rightful decision [ mulanje ] . we test her [ a patient ] today and she is found hiv positive , we immediately initiate her onto art whilst she is still thinking about how she contracted the virus and she hasn't yet accepted the reality about her serostatus , and we give her the drugs for her unborn child at the same time . so it 's like she is getting too much information at once before she makes the rightful decision [ mulanje ] . another challenge experienced by hcws is that , because of staff shortages , some mothers are asked to come back another day to be tested . similarly to patients , hcws highlighted the lack of privacy and confidentiality when patients came for testing or to pick up art : the space where vct [ voluntary counseling and testing ] and art clinics are conducted is small , so on art day the space is overcrowded . [ nsipe ] the space where vct [ voluntary counseling and testing ] and art clinics are conducted is small , most women interviewed self - reported that they were adhering to treatment by taking art as prescribed and understood that they should take treatment for life : they explained to me that i would be taking these drugs daily for the rest of my life . [ nsipe ] they explained to me that i would be taking these drugs daily for the rest of my life . [ nsipe ] despite declaring that they adhered to treatment , and generally understanding well how to take art , nearly all mothers and hcws interviewed reported problems linked to retention in care and adherence to art ; some patients reported missing doses of art while they continued to collect treatment regularly from the clinic . hcws reported that several mothers did not come back after delivery or after their first visit to the health facility . hcws expressed that patients feel discouraged when they see people they know queuing for art . hcws reported that the ltfu may be explained by the fact that women are expected to start treatment immediately , sometimes while they are still in the labor ward : once we diagnose them [ patients ] positive , we immediately initiate them on art . so you find that most of them do n't come for subsequent visits because they were introduced to arvs while in pain . this is a common problem and most of the defaulters belong to this category of ladies initiated at labour ward . [ trinity ] once we diagnose them [ patients ] positive , we immediately initiate them on art . so you find that most of them do n't come for subsequent visits because they were introduced to arvs while in pain . this is a common problem and most of the defaulters belong to this category of ladies initiated at labour ward . [ trinity ] a key concern expressed by hcws was that they do not always know how to follow - up with the mothers who may be defaulting . some hcws noted that the loss to follow - up was also because of the lack of involvement of male partners in pmtct care , for instance , because men did not accompany women to anc or because they were not routinely tested for hiv and followed - up within pmtct services : because men do n't accompany their spouses to antenatal clinic , it happens that once the lady delivers and she is not suffering from any disease , she does not see the reasons for coming to the clinic to continue art treatment . [ trinity ] because men do n't accompany their spouses to antenatal clinic , it happens that once the lady delivers and she is not suffering from any disease , she does not see the reasons for coming to the clinic to continue art treatment . when asked how one can improve the current pmtct care in malawi , the main suggestion from women and hcws was to encourage more men to come forward for hiv testing and to start treatment when required , for example , by improving couple counseling and testing initiatives . many patients would have liked additional life skills advice ( for example , skills to be empowered to improve their day - to - day situation ) to be included in the care package that they received at health facilities . family planning and contraception as well as nutrition and income - generating activities were cited as important aspects of pmtct services to be considered . another suggestion from patients to improve pmtct care was to address the issues around privacy and confidentiality . some suggested having a specific day for art visits to get drugs as a group:[in order to improve confidentiality ] we should have a special day for art visits to get drugs as a group . [ nsipe ] [ in order to improve confidentiality ] we should have a special day for art visits to get drugs as a group . [ nsipe ] from the hcws ' perspective , more training was necessary to help patients enrolled in option b+ . distance , especially among rural health facilities , was an obstacle to trace patients ltfu . hcws suggested that task sharing with health surveillance assistants ( hsas ) was important to link patients back to care and that bikes and motorbikes could help them to address this issue : we have many clients who come from far and we do not know if their reason for defaulting might be that they are dead or not , so if we can have another motorbike and make the effective follow - ups we can have a successful art . [ lobi ] we have many clients who come from far and we do not know if their reason for defaulting might be that they are dead or not , so if we can have another motorbike and make the effective follow - ups we can have a successful art . this study explored the experiences of women and hcws in the context of the early rollout of option b+ in malawi . our findings illustrate that patients and hcws identified the lack of male involvement as a barrier to retention in care and expressed concerns at the rapidity of the test - and - treat process . fear regarding the breach of privacy and confidentiality were also identified as contributing to loss to follow - up of women initiated under the option b+ strategy . similar to recent studies exploring the challenges to implementing option b+ in malawi , we note that stopping treatment within the first few months of art initiation may be linked to lack of means of travel and transport money , not understanding how to take art , informal transfers between health facilities , or being too weak / sick and drugs side effects . in addition to health systems barriers , such as the irregular availability of cpt and hiv test kits , and the lack of infrastructure and human resources for health , our findings illustrate that stigma , confidentiality , and privacy remain central concerns for women accessing pmtct services in the context of option b+ . as described by others , despite the broader availability of art , women continue to fear stigma in relation to being publicly identified as living with hiv . to understand the experience of receiving care and support under option b+ , it is crucial to consider the different cadres of hcws involved in providing pmtct care ; hsas , in particular , have an important role to play , especially outside health facilities . hsas perform a number of tasks within the pmtct cascade , often without receiving additional specialized training , for example , to carry out tracing activities . hsas are required to spend a considerable portion of their time in health facilities to assist hcws ; as their roles and responsibilities continue to expand in the context of option b+ , it is important for their roles to be rationalized to ensure that they are not overwhelmed by the large number of tasks that are shifted to them . some of the key reasons identified by hcws as contributing to early loss to follow - up include lack of support from family members , fear for breach of confidentiality , feeling discouraged at the prospect of lifelong art , and lack of partner involvement . male partner involvement is viewed as a key intervention by patients and hcws and remains inadequately addressed in current pmtct interventions . however , as others have noted , male partner involvement in pmtct care is fragmented and , at best , associated with first anc and couple hiv counseling and testing , suggesting poor integration of male involvement into existing programs . similar to a study from nigeria , our results indicate that nondisclosure is a factor that may contribute to nonretention in option b+ . in addition , our data support the results described in other studies that explored couple testing and disclosure ; although male involvement is associated with better early retention on art , a majority of pregnant women living with hiv face difficulties in sharing their serostatus with their partners and fear being rejected by their partners . importantly , patients and hcws were concerned about the rapidity of the process for same day test - and - treat . our study highlights key concerns regarding the immediate nature of this approach , where patients are given little time to digest a new diagnosis and start treatment . this was highlighted by patients and hcws as a crucial concern regarding the implementation of option b+ . our results confirm that some women may find pmtct intervention coercive or that they are not adequately prepared to start art . in addition , in rural malawi , hiv testing may , in some cases , be incompatible with marriage because it may be perceived as against ideals of trust , love , and intimacy that couples strive for in their relationships . hence , the use of same - day hiv testing and art initiation may be contributing to the inadequate uptake of treatment under option b+ , which points to the need to identify alternatives or enhancements to this approach for women who are unable to start treatment right away for instance , enhancing support for women after testing , tracing male partners early , improve couple testing procedures , and help women overcome the hurdle of disclosure . although study sites were carefully selected in relation to criteria described in the methods section , the purposive selection of patients and hcws at each site may not reflect some of the challenges of the current implementation of option b+ . in addition , this study focused on patients currently retained in care , who may have a different perspective on option b+ services to that of patients who have been ltfu . although study sites were carefully selected in relation to criteria described in the methods section , the purposive selection of patients and hcws at each site may not reflect some of the challenges of the current implementation of option b+ . in addition , this study focused on patients currently retained in care , who may have a different perspective on option b+ services to that of patients who have been ltfu . as option b+ continues to be rolled out across malawi and in several other sub - saharan african countries , new interventions to support women to be retained into care must be implemented . novel types of interventions , for instance , providing care through other mothers who are also enrolled in option b+ , are seen by some patients and hcws as providing more personal and family - centered peer - supported care , but key questions remain as to how these interventions will cope with patients ' perception of the quality of care received and with expectations placed on peer support mothers . current option b+ interventions should consider the lack of patient agency during treatment initiation in relation to early loss to follow - up ; interventions should improve processes to providing appropriate space , time , and support for patients to accept a positive hiv diagnosis before starting treatment , engaging male partners and families into pmtct care , and addressing the need for peer support and confidentiality expressed by women who are already accessing pmtct care .

introduction : malawi has embarked on a test - and - treat approach to prevent mother - to - child transmission ( pmtct ) of hiv , known as option b+ , offering all hiv - infected pregnant and breastfeeding women lifelong antiretroviral therapy ( art ) regardless of cd4 count or clinical stage . a cross - sectional qualitative study was conducted to explore early experiences surrounding option b+ for patients and health care workers ( hcws ) in malawi.methods:study participants were purposively selected across 6 health facilities in 3 regional health zones in malawi . semi - structured interviews were conducted with women enrolled in option b+ ( n = 24 ) , and focus group discussions were conducted with hcws providing option b+ services ( n = 6 groups of 8 hcws ) . data were analyzed using a qualitative thematic coding framework.results:patients and hcws identified the lack of male involvement as a barrier to retention in care and expressed concerns at the rapidity of the test - and - treat process , which makes it difficult for patients to digest a positive diagnosis before starting art . fear regarding the breach of privacy and confidentiality were also identified as contributing to loss to follow - up of women initiated under the option b+ . disclosure remains a difficult process within families and couples . lifelong art was also perceived as an opportunity to plan future pregnancies.conclusions:as option b+ continues to be rolled out , novel interventions to support and retain women into care must be implemented . these include providing space , time , and support to accept a diagnosis before starting art , engaging partners and families , and addressing the need for peer support and confidentiality .

INTRODUCTION METHODS Study Design Study Sites Participant Selection Data Collection Data Analysis Ethical Considerations RESULTS Perception of Option B+ by Patients and HCWs Adherence and Loss to Follow-up in Option B+ Perspectives on Improving Option B+ DISCUSSION Limitations of the Study CONCLUSIONS

in malawi , a country of 15.9 million people in southern africa , an estimated 10.3% ( 9.6%10.8% ) of adults are hiv positive , and an estimated 170,000 children younger than 15 years are living with hiv , most of whom were infected through vertical transmission . since july 2011 , the malawi ministry of health has embarked on a program to prevent mother - to - child transmission ( pmtct ) of hiv known as option b plus malawi became the first country to implement a test - and - treat approach in a national pmtct program : option b+ offers all hiv - infected pregnant and breastfeeding women lifelong antiretroviral therapy ( art ) , regardless of cd4 count or clinical stage . the overall goal of the program was to improve art uptake and retention , and thus the outcomes of hiv - infected pregnant women , their uninfected partners , and their infants within the continuum of hiv testing and treatment services . the option b+ strategy offers an attractive rapid art scale - up and was adopted in malawi ahead of the recommendations from the world health organization . although it has the potential to profoundly impact maternal and infant outcomes , operational challenges throughout the cascade of pmtct services may affect uptake and adherence to art by pregnant women , the follow - up of hiv - exposed infants , and the long - term retention of this population . from the onset , potential threats to the implementation and scale - up of option b+ were identified and concerns were raised in relation to initiation into care , retention in care , adherence to art , and the lack of psycho - social support in the context of lifelong retention of a relatively asymptomatic population group . during its first year of implementation in malawi , option b+ rapidly expanded uptake and access to pmtct services , with a 7-fold increase in the number of women starting art for pmtct . in the first year of implementation , a large proportion of women ( 17% ) seemed to be lost to follow - up ( ltfu ) 6 months after art initiation , with most losses occurring in the first 3 months of therapy . early assessments into the reasons associated with stopping art among women who initiated treatment during option b+ pointed at difficulties linked to traveling to health facilities and lack of transport money , not understanding initial treatment guidance , and being too weak , sick , or experiencing treatment side effects . in light of these potential challenges to the implementation of option b+ in malawi , a cluster randomized control trial , pure ( pmtct uptake and retention in malawi ) , was designed to evaluate and compare strategies to strengthen uptake and retention of mothers and families in pmtct care . the trial compares facility - based peer support and community - based peer support to standard of care across the south east , south west , and central west regions . interventions include the introduction of trained expert patients ( mentor mothers and expert mothers ) , who are working within and outside health facilities to support women involved in option b+ and their families . the aim of this study was to explore the experience of patients and health care workers ( hcws ) in relation to the implementation of option b+ . the specific objectives were to elicit local perceptions of option b+ among hcws and women who have recently been enrolled in this program and to document the nature of the support provided to these women . this was descriptive , cross - sectional formative research , drawing on a qualitative approach to collect data through semi - structured interviews and focus group discussions with both option b+ patients and hcws . the study was conducted in 6 of 21 facilities within the pure trial sites across 3 health zones ( central west , south east , and south west ) . among the 21 study sites included in the pure trial , in addition , in each health zone , we selected 2 sites that were different according to their location ( rural vs urban settings ) , number of pregnant and/or breastfeeding women attending services in the past quarter ( large vs smaller facilities ) , and type of ownership of the health facility ( ministry of health vs christian health association malawi ) . in each site , the research team identified 4 women newly initiating art services and enrolled in option b+ , and all available hcws involved in providing services as part of option b+ . based on previous studies conducted in the same setting in relation to pmtct services , we expected that saturation would be achieved at 2030 interviews ; we therefore selected 24 interviews ( 4 per site ) as a suitable number of women to be interviewed . hcws recruited for the study included nurses , medical assistants , and community health workers ( health surveillance assistants ) who routinely provided pmtct care in the selected health facilities . patients involved in the study included hiv - infected pregnant or breastfeeding women seeking antenatal care ( anc ) or initiating art services at 1 of the 6 study sites . in total , 48 hcws and 24 women receiving care as part of option b+ all data were collected at the health facilities at a time identified as convenient by research participants themselves . semi - structured interviews were conducted with 4 patients in each study site ( n = 24 ) . one focus group discussion was conducted with 8 hcws per facility ( n = 6 groups of 8 individuals ) . recordings of interviews and focus group discussions were transcribed in chichewa and later translated verbatim to english . using a framework analysis approach , themes were developed to capture common issues , such as perception and acceptability of current care , adherence and loss to follow - up , care relationships , disclosure , and task - shifting involving community hcws . study participants did not receive an incentive to be part of the study but they received a transport reimbursement ( equivalent usd 1.40 ) for taking part in the interviews and focus group discussions . this was descriptive , cross - sectional formative research , drawing on a qualitative approach to collect data through semi - structured interviews and focus group discussions with both option b+ patients and hcws . the study was conducted in 6 of 21 facilities within the pure trial sites across 3 health zones ( central west , south east , and south west ) . among the 21 study sites included in the pure trial , in addition , in each health zone , we selected 2 sites that were different according to their location ( rural vs urban settings ) , number of pregnant and/or breastfeeding women attending services in the past quarter ( large vs smaller facilities ) , and type of ownership of the health facility ( ministry of health vs christian health association malawi ) . geographic distribution of the 6 pure formative sites in the south east , south west , and central west zones of the country ( mulanje district hospital and muloza health centre in the south east zone ; trinity mission hospital and makhetha health centre in the south west zone ; and nsipe health centre and lobi rural hospital in the central east / central west zone ) . in each site , the research team identified 4 women newly initiating art services and enrolled in option b+ , and all available hcws involved in providing services as part of option b+ . based on previous studies conducted in the same setting in relation to pmtct services , we expected that saturation would be achieved at 2030 interviews ; we therefore selected 24 interviews ( 4 per site ) as a suitable number of women to be interviewed . hcws recruited for the study included nurses , medical assistants , and community health workers ( health surveillance assistants ) who routinely provided pmtct care in the selected health facilities . patients involved in the study included hiv - infected pregnant or breastfeeding women seeking antenatal care ( anc ) or initiating art services at 1 of the 6 study sites . in total , 48 hcws and 24 women receiving care as part of option b+ all data were collected at the health facilities at a time identified as convenient by research participants themselves . semi - structured interviews were conducted with 4 patients in each study site ( n = 24 ) . one focus group discussion was conducted with 8 hcws per facility ( n = 6 groups of 8 individuals ) . recordings of interviews and focus group discussions were transcribed in chichewa and later translated verbatim to english . using a framework analysis approach , themes were developed to capture common issues , such as perception and acceptability of current care , adherence and loss to follow - up , care relationships , disclosure , and task - shifting involving community hcws . study participants did not receive an incentive to be part of the study but they received a transport reimbursement ( equivalent usd 1.40 ) for taking part in the interviews and focus group discussions . for those who had an economic activity , this ranged from farming ( 6 of 24 ) , small business ( 7 of 24 ) , and small labor or seasonal work in tea plantations ( 4 of 24 ) . results are presented across 3 main themes : the perception of option b+ by patients and hcws ; experiences of adherence and loss to follow - up in the context of option b+ ; and patients ' and hcws ' perspectives on improving option b+ . distance , privacy , and confidentiality remain problematic issues for women , especially for those living in rural areas ; they expressed feeling stigmatized in relation to living with hiv , within their social network and during their visits to the health centers . [ makhetha ] across several health centers , women criticized the lack of privacy in hiv and art services , with confidentiality around their serostatus compromised each time they go to the clinic : if you meet a person who knows you and is seeing you enter the room where we get arvs [ antiretrovirals ] , they tend to tell people that so and so are on arvs . [ nsipe ] the lack of inclusion of male partners , and their reluctance to be tested for hiv , was cited as an ongoing problem by women ; some feared for the stability of their relationship if their partner did not test . across all sites , hcws complained about the lack of infrastructure and the recurrent shortage of hiv test kits . hcws were concerned that the test - and - treat approach is too fast and too much information is given to women at once , with no time to digest and communicate a new positive hiv diagnosis : we test her [ a patient ] today and she is found hiv positive , we immediately initiate her onto art whilst she is still thinking about how she contracted the virus and she hasn't yet accepted the reality about her serostatus , and we give her the drugs for her unborn child at the same time . similarly to patients , hcws highlighted the lack of privacy and confidentiality when patients came for testing or to pick up art : the space where vct [ voluntary counseling and testing ] and art clinics are conducted is small , so on art day the space is overcrowded . [ nsipe ] despite declaring that they adhered to treatment , and generally understanding well how to take art , nearly all mothers and hcws interviewed reported problems linked to retention in care and adherence to art ; some patients reported missing doses of art while they continued to collect treatment regularly from the clinic . [ trinity ] a key concern expressed by hcws was that they do not always know how to follow - up with the mothers who may be defaulting . some hcws noted that the loss to follow - up was also because of the lack of involvement of male partners in pmtct care , for instance , because men did not accompany women to anc or because they were not routinely tested for hiv and followed - up within pmtct services : because men do n't accompany their spouses to antenatal clinic , it happens that once the lady delivers and she is not suffering from any disease , she does not see the reasons for coming to the clinic to continue art treatment . life skills advice ( for example , skills to be empowered to improve their day - to - day situation ) to be included in the care package that they received at health facilities . another suggestion from patients to improve pmtct care was to address the issues around privacy and confidentiality . [ nsipe ] from the hcws ' perspective , more training was necessary to help patients enrolled in option b+ . distance , privacy , and confidentiality remain problematic issues for women , especially for those living in rural areas ; they expressed feeling stigmatized in relation to living with hiv , within their social network and during their visits to the health centers . [ makhetha ] across several health centers , women criticized the lack of privacy in hiv and art services , with confidentiality around their serostatus compromised each time they go to the clinic : if you meet a person who knows you and is seeing you enter the room where we get arvs [ antiretrovirals ] , they tend to tell people that so and so are on arvs . [ nsipe ] the lack of inclusion of male partners , and their reluctance to be tested for hiv , was cited as an ongoing problem by women ; some feared for the stability of their relationship if their partner did not test . across all sites , hcws complained about the lack of infrastructure and the recurrent shortage of hiv test kits . hcws were concerned that the test - and - treat approach is too fast and too much information is given to women at once , with no time to digest and communicate a new positive hiv diagnosis : we test her [ a patient ] today and she is found hiv positive , we immediately initiate her onto art whilst she is still thinking about how she contracted the virus and she hasn't yet accepted the reality about her serostatus , and we give her the drugs for her unborn child at the same time . similarly to patients , hcws highlighted the lack of privacy and confidentiality when patients came for testing or to pick up art : the space where vct [ voluntary counseling and testing ] and art clinics are conducted is small , so on art day the space is overcrowded . [ nsipe ] despite declaring that they adhered to treatment , and generally understanding well how to take art , nearly all mothers and hcws interviewed reported problems linked to retention in care and adherence to art ; some patients reported missing doses of art while they continued to collect treatment regularly from the clinic . [ trinity ] a key concern expressed by hcws was that they do not always know how to follow - up with the mothers who may be defaulting . some hcws noted that the loss to follow - up was also because of the lack of involvement of male partners in pmtct care , for instance , because men did not accompany women to anc or because they were not routinely tested for hiv and followed - up within pmtct services : because men do n't accompany their spouses to antenatal clinic , it happens that once the lady delivers and she is not suffering from any disease , she does not see the reasons for coming to the clinic to continue art treatment . many patients would have liked additional life skills advice ( for example , skills to be empowered to improve their day - to - day situation ) to be included in the care package that they received at health facilities . another suggestion from patients to improve pmtct care was to address the issues around privacy and confidentiality . [ nsipe ] from the hcws ' perspective , more training was necessary to help patients enrolled in option b+ . this study explored the experiences of women and hcws in the context of the early rollout of option b+ in malawi . our findings illustrate that patients and hcws identified the lack of male involvement as a barrier to retention in care and expressed concerns at the rapidity of the test - and - treat process . fear regarding the breach of privacy and confidentiality were also identified as contributing to loss to follow - up of women initiated under the option b+ strategy . similar to recent studies exploring the challenges to implementing option b+ in malawi , we note that stopping treatment within the first few months of art initiation may be linked to lack of means of travel and transport money , not understanding how to take art , informal transfers between health facilities , or being too weak / sick and drugs side effects . in addition to health systems barriers , such as the irregular availability of cpt and hiv test kits , and the lack of infrastructure and human resources for health , our findings illustrate that stigma , confidentiality , and privacy remain central concerns for women accessing pmtct services in the context of option b+ . to understand the experience of receiving care and support under option b+ , it is crucial to consider the different cadres of hcws involved in providing pmtct care ; hsas , in particular , have an important role to play , especially outside health facilities . hsas are required to spend a considerable portion of their time in health facilities to assist hcws ; as their roles and responsibilities continue to expand in the context of option b+ , it is important for their roles to be rationalized to ensure that they are not overwhelmed by the large number of tasks that are shifted to them . some of the key reasons identified by hcws as contributing to early loss to follow - up include lack of support from family members , fear for breach of confidentiality , feeling discouraged at the prospect of lifelong art , and lack of partner involvement . male partner involvement is viewed as a key intervention by patients and hcws and remains inadequately addressed in current pmtct interventions . in addition , our data support the results described in other studies that explored couple testing and disclosure ; although male involvement is associated with better early retention on art , a majority of pregnant women living with hiv face difficulties in sharing their serostatus with their partners and fear being rejected by their partners . importantly , patients and hcws were concerned about the rapidity of the process for same day test - and - treat . our study highlights key concerns regarding the immediate nature of this approach , where patients are given little time to digest a new diagnosis and start treatment . this was highlighted by patients and hcws as a crucial concern regarding the implementation of option b+ . hence , the use of same - day hiv testing and art initiation may be contributing to the inadequate uptake of treatment under option b+ , which points to the need to identify alternatives or enhancements to this approach for women who are unable to start treatment right away for instance , enhancing support for women after testing , tracing male partners early , improve couple testing procedures , and help women overcome the hurdle of disclosure . although study sites were carefully selected in relation to criteria described in the methods section , the purposive selection of patients and hcws at each site may not reflect some of the challenges of the current implementation of option b+ . in addition , this study focused on patients currently retained in care , who may have a different perspective on option b+ services to that of patients who have been ltfu . although study sites were carefully selected in relation to criteria described in the methods section , the purposive selection of patients and hcws at each site may not reflect some of the challenges of the current implementation of option b+ . in addition , this study focused on patients currently retained in care , who may have a different perspective on option b+ services to that of patients who have been ltfu . as option b+ continues to be rolled out across malawi and in several other sub - saharan african countries , new interventions to support women to be retained into care must be implemented . novel types of interventions , for instance , providing care through other mothers who are also enrolled in option b+ , are seen by some patients and hcws as providing more personal and family - centered peer - supported care , but key questions remain as to how these interventions will cope with patients ' perception of the quality of care received and with expectations placed on peer support mothers . current option b+ interventions should consider the lack of patient agency during treatment initiation in relation to early loss to follow - up ; interventions should improve processes to providing appropriate space , time , and support for patients to accept a positive hiv diagnosis before starting treatment , engaging male partners and families into pmtct care , and addressing the need for peer support and confidentiality expressed by women who are already accessing pmtct care .

development of transesterification process for production of biodiesel as an alternative , green , and sustainable fuel has become an important issue due to diminishing fossil fuel reserves , rising crude oil price , and the stringent exhaust emission regulations . biodiesel produced by transesterification reaction can be catalyzed with alkali , acid , or enzyme , in which a primary alcohol reacts with the triglycerides of fatty acids form glycerol and esters . triglyceride for biodiesel production comes from various sources edible and nonedible oil , waste and used oil , and fats . the major hurdle of applicability of biodiesel is the operational cost of its production process , approximately 7095% of the production cost arose from the feedstock . homogenous chemical catalyst processes , including alkali or acid , are more practical compared with the enzymatic method . the use of acid catalysts has been found to be used for pretreating high free fatty acid feedstock but the reaction rates for converting triglycerides to methyl esters are very slow . enzymes have shown good tolerance for the free fatty acid level of the feedstock but the enzymes are expensive . the use of homogenous basic catalytic process overcome the aforementioned problems but suffers some drawbacks : production of large amount of wastewater from washing process of catalyst residues and neutralization step , difficulty of the product separation and purification , and unreusability of the catalysts . calcium oxide cao has attracted attention due to the elimination of neutralization step , high activity , being active in mild reaction conditions , long catalyst life time , low solubility in methanol , lack of toxicity , ability to withstand high temperatures , ease of recycling , low cost , and being abundantly available in nature as limestone and its performance for biodiesel production is comparable to several homogenous catalysts . in egypt , millions of liters of waste cooking oil wco are discarded each year into sewage systems . thus , it pollutes water streams , causing a lot of waste management problems and , consequently , adds to the cost of treating effluent . solid wastes density in egypt averages about 300 kg / m , where 60% is organic wastes . thus , production of biodiesel from wco using cao prepared from organic wastes ( e.g. , eggshells , mollusks shells , crabs shells , etc . ) would offer a triple - fact solution : economic , environmental , and waste management . the aim of this work is to optimize biodiesel transesterification process using waste cooking oil and calcium oxide catalyst prepared from waste mollusks and crabs shells collected from seafood restaurants in an attempt to reach an effective process for practical , low cost industrial biodiesel production . pure calcium oxide as heterogeneous catalyst and methanol ( ar grade ) were purchased from fluka chemical corp . , gillingham , uk . novozym 435 ( candida antarctica lipase b ) was a gift from novozyme a / s , bagsvaerd , denmark , and was supplied as an immobilized enzyme on macroporous acrylic resin . the waste frying oil wfo , mollusks , and crabs shells ( ms and cs ) were collected from local seafood restaurants and prepared according to el - gendy et al . . the wco was characterized by high total acid number , density , and viscosity , recording 3 mg koh / g oil , 0.9208 g / cm , and 50 cst , respectively , and its saponification and iodine value were 197 mg koh / g oil and 119 mgi2/100 g oil , respectively . the wco consists of 24.36 , 36.35 , 28.68 , and 10.61% palmitic ( c16:0 ) , stearic ( c18:0 ) , oleic ( c18:1 ) , and linoleic ( c18:2 ) acid , respectively . the prepared catalysts were characterized according to el - gendy et al . using differential scanning calorimetric - thermal gravimetric analysis ( dsc - tga ) and were performed by q600 sdt simultaneous dsc - tga ( new castle , de usa ) , a high - resolution x - ray diffractometer ( xrd , panalytical x'pert pro mpd , netherland ) coupled with cu k radiation source ( = 1.5418 ) , dispersive raman spectrometer ( bruker - senterra , germany ) equipped with an integral microscope ( olympos ) , energy dispersive x - ray analysis ( edx , oxford x - max , england ) conjugated with transmission electron microscope tem ( jem 2100 , jeol , japan ) , analytical fourier transforms infrared ( ft - ir , perkin elmer spectrum one , usa ) instrument , scanning electron microscope ( sem , jeol - model jsm-53000 , japan ) . the specific surface area of the prepared biocatalysts was measured by brunauer - emmett - teller bet method using low temperature n2 adsorption - desorption ( non a3200e , quantachrome , usa ) . the samples were tested for pore volume and pore size distribution using barrett - joyner - halenda bjh method . temperature programmed desorption using co2 as a probe molecules ( co2-tpd ) was used to study basic properties of the prepared biocatalysts and it was done according to viriya - empikul et al . . the transesterification reactions were conducted in a laboratory - scale setup , according to el - gendy et al . , and the biodiesel yield was calculated according to boro et al . . the activity of the prepared biocatalysts was compared with that of commercially available , most effective heterogeneous basic chemical catalyst cao and immobilized enzyme novozym 435 . based on d - optimal design of experiments , twenty runs of experiments have been conducted for three levels of four independent variables : methanol : oil m : o ( molar ratio ; a ) , catalyst concentration ( wt% ; b ) , reaction time ( min ; c ) , and mixing rate ( rpm ; d ) , to study their effect on the % yield of the produced biodiesel at constant temperature 60c . once the experiments were preformed , the next step was to perform a response surface experiment to produce a prediction model to determine curvature , detect interactions among the design factors ( independent variables ) , and optimize the process , that is , to determine the local optimum independent variables with maximum yield of biodiesel . the model used in this study to estimate the response surface is the quadratic polynomial represented by the following equation : ( 1)y=o+i=1nixi+i=1n1j = i+1nijxixj+i=1niixi2 , where y is the biodiesel yield ( wt% ) , n is the number of factors , o is the intercept term , i , ij , and ii are the linear , interactive , and quadratic coefficients , respectively . minneapolis , usa ) was used for regression and graphical analyses of the data obtained and statistical analysis of the model to evaluate the analysis of variance ( anova ) . the produced biodiesel was tested for estimating and evaluating its fuel properties , using the standard methods of analysis for petroleum products , american society for testing and materials astm standards methods . the results were compared with the egyptian standards for petro - diesel and european and american standards of biodiesel ( en14214 and d-6751 , resp . ) all the properties were analyzed in two replicates and the final results given below were obtained as the average values . the thermal transition during the calcination process of each collected waste shells was investigated with tga / dsc . figure 1 shows the thermal analysis results along with the weight loss when the temperature was raised from room temperature to 1100c . the thermogravimetric analysis of ms shows that there was no significant decomposition occurred upon heating up to 600c , recording weight loss of 5% . the dominant decomposition 41% weight loss occurred within 700800c ( peaked at 778 ) and remained sustained thereafter up to 1100c . the dsc curve supports the tga curve , where the heat flow chart illustrates endothermic peak at 778c . the tga analysis reveals three phases of mass loss with a total weight loss of 56.77% . the first mass loss was observed at temperatures below 200c , recording weight loss of 5% ( peaked at 170c ) . the second mass loss occurred within 250400c , recording weight loss of 20% ( peaked at 320c ) . the dominant decomposition with 30% weight loss occurred within 650770c ( peaked at 750c ) and remained sustained thereafter up to 1100c . the dsc curve shows an exothermic peak at 130c . according to boro et al . , this can be attributed to evaporation of water , crystallization , decomposition of organic components , or possible rearrangement in the structural arrangement within the compound itself . the dsc curve shows also a weak broad endothermic peak located at the range 320370c and another sharp endothermic peak at 753c . the two endothermic peaks might indicate the decomposition of compounds and formation of a new one . according to n. b. singh and n. p. singh , the weight loss might be attributed to the decomposition of caco3 through the loss of carbon dioxide co2 and production of calcium oxide cao and also due to the possible removal of absorbed water molecules which occurs according to the following dissociation equation : ( 2)caco3(s)cao(s)+co2(g ) it was observed that during calcination , the ms and cs turned completely pale grey and white , respectively . according to engin et al . , this indicates that calcium carbonate caco3 is converted to cao with elevating calcination temperatures . the xrd patterns of natural ms ( figure 2 ) are mainly aragonite caco3 ( jcpds card number : 024 - 0025 ) and upon calcination at 400c , it was changed to calcite caco3 ( jcpds card number : 005 - 0586 ) and remained unchanged up to calcination at 600c , while calcination at 700c showed mixture of calcite caco3 ( jcpds card number : 005 - 0586 ) and lime cao ( jcpds card number : 043 - 1001 ) . the xrd patterns showed a pure crystalline cao ( jcpds card number : 043 - 1001 ) for calcination temperatures 8001100c , while the xrd patterns of cs revealed that the natural cs is composed mainly of crystalline calcite caco3 ( jcpds card number : 005 - 0586 ) and also to a minor extent of monohydrocalcite caco3h2o ( jcpds card number : 022 - 0147 ) . upon calcination at 400c , caco3h2o was lost and only calcite caco3 ( jcpds card number : 005 - 0586 ) detected and remained unchanged up to calcination at 600c . the xrd patterns showed a pure crystalline calcia or burnt lime cao ( jcpds card number : 004 - 0777 ) for calcination temperatures 7001100c . the obtained xrd patterns could explain the tga / dsc flowcharts of cs , where the weak broad endothermic peak at the range of 320370c might be due to the loss of water and decomposition of caco3h2o and the sharp one at 753c might be due to the decomposition of caco3 and formation of cao . narrow and high intense peaks of the calcinated ms and cs ( at temperature 800 and 700c , resp . ) could define the well - crystallized nature of the prepared biocatalyst similar observation which was reported by boey et al . and viriya - empikul et al . on preparation of biocatalyst from waste mud crab shells ( scylla serrata ) and the crystal size was calculated from the xrd data ( table 1 ) , recording 42.15 nm for natural ms , which decreased upon calcination at 800c , recording 38.77 nm . similar observation was reported by yoosuk et al . who attributed this to the presence of water molecules during calcination of caco3 to cao . but , the crystalline size of the uncalcinated cs was found to be 32.15 nm and it was slightly increased by calcination at 700c , recording 32.82 nm . borgwardt and liu et al . when caco3 decomposes at high temperatures , small cao grains formed and then the contact grains form necks and begin to grow resulting in an increase in the average grain size . raman spectra ( figure 3 ) are consistent with the xrd patterns . in case of natural ms , the vibration bands at 155 , 197 , 650 , 703 , and 1086 cm correspond to those of aragonite caco3 . the vibration bands at 360 , 1075 , 1334 , and 1463 cm of calcined ms at 800c correspond to pure lime cao , while the vibration bands at 155 , 282 , 714 , and 1087 cm of natural cs correspond to calcite caco3 . the high vibration bands at 360 , 960 , and 1075 cm of calcined cs at 700c correspond to pure burnt lime cao . the edx analysis ( table 2 ) revealed that the chemical composition of the shells was highly affected by calcination . the uncalcinated shells exhibit oxygen as the main component , 56.62 and 53.69% , wt% , while in calcinated shells at 800c and 700c , calcium represents the major component ( 62.32 and 49.89% , wt% ) for ms and cs , respectively . similar observation was reported by viriya - empikul et al . and birla et al . , where the main component in the calcinated shells was calcium and other elements , for example , na , mg , and so forth , stoichiometrically , this is true , as in cao , ca is the main constituent ( 71% ) , while oxygen is the major one in caco3 ( 48% ) . the basicity of the prepared cao from ms and cs recorded 52.18 and 27 mmol co2/g , respectively . this might affect the activity of the catalyst in the transesterification reaction , where the mechanism of cao in transesterification reactions would be started by dissociation of cao ( scheme 1 ) . then oxide anion attacks the methanol to form methoxide anion , which is why excess methanol is required to drive the reaction in the forward direction . the methoxide anion attacks the carbonyl carbon of the triglyceride to form a tetrahedral intermediate . another methoxide anion attacks the carbonyl carbon in the formed diglyceride , forming another mole of methyl ester and monoglyceride . finally , a new methoxide anion attacks the monoglyceride , producing a total of three moles of methyl esters and a mole of glycerol . the ftir patterns of ms and cs with respect to calcination process ( figure 4 ) were nearly the same , showing major peaks around 14201471 , 860874 , and 709 cm in patterns of natural shells , which disappeared in that of calcinated shells . according to engin et al . , these peaks are attributed to asymmetric stretch ; out - of - plane bend and in - plane bend vibration modes for co3 molecules . the strong sharp peak at 3643 cm in uncalcinated cs confirms the xrd pattern and presence of caco3h2o . also , upon calcination , weak bands around 2509 and 17861793 cm disappeared and new weak peak appeared at 1111 cm for calcinated ms and 1089 and 1056 cm for calcinated cs . the observed changes in ir patterns might indicate the complete transformation of caco3 to cao . in the viewpoint of preparation time , energy consumption , and cost of catalyst preparation , the temperatures of 800c and 700c were selected as perfect calcination temperature to prepare biocatalysts from natural waste mollusks and crabs shells , respectively . the morphology of natural and calcined shells was investigated by sem at equal magnification of 500x . sem micrograph of catalyst derived from ms ( figure 5(b ) ) shows that , upon calcination , the morphology of ms changed from layered bulky substances without any clear pores on its surface ( figure 5(a ) ) to porous particles of various sizes and shapes , with higher specific surface area . the sem micrographs of the natural crabs shells ( figure 5(c ) ) showed bulky and nonuniform clustered substances with clear pores on its surface which transformed to relatively similar aggregates of porous smaller particles with higher specific surface area upon calcination at 700c ( figure 5(d ) ) . this porosity is probably due to the fact that a large number of gaseous water molecules are released upon the decomposition of caco3h2o . hu et al . reported that the gaseous water molecules create high porosity in the catalyst , that is , to act as porogens . the particles size distribution ( table 1 ) proved the sem analysis ( figure 5 ) , where a large part of the particles size distribution of the natural ms and cs was within the size range of 8.5817.23 and 9.0118.43 m , while the rest was within the range of 6.487.13 and 6.527.25 m , with overall average particles diameter of 11.07 and 10.27 m , respectively . but upon calcination at 800 and 700c the particles size decreased , where a large part of the particles size distribution was in the range of 7.9314.50 and 7.7110.74 m and the rest was within the range of 6.376.80 and 6.386.81 m , with overall average particles diameter of 9.86 and 8.24 m , respectively . the smaller size of the grains and aggregates would provide higher specific surface areas . because the prepared biocatalyst has relatively large particle sizes , it is easy to separate the catalyst from the products after the reaction , by filtration or centrifugation . the bet surface area sbet of the prepared catalysts ( table 1 ) recorded 12.63 and 43.73 m / g for cao prepared from ms and cs , respectively . the bjh method was used for calculations of the pore size distributions ( table 1 ) and the prepared biocatalyst recorded total pore volume of 0.046 and 0.211 cm / g , respectively , and the pore size , distributed between 0.83 and 17.18 nm and between 1.04 and 15.58 nm , with average pore diameter of 0.94 and 1.84 nm , respectively , indicating that most of the pore size distributions are found in the microporous range . according to roschat et al . , a high porosity catalyst is a key requirement to achieve high conversion efficiency for heterogeneous process , thereby high surface area or high catalytic sites are necessary . sharma et al . also reported a high pore size to be desirable for better diffusion of reactants and product molecules . thus , this would recommend the biocatalyst prepared from ms and cs for application in biodiesel production . the n2 adsorption - desorption isotherms of the prepared cao from ms and cs are shown in figures 6(a ) and 6(b ) and they are of type ii isotherm ( based on iupac 's classification ) with a low slope in the middle region of the isotherm and a desorption curve almost overlapping with adsorption curve . applying bet equation , p / po = 0.9590 and 0.9482 ( < 1 ) and c = 7.152 and 17.012 ( > 1 ) , respectively , indicating monolayer formation . the isotherms have a hysteresis loop of h3 and slopping adsorption and desorption branches covering a large range of p / po with underlying type ii isotherm . the complete design matrix with experimental and predicted values of % yield of biodiesel using different cao catalyst prepared from mollusks and crabs shells is presented in table 3 . based on d - optimal design and experimental data , two second - order quadratic models for the applied catalysts have been predicted and can be given as follows : ( 3)y1=82.432.21a0.98b+3.12c+0.80d+4.48a2 + 1.89b27.51c22.56d22.66ab1.16ac+5.79ad+2.50bc+8.42bd+0.87cd,(4)y2=85.05 + 3.41a3.28b5.05c0.6d2.19a2 + 4.63b2 + 1.90c2 + 0.04d2 + 4.89ab0.56ac+0.32ad3.194bc5.45bd+2.53cd , where y1 and y2 are the biodiesel yields from transesterification processes using biocatalyst prepared from ms and cs , respectively . positive sign in front of the terms indicate synergetic effect , whereas negative sign indicates antagonistic effect . pareto charts , which are very useful in design of experiments , were used in this work to make it much easier to visualize the main and interaction effects of all factors to the response variable that is biodiesel yield ( figure 7 ) . in case of using biocatalyst prepared from ms in the transesterification process , reaction time has a high positive impact on the reaction yield , followed by the mixing rate . but , m : o molar ratio has a high negative impact followed by catalyst loading . the interaction effects of m : o molar ratio and catalyst loading have a higher negative impact than m : o and reaction time on the biodiesel yield , while the interactive effect of catalyst loading and mixing rate expresses a very high positive impact on the biodiesel yield , coming after it the interaction between m : o ratio and mixing rate , catalyst loading , and reaction time , in a decreasing order , and the interactive effect of reaction time and mixing rate has a low positive impact on the reaction yield . in case of using biocatalyst prepared from cs in the transesterification process , only the m : o molar ratio has positive impact ; that is , increasing the m : o would increase the biodiesel yield . but , reaction time has a high negative impact followed by catalyst concentration and mixing rate , in a decreasing order ; that is , their increase would lower the biodiesel yield . the positive interactive impact on the biodiesel yield was expressed by m : o and catalyst loading , reaction time , and mixing rate and m : o and mixing rate with a decreasing order . while the interaction between catalyst loading and mixing rate , catalyst loading , and reaction time and m : o and reaction time has negative impact on the biodiesel yield in a decreasing order . the validity of the fitted models ( 3 ) and ( 4 ) was evaluated and their statistical significance was controlled by f - test . the analyses of variance ( anova ) for the response surface full quadratic models are given in tables 4 and 5 . it can be indicated that the models are highly statistically significant at 95% confidence level , with f value of 940.12 and 99.58 with very low probability p value of < 0.0001 , respectively ; that is , there is less than 0.01% chance that this error is caused by noise . the values of the determination coefficients , r and radj , which measure the model fitting reliability for the models ( 3 ) and ( 4 ) were calculated to be ( 0.9996 , 0.9986 ) and ( 0.9964 and 0.9864 ) , respectively . this suggests that approximately 99.96% and 99.64% of the variance is attributed to the variables and indicated a high significance of the predicted models . thus , only 0.04% and 0.36% of the total variations can not be explained by the models , respectively , which ensures the good adjustment of the above models to the experimental data . confirmation of the adequacy of the regression models was reflected also by the good agreement between experimental and predicted values of response variables as shown in table 3 , where the actual biodiesel yield for mollusks and crabs shells biocatalyst ranged from 71.80 to 92% and from 74 to 97% and there corresponding predicted values are 71.80 and 91.75% and 74 and 96.50% , respectively . the relationship between predicted and experimental values of biodiesel yield for cao prepared from ms and cs is shown in figures 8(a ) and 8(b ) . it can be seen that there is a high correlation ( r 1 ) between the predicted and experimental values indicating that the predicted and experimental values were in high reasonable agreement . it means that the data fit well with the model and give a convincingly good estimate of response for the system in the experimental range studied . the perturbation plots in figures 9(a ) and 9(b ) show the comparative effects of all independent variables on the biodiesel yield . the curvatures of the four factors from the center point confirm the statistical data obtained from analysis of variance ( anova , tables 4 and 5 ) , that is , the significance of each parameter ( coefficient ) . in case of cao prepared from ms ( figure 9(a ) ) , the sharp curvature of the two factors , m : o ( a ) and reaction time ( c ) , shows that the response biodiesel yield was very sensitive to these two variables . the comparatively low curvature of catalyst concentration ( b ) and mixing rate ( d ) curves shows less sensitivity of biodiesel yield towards the change in these two factors . the curvatures also confirm the data illustrated in pareto chart ( figure 7(a ) ) , where the increase of m : o molar ratio decreases the biodiesel yield , while the increase in reaction time increases the biodiesel yield . the sensitivity of biodiesel yield towards the four variables can be ranked in the following decreasing order reaction time > m : o > catalyst concentration > mixing rate . in case of cao prepared from cs ( figure 9(b ) ) , all the parameters , within the studied range , have highly significant effect on the biodiesel yield except the mixing rate ( d ) which has relatively nonsignificant effect . the curvatures also confirm the data illustrated in pareto chart ( figure 7(b ) ) , where the increase of m : o molar ratio increases the biodiesel yield , while the increase of reaction time , catalyst loading , and mixing rate decrease the yield , with decrease of sensitivity ( reaction time > m : o catalyst concentration mixing rate ) . these observations are well matched to the model mathematical equations ( 3 ) and ( 4 ) . three - dimensional response surface graphical diagrams of the regression equations ( 3 ) and ( 4 ) were plotted ( figures 10 and 11 ) to understand the interactive relationship between the independent variables and % yield of biodiesel and determine the optimum conditions for maximum biodiesel production , using cao prepared from ms and cs , respectively . figure 10(a ) illustrates the effect of m : o molar ratio and the prepared ms - cao concentration on biodiesel production at a constant reaction time of 30 min and mixing rate of 200 rpm . the increase of m : o ratio and catalyst loading decreased the biodiesel yield . the maximum yield 92% occurred within the range 6 : 16.5 : 1 m : o and 34.5 wt% catalyst concentration , but further increase in m : o and catalyst slightly decreased the biodiesel yield to 84% at 6 : 1 and 9 wt% , 89% at 12 : 1 and 6 wt% , and 81% at 12 : 1 and 9 wt% . m : o higher than the stoichiometric ratio has generally been adopted for biodiesel production . but further increase in m : o would lower the biodiesel yield as it might have dilution effect on the catalyst concentration . glycerol would dissolve in excessive methanol and subsequently inhibit the reaction of methanol with oil and catalyst . also the separation of glycerol would be difficult , which would consequently shift the equilibrium in the reverse direction . figure 10(b ) represents the effect of m : o molar ratio and reaction time on biodiesel production at a constant catalyst concentration 3 wt% and mixing rate 200 rpm . it seems within the studied range of experiments , the increase of reaction time increased the biodiesel yield , recording 92% at 6 : 16.5 : 1 m : o and 7593 min molar ratio . but the biodiesel yield slightly decreased with further increment of m : o molar ratio and time reaching 86% at 12 : 1 m : o and 120 min . the decrease in biodiesel production at higher reaction time might be due to the saturation of the active sites of the catalyst . figure 10(c ) represents the effect of m : o and mixing rate on biodiesel production at a constant reaction time 30 min and ms - cao concentration 3 wt% . biodiesel production recorded its maximum yield 92% at 6 : 16.5 : 1 m : o and 200250 rpm , further increment to 12 : 1 m : o and 400 rpm , decreased the yield to 86% . it is obvious from the 3d plot the high positive interaction of these factors , at low catalyst loading and high rpm , the biodiesel yield increased recording 81% at 3 wt% and 400 rpm and also at high catalyst loading 9 wt% and low mixing rate 200 rpm . but at low mixing rate 200250 rpm and catalyst loading 3 wt% , the biodiesel yield was high 92% . figure 10(e ) shows the interactive effect of mixing rate and reaction time , at a constant 6 : 1 m : o and ms - cao concentration 3 wt% . it is obvious that increasing the mixing rate and reaction time increased the yield to a certain extent recording 92% at 200250 rpm and 7593 min . further increment of mixing rate decreased the yield recording 81% at 400 rpm and 30 min and 84% at 400 rpm and 120 min . the increases of both factors decrease the biodiesel yield , recording maximum yield 92% at 34.5 wt% and 7593 min . from the rsm analysis it can be concluded that the maximum biodiesel production 8992% using cao prepared from mollusks shells ms - cao can be achieved over a wide range of experimental parameters , at 6 : 16.5 m : o molar ratio , 34.5 wt% catalyst concentration , 7593 min reaction time , and mixing rate of 200250 rpm , at 60c . figure 11(a ) illustrates the effect of m : o molar ratio and cs - cao catalyst loading on biodiesel production at a constant reaction time of 120 min and mixing rate 400 rpm . at low m : o ratio the increase of catalyst concentration decreased the biodiesel yield , recording 79.6% yield at 6 : 1 m : o and 9 wt% catalyst concentration . this can be explained by the effect of mass transfer limitation with the presence of excess solid catalyst . but at high m : o ratio , the biodiesel yield increased with increase of catalyst loading , recording maximum biodiesel yield of approximately 97% at 10.5 : 112 : 1 m : o and 7.59% catalyst loading ( w : w ) . according to son et al . , a molar ratio of methanol to oil m : o higher than the stoichiometric ratio has generally been adopted for biodiesel production , as methanol would promote the formation of methoxy anions on the catalyst surface , leading to a shift in the equilibrium in the forward direction , thus increasing the biodiesel yield . figure 11(b ) represents the effect of m : o molar ratio and reaction time on biodiesel production at a constant catalyst concentration 3 wt% and mixing rate 400 rpm . it seems that at low and high m : o , the increase in reaction time decreases the yield , recording 80% at 6 : 1 m : o and 120 min and 93% at 12 : 1 m : o and 120 min but 97% at 12 : 1 m : o and 30 min . figure 11(c ) represents the effect of m : o and mixing rate on biodiesel production at a constant reaction time 120 min and catalyst loading 3% . the 3d plot shows that , at low and high m : o , the increase in mixing rate decreases the yield , recording 97% at 12 : 1 m : o and 200 rpm but 93% and 80% at 400 rpm using 12 : 1 and 6 : 1 m : o , respectively . figure 11(d ) shows that there is a negative interactive effect between these two factors , where at low mixing rate , increasing the catalyst concentration increased the biodiesel yield , recording 80 and 97% at 200 rpm using 3 and 9 wt% catalyst , respectively . but , at high mixing rate 400 rpm , the opposite occurred ; increasing the catalyst loading decreased the yield , recording 93 and 97% at 9 and 3 wt% catalyst loading , respectively . the separation phase between hydrophilic methanol and hydrophobic oil and between the two liquid reactants and solid catalyst is generally known to be major problem , lowering the biodiesel yield in heterogeneous process . thus , increasing the stirring rate would increase the mixing of the reactants , which would consequently promote the transesterification process , as the mass transfer of the reactants to the catalyst surface can be enhanced . further increase in the stirring velocity would lead to increase in turbulence in the reaction mixture , which would lower the biodiesel yield . also the increase of catalyst concentration would increase the active sites of the solid catalyst available for the transesterification process . it is obvious that increasing the mixing rate to a certain limit 200300 rpm , with increase of reaction time 3050 min , yields larger amount of biodiesel , recording its maximum 97% . further increase of mixing rate slightly decreased the biodiesel yield , recording 93% at 400 rpm and 120 min and 89% at 400 rpm and 30 min . the increase of catalyst loading and reaction time decreased the biodiesel yield to reach 80% at 9 wt% catalyst concentration and 120 min . the maximum biodiesel yield 97% occurred at 9% catalyst concentrations and 30 min reaction time and 93% at 3% catalyst concentrations and 120 min reaction time . according to zhang et al . , the reason for the reduction in biodiesel yield with excess catalyst may be attributed to increase in viscosity of reaction mixture which might result in mass transfer limitation . from the rsm analysis it can be concluded that the maximum biodiesel production using cao prepared from crabs shells cs - cao can be achieved at 10.5 : 112 : 1 m : o , 7.59% catalyst concentration , 3050 min reaction time , and mixing rate of 200300 rpm , at 60c . the optimization process was carried out to determine the optimum value of biodiesel production efficiency , using the design expert 6.0.7 software . according to the software optimization step , the desired goal for each operational condition ( a m : o , b catalyst concentration , c reaction time , and d mixing rate ) was chosen within the studied range . the responses ( % biodiesel yield ) were defined as maximum to achieve the highest performance . the program combines the individual desirability into a single number and then searches to optimize this function based on the response goal . the maximum predicted biodiesel yields 94 and 100% were found to be achieved at 6 : 1 and 12 : 1 m : o , 4.5 and 7.3 catalyst wt% , 82 and 30 min reaction time , and 220 and 214 rpm mixing rate , in case of using biocatalyst prepared from mollusks and crabs shells , respectively . the laboratory experiment agrees well with the predicted response values 96% and 98% , respectively . that indicates the process optimization based on d - optimal design of experiments was capable and reliable to optimize biodiesel production from waste cooking oil using biocatalyst prepared from ms and cs . the lower required amounts of methanol and catalyst concentrations in case of transesterification using ms - cao relative to those using cs - cao might be attributed to the recorded higher basicity of ms - cao relative to that of cs - cao . reported the optimum conditions for production of biodiesel ( 98% ) from palm olein oil using cao prepared from waste mud crab shells by calcination above 700c for 2 h to be 0.5 : 1 m : o g : g , 5 wt% catalyst concentration , 2.5 h , 500 rpm , and 65c . reported the production of 83.1% biodiesel yield from sunflower oil using cao prepared from waste crabs shells calcinated at 900c for 2 h , using the following conditions : 6 : 1 m : o molar ratio , 3 wt% catalyst concentration , 4 h , 1000 rpm , and 60c . reported 92% biodiesel yield from palm olein oil using cao prepared from waste mollusks shells by calcination at 800c for 2 h to be 18 : 1 m : o molar ratio , 10 wt% catalyst concentration , 2 h , and 60c . the activity of the prepared ms - cao and cs - cao , using the obtained optimum conditions for production of biodiesel , was compared with that of commercially available most effective heterogeneous basic catalysts cao and novozym 435 . at optimum conditions of ms - cao , 6 : 1 m : o , 4.5% catalyst wt% , 82 min , 220 rpm , and 60c , the achieved biodiesel yield was 95 , 83 , and 40% using ms - cao , chemical cao , and novozym 435 , respectively . while applying the optimum operating conditions for cs - cao , 12 : 1 m : o , 7.3 catalyst wt% , 30 min , 214 rpm , and 60c , the biodiesel yield recorded 97 , 78 , and 35% using cs - cao , chemical cao , and novozym 435 , respectively . that indicates the higher efficiency of the produced ms - cao and cs - cao for production of biodiesel in comparison with the commercial chemical cao and the most widely used enzyme novozym 435 . the produced biodiesel using the obtained optimum transesterification conditions of each of the prepared biocatalysts was evaluated on the basis of its fuel properties compared to egyptian petro - diesel and international biodiesel standards as shown in table 6 . all the properties of the produced biodiesel are completely acceptable and meet most of the specifications . so it can be ranked as a realistic fuel and as an alternative to petro - diesel . this recommends the applicability of waste mollusks and crabs shells for production of cheap catalyst to produce biodiesel in a cost effective process . the iodine value of the produced biodiesel fuel bdf was in the range of 105108 mg i2/100 g oil . , methyl esters used as diesel fuel must have an iodine value less than 120 mg i2/100 g sample . the acid value measures the content of free acids in the sample , which has influence on fuel aging . the acid value of produced biodiesel was 0.6 and 0.7 mg koh / g , with average lowering of 80 and 76.67% from the used wco , indicating better transesterification efficiency , using cao prepared from ms than that prepared from cs . the tan of the produced biodiesel is relatively high , but within the astm d6751 biodiesel standards . reported that the biodiesel with high tan causes operational problems , such as corrosion and pump plugging , caused by corrosion and deposit formation . reported that density at 15c and kinematic viscosity at 40c are important properties , mainly in airless combustion systems because they influence the efficiency of atomization of the fuel , flow , and distribution . the density of the produced biodiesel fuel using ms - cao and cs - cao recorded 0.8914 and 0.9003 g / cm compared to that of petro - diesel 0.8421 g / cm . the produced biodiesel was characterized by higher specific gravity ( 0.8924 and 0.9011 , resp . ) and lower api value ( 27.06 and 25.53 , resp . ) compared to those of the egyptian petro - diesel sample ( 0.8428 and 36.39 , resp . ) . the viscosity of the produced biodiesel recorded 5.5 and 7.6 cst with a remarkable decrease from that of wco of 89 and 84.8% through transesterification using cao prepared from ms and cs , respectively . the better recorded tan , density , and viscosity of the produced biodiesel using ms - cao would indicate better transesterification efficiency , using cao prepared from ms than that prepared from cs . this might be attributed to the recorded higher basicity of ms - cao relative to that of cs - cao . the produced biodiesel has acceptable cold flow properties pour point pp 2c and cloud point 1c and is characterized by lower cv ( 38.1535.55 mj / kg , resp . ) relevant to that of the egyptian petro - diesel sample ( 45.49 mj / kg ) . the water content of the produced biodiesel is higher than that of the egyptian petro - diesel sample recording 312300 and 84 ppm , respectively . the produced biodiesel has three major advantages ; it is ultralow sulfur biofuel with sulfur content of 0.0030.006% , while petro - diesel has 0.2% sulfur . so it meets the aim of petroleum industry for ultralow sulfur diesel fuel and the biodiesel combustion will not produce large amount of sulfur oxides which lead to corrosion of the engine parts and environmental pollution . the produced biodiesel has a higher fp 148143c , compared to 63c for petro - diesel . so biodiesel is much less flammable fuel than petro - diesel and hence it is much safer in handling , storage , and transport . in addition , the viscosity of the produced biodiesel 5.57.6 cst is competitive to regular egyptian standards for petro - diesel 1.67 cst . hence , no hardware modifications are required for handling the produced bdf in the existing engine . the better qualifications of the produced biodiesel , using cao prepared from ms than those of biodiesel produced using cao prepared from cs , might be attributed to the higher basicity of ms - cao than that of cs - cao which would positively impact the transesterification reaction . in the viewpoint of preparation time , energy consumption , cost of catalyst , and high quality biodiesel yield , the cao catalyst prepared from waste mollusks and crabs shells can be recommended for biodiesel production from waste cooking oil collected from seafood restaurants . this would have a triple positive impact on environment , economic , and energy sectors . further work is undertaken now in epr - biotechnology laboratory concerning the kinetics and mechanism of the transesterification process , the reusability , and stability of the prepared biocatalysts compared to those of the chemical cao and novozym 435 , and their effects on the biodiesel yield , its purity , and quality .

this work illustrates a comparative study on the applicability of the basic heterogeneous calcium oxide catalyst prepared from waste mollusks and crabs shells ( ms and cs , resp . ) in the transesterification of waste cooking oil collected from seafood restaurants with methanol for production of biodiesel . response surface methodology rsm based on d - optimal deign of experiments was employed to study the significance and interactive effect of methanol to oil m : o molar ratio , catalyst concentration , reaction time , and mixing rate on biodiesel yield . second - order quadratic model equations were obtained describing the interrelationships between dependent and independent variables to maximize the response variable ( biodiesel yield ) and the validity of the predicted models were confirmed . the activity of the produced green catalysts was better than that of chemical cao and immobilized enzyme novozym 435 . fuel properties of the produced biodiesel were measured and compared with those of egyptian petro - diesel and international biodiesel standards . the biodiesel produced using ms - cao recorded higher quality than that produced using cs - cao . the overall biodiesel characteristics were acceptable , encouraging application of cao prepared from waste ms and cs for production of biodiesel as an efficient , environmentally friendly , sustainable , and low cost heterogeneous catalyst .

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

development of transesterification process for production of biodiesel as an alternative , green , and sustainable fuel has become an important issue due to diminishing fossil fuel reserves , rising crude oil price , and the stringent exhaust emission regulations . the aim of this work is to optimize biodiesel transesterification process using waste cooking oil and calcium oxide catalyst prepared from waste mollusks and crabs shells collected from seafood restaurants in an attempt to reach an effective process for practical , low cost industrial biodiesel production . the waste frying oil wfo , mollusks , and crabs shells ( ms and cs ) were collected from local seafood restaurants and prepared according to el - gendy et al . the activity of the prepared biocatalysts was compared with that of commercially available , most effective heterogeneous basic chemical catalyst cao and immobilized enzyme novozym 435 . based on d - optimal design of experiments , twenty runs of experiments have been conducted for three levels of four independent variables : methanol : oil m : o ( molar ratio ; a ) , catalyst concentration ( wt% ; b ) , reaction time ( min ; c ) , and mixing rate ( rpm ; d ) , to study their effect on the % yield of the produced biodiesel at constant temperature 60c . the model used in this study to estimate the response surface is the quadratic polynomial represented by the following equation : ( 1)y=o+i=1nixi+i=1n1j = i+1nijxixj+i=1niixi2 , where y is the biodiesel yield ( wt% ) , n is the number of factors , o is the intercept term , i , ij , and ii are the linear , interactive , and quadratic coefficients , respectively . the results were compared with the egyptian standards for petro - diesel and european and american standards of biodiesel ( en14214 and d-6751 , resp . ) according to n. b. singh and n. p. singh , the weight loss might be attributed to the decomposition of caco3 through the loss of carbon dioxide co2 and production of calcium oxide cao and also due to the possible removal of absorbed water molecules which occurs according to the following dissociation equation : ( 2)caco3(s)cao(s)+co2(g ) it was observed that during calcination , the ms and cs turned completely pale grey and white , respectively . narrow and high intense peaks of the calcinated ms and cs ( at temperature 800 and 700c , resp . ) this might affect the activity of the catalyst in the transesterification reaction , where the mechanism of cao in transesterification reactions would be started by dissociation of cao ( scheme 1 ) . in the viewpoint of preparation time , energy consumption , and cost of catalyst preparation , the temperatures of 800c and 700c were selected as perfect calcination temperature to prepare biocatalysts from natural waste mollusks and crabs shells , respectively . the bet surface area sbet of the prepared catalysts ( table 1 ) recorded 12.63 and 43.73 m / g for cao prepared from ms and cs , respectively . the bjh method was used for calculations of the pore size distributions ( table 1 ) and the prepared biocatalyst recorded total pore volume of 0.046 and 0.211 cm / g , respectively , and the pore size , distributed between 0.83 and 17.18 nm and between 1.04 and 15.58 nm , with average pore diameter of 0.94 and 1.84 nm , respectively , indicating that most of the pore size distributions are found in the microporous range . the n2 adsorption - desorption isotherms of the prepared cao from ms and cs are shown in figures 6(a ) and 6(b ) and they are of type ii isotherm ( based on iupac 's classification ) with a low slope in the middle region of the isotherm and a desorption curve almost overlapping with adsorption curve . the complete design matrix with experimental and predicted values of % yield of biodiesel using different cao catalyst prepared from mollusks and crabs shells is presented in table 3 . based on d - optimal design and experimental data , two second - order quadratic models for the applied catalysts have been predicted and can be given as follows : ( 3)y1=82.432.21a0.98b+3.12c+0.80d+4.48a2 + 1.89b27.51c22.56d22.66ab1.16ac+5.79ad+2.50bc+8.42bd+0.87cd,(4)y2=85.05 + 3.41a3.28b5.05c0.6d2.19a2 + 4.63b2 + 1.90c2 + 0.04d2 + 4.89ab0.56ac+0.32ad3.194bc5.45bd+2.53cd , where y1 and y2 are the biodiesel yields from transesterification processes using biocatalyst prepared from ms and cs , respectively . pareto charts , which are very useful in design of experiments , were used in this work to make it much easier to visualize the main and interaction effects of all factors to the response variable that is biodiesel yield ( figure 7 ) . in case of using biocatalyst prepared from ms in the transesterification process , reaction time has a high positive impact on the reaction yield , followed by the mixing rate . the interaction effects of m : o molar ratio and catalyst loading have a higher negative impact than m : o and reaction time on the biodiesel yield , while the interactive effect of catalyst loading and mixing rate expresses a very high positive impact on the biodiesel yield , coming after it the interaction between m : o ratio and mixing rate , catalyst loading , and reaction time , in a decreasing order , and the interactive effect of reaction time and mixing rate has a low positive impact on the reaction yield . in case of using biocatalyst prepared from cs in the transesterification process , only the m : o molar ratio has positive impact ; that is , increasing the m : o would increase the biodiesel yield . but , reaction time has a high negative impact followed by catalyst concentration and mixing rate , in a decreasing order ; that is , their increase would lower the biodiesel yield . the positive interactive impact on the biodiesel yield was expressed by m : o and catalyst loading , reaction time , and mixing rate and m : o and mixing rate with a decreasing order . while the interaction between catalyst loading and mixing rate , catalyst loading , and reaction time and m : o and reaction time has negative impact on the biodiesel yield in a decreasing order . confirmation of the adequacy of the regression models was reflected also by the good agreement between experimental and predicted values of response variables as shown in table 3 , where the actual biodiesel yield for mollusks and crabs shells biocatalyst ranged from 71.80 to 92% and from 74 to 97% and there corresponding predicted values are 71.80 and 91.75% and 74 and 96.50% , respectively . the relationship between predicted and experimental values of biodiesel yield for cao prepared from ms and cs is shown in figures 8(a ) and 8(b ) . the perturbation plots in figures 9(a ) and 9(b ) show the comparative effects of all independent variables on the biodiesel yield . in case of cao prepared from ms ( figure 9(a ) ) , the sharp curvature of the two factors , m : o ( a ) and reaction time ( c ) , shows that the response biodiesel yield was very sensitive to these two variables . the comparatively low curvature of catalyst concentration ( b ) and mixing rate ( d ) curves shows less sensitivity of biodiesel yield towards the change in these two factors . the curvatures also confirm the data illustrated in pareto chart ( figure 7(a ) ) , where the increase of m : o molar ratio decreases the biodiesel yield , while the increase in reaction time increases the biodiesel yield . the sensitivity of biodiesel yield towards the four variables can be ranked in the following decreasing order reaction time > m : o > catalyst concentration > mixing rate . in case of cao prepared from cs ( figure 9(b ) ) , all the parameters , within the studied range , have highly significant effect on the biodiesel yield except the mixing rate ( d ) which has relatively nonsignificant effect . the curvatures also confirm the data illustrated in pareto chart ( figure 7(b ) ) , where the increase of m : o molar ratio increases the biodiesel yield , while the increase of reaction time , catalyst loading , and mixing rate decrease the yield , with decrease of sensitivity ( reaction time > m : o catalyst concentration mixing rate ) . three - dimensional response surface graphical diagrams of the regression equations ( 3 ) and ( 4 ) were plotted ( figures 10 and 11 ) to understand the interactive relationship between the independent variables and % yield of biodiesel and determine the optimum conditions for maximum biodiesel production , using cao prepared from ms and cs , respectively . figure 10(a ) illustrates the effect of m : o molar ratio and the prepared ms - cao concentration on biodiesel production at a constant reaction time of 30 min and mixing rate of 200 rpm . the maximum yield 92% occurred within the range 6 : 16.5 : 1 m : o and 34.5 wt% catalyst concentration , but further increase in m : o and catalyst slightly decreased the biodiesel yield to 84% at 6 : 1 and 9 wt% , 89% at 12 : 1 and 6 wt% , and 81% at 12 : 1 and 9 wt% . but further increase in m : o would lower the biodiesel yield as it might have dilution effect on the catalyst concentration . figure 10(b ) represents the effect of m : o molar ratio and reaction time on biodiesel production at a constant catalyst concentration 3 wt% and mixing rate 200 rpm . it seems within the studied range of experiments , the increase of reaction time increased the biodiesel yield , recording 92% at 6 : 16.5 : 1 m : o and 7593 min molar ratio . but the biodiesel yield slightly decreased with further increment of m : o molar ratio and time reaching 86% at 12 : 1 m : o and 120 min . figure 10(c ) represents the effect of m : o and mixing rate on biodiesel production at a constant reaction time 30 min and ms - cao concentration 3 wt% . figure 10(e ) shows the interactive effect of mixing rate and reaction time , at a constant 6 : 1 m : o and ms - cao concentration 3 wt% . from the rsm analysis it can be concluded that the maximum biodiesel production 8992% using cao prepared from mollusks shells ms - cao can be achieved over a wide range of experimental parameters , at 6 : 16.5 m : o molar ratio , 34.5 wt% catalyst concentration , 7593 min reaction time , and mixing rate of 200250 rpm , at 60c . figure 11(a ) illustrates the effect of m : o molar ratio and cs - cao catalyst loading on biodiesel production at a constant reaction time of 120 min and mixing rate 400 rpm . at low m : o ratio the increase of catalyst concentration decreased the biodiesel yield , recording 79.6% yield at 6 : 1 m : o and 9 wt% catalyst concentration . but at high m : o ratio , the biodiesel yield increased with increase of catalyst loading , recording maximum biodiesel yield of approximately 97% at 10.5 : 112 : 1 m : o and 7.59% catalyst loading ( w : w ) . , a molar ratio of methanol to oil m : o higher than the stoichiometric ratio has generally been adopted for biodiesel production , as methanol would promote the formation of methoxy anions on the catalyst surface , leading to a shift in the equilibrium in the forward direction , thus increasing the biodiesel yield . figure 11(b ) represents the effect of m : o molar ratio and reaction time on biodiesel production at a constant catalyst concentration 3 wt% and mixing rate 400 rpm . figure 11(c ) represents the effect of m : o and mixing rate on biodiesel production at a constant reaction time 120 min and catalyst loading 3% . figure 11(d ) shows that there is a negative interactive effect between these two factors , where at low mixing rate , increasing the catalyst concentration increased the biodiesel yield , recording 80 and 97% at 200 rpm using 3 and 9 wt% catalyst , respectively . from the rsm analysis it can be concluded that the maximum biodiesel production using cao prepared from crabs shells cs - cao can be achieved at 10.5 : 112 : 1 m : o , 7.59% catalyst concentration , 3050 min reaction time , and mixing rate of 200300 rpm , at 60c . according to the software optimization step , the desired goal for each operational condition ( a m : o , b catalyst concentration , c reaction time , and d mixing rate ) was chosen within the studied range . the maximum predicted biodiesel yields 94 and 100% were found to be achieved at 6 : 1 and 12 : 1 m : o , 4.5 and 7.3 catalyst wt% , 82 and 30 min reaction time , and 220 and 214 rpm mixing rate , in case of using biocatalyst prepared from mollusks and crabs shells , respectively . that indicates the process optimization based on d - optimal design of experiments was capable and reliable to optimize biodiesel production from waste cooking oil using biocatalyst prepared from ms and cs . the lower required amounts of methanol and catalyst concentrations in case of transesterification using ms - cao relative to those using cs - cao might be attributed to the recorded higher basicity of ms - cao relative to that of cs - cao . reported the optimum conditions for production of biodiesel ( 98% ) from palm olein oil using cao prepared from waste mud crab shells by calcination above 700c for 2 h to be 0.5 : 1 m : o g : g , 5 wt% catalyst concentration , 2.5 h , 500 rpm , and 65c . reported the production of 83.1% biodiesel yield from sunflower oil using cao prepared from waste crabs shells calcinated at 900c for 2 h , using the following conditions : 6 : 1 m : o molar ratio , 3 wt% catalyst concentration , 4 h , 1000 rpm , and 60c . reported 92% biodiesel yield from palm olein oil using cao prepared from waste mollusks shells by calcination at 800c for 2 h to be 18 : 1 m : o molar ratio , 10 wt% catalyst concentration , 2 h , and 60c . the activity of the prepared ms - cao and cs - cao , using the obtained optimum conditions for production of biodiesel , was compared with that of commercially available most effective heterogeneous basic catalysts cao and novozym 435 . at optimum conditions of ms - cao , 6 : 1 m : o , 4.5% catalyst wt% , 82 min , 220 rpm , and 60c , the achieved biodiesel yield was 95 , 83 , and 40% using ms - cao , chemical cao , and novozym 435 , respectively . while applying the optimum operating conditions for cs - cao , 12 : 1 m : o , 7.3 catalyst wt% , 30 min , 214 rpm , and 60c , the biodiesel yield recorded 97 , 78 , and 35% using cs - cao , chemical cao , and novozym 435 , respectively . that indicates the higher efficiency of the produced ms - cao and cs - cao for production of biodiesel in comparison with the commercial chemical cao and the most widely used enzyme novozym 435 . the produced biodiesel using the obtained optimum transesterification conditions of each of the prepared biocatalysts was evaluated on the basis of its fuel properties compared to egyptian petro - diesel and international biodiesel standards as shown in table 6 . this recommends the applicability of waste mollusks and crabs shells for production of cheap catalyst to produce biodiesel in a cost effective process . the density of the produced biodiesel fuel using ms - cao and cs - cao recorded 0.8914 and 0.9003 g / cm compared to that of petro - diesel 0.8421 g / cm . compared to those of the egyptian petro - diesel sample ( 0.8428 and 36.39 , resp . ) the viscosity of the produced biodiesel recorded 5.5 and 7.6 cst with a remarkable decrease from that of wco of 89 and 84.8% through transesterification using cao prepared from ms and cs , respectively . the better recorded tan , density , and viscosity of the produced biodiesel using ms - cao would indicate better transesterification efficiency , using cao prepared from ms than that prepared from cs . this might be attributed to the recorded higher basicity of ms - cao relative to that of cs - cao . relevant to that of the egyptian petro - diesel sample ( 45.49 mj / kg ) . the water content of the produced biodiesel is higher than that of the egyptian petro - diesel sample recording 312300 and 84 ppm , respectively . in addition , the viscosity of the produced biodiesel 5.57.6 cst is competitive to regular egyptian standards for petro - diesel 1.67 cst . the better qualifications of the produced biodiesel , using cao prepared from ms than those of biodiesel produced using cao prepared from cs , might be attributed to the higher basicity of ms - cao than that of cs - cao which would positively impact the transesterification reaction . in the viewpoint of preparation time , energy consumption , cost of catalyst , and high quality biodiesel yield , the cao catalyst prepared from waste mollusks and crabs shells can be recommended for biodiesel production from waste cooking oil collected from seafood restaurants . further work is undertaken now in epr - biotechnology laboratory concerning the kinetics and mechanism of the transesterification process , the reusability , and stability of the prepared biocatalysts compared to those of the chemical cao and novozym 435 , and their effects on the biodiesel yield , its purity , and quality .

this study provides class ii evidence that rc - ifa accurately detects anti - tr as compared to conventional ifa . anonymized sera from 38 patients with pcd were sent under code to euroimmun by centers in the netherlands , france , spain , germany , and italy for confirmatory anti - dner testing . all sera had previously tested positive for anti - tr in the contributing laboratories , either by conventional immunohistochemistry or by tissue - based ifa using cryosections of rat cerebellum , according to published criteria for anti - tr staining . clinical information was obtained from forms filled out by the referring neurologists , telephone interviews , and review of the clinical records . the remaining 5 samples included 1 patient with pcd without a confirmed tumor , 1 with limbic encephalitis and hl , 1 with paraneoplastic encephalomyelitis and hl , 1 with hl only , and 1 with unknown diagnosis . five samples from patients with cerebellar ataxia were originally included as disease controls in a study evaluating the utility of antineuronal antibody testing in neuromyelitis optica ( s.j . and b.w . , unpublished data ) ; these samples were tested in an anonymized fashion . in addition , serum samples from 207 controls were tested . controls included 66 patients with pcd with a variety of tumors but no anti - tr antibodies : 25 with non - hl , 12 with hl , 7 with breast cancer , 6 with non - sclc , 3 with ovarian cancer , and 13 with other tumors . furthermore , 53 patients with hl without neurologic symptoms were enrolled , as were 46 patients with other diseases ( 20 rheumatoid arthritis , 10 systemic lupus erythematosus , 10 sjgren syndrome , 4 encephalitis , 2 neuropathy ) and 42 healthy individuals . the cdnas for human dner ( uniprot q8nft8 ) , its extracellular domain ( dner[ec ] : amino acids 1640 ) , cdr2 ( q01850 ) , or cdr2l ( q86x02 ) were obtained by performing pcr on a commercially available cdna ( biosource , solingen , germany , genbank bc035009 ; bc017503 ; bc047534 ) . the amplification products were ligated with ptriex-1 ( merck biosciences , darmstadt , germany ) . dner , dner(ec ) , cdr2/yo , or cdr2l ( each with c - terminal octahistidine - tags ) was expressed in human hek293 cells after exgen500-mediated transfection ( fermentas , st . leon - rot , germany ) according to the manufacturer 's instructions . to prepare substrates for the ifa , hek293 cells were grown on sterile cover glasses , transfected , and allowed to express the recombinant protein for 48 hours . cover glasses were washed with phosphate - buffered saline ( pbs ) , fixed with either acetone or 1% ( w / v ) formalin for 10 minutes at room temperature , air dried , cut into millimeter - sized fragments ( biochips ) , and used as substrates in ifa ( see below ) . for selected experiments , cells were transfected in standard t - flasks and the cell culture medium was harvested after 5 days . the medium was centrifuged at 20,000 g at 4c for 20 minutes , and the resulting supernatant was stored in aliquots at 80c until further use . ifa was conducted using slides with a mosaic of 9 biochip substrates : hek293-dner , hek293-cdr2/yo , hek293-cdr2l , and mock - transfected hek293 , each acetone- and formalin - fixed , as well as 4 m unfixed cryosections of monkey cerebellum ( figure 1a ) . each biochip mosaic was incubated with 70 l of pbs - diluted sample at room temperature for 30 minutes , washed with pbs - tween , and immersed in pbs - tween for 5 minutes . in the second step , fluorescein isothiocyanate labeled goat anti - human igg ( euroimmun , lbeck , germany ) was applied and incubated at room temperature for 30 minutes . slides were washed again with a flush of pbs - tween and then immersed in pbs - tween for 5 minutes . slides were embedded in pbs - buffered dabco containing glycerol ( approximately 20 l per field ) and examined by fluorescence microscopy with 460490 nm led excitation ( eurostar , euroimmun ) . results were evaluated by 2 independent observers with more than 10 years of professional experience . samples were classified according to the fluorescence intensity of the transfected cells in direct comparison with cdr2/yo , crd2l , and mock - transfected cells and control samples . a specific fluorescence of the transfected cells at a dilution of 1:10 or higher was considered positive . a staining of monkey cerebellum was scored as having an anti - tr pattern when a distinct punctate immunoreactivity was detected in purkinje cell dendritic tree and soma , as previously described . endpoint titers refer to the last dilution showing visible fluorescence . in competitive inhibition experiments , recombinant antigens were mixed with diluted serum sample 30 minutes prior to the ifa , as described in stcker et al . each reaction field contained 9 biochips with hek293 cells expressing delta / notch - like epidermal growth factor - related receptor ( dner ) , cdr2/yo , or cdr2l , and mock - transfected control cells fixed with either acetone or formalin as well as cryosections of monkey cerebellum . ( b ) binding of serum igg of a patient with paraneoplastic cerebellar degeneration and hodgkin lymphoma ( a c ) to purkinje cell somata and dendritic trees ( anti - tr ) pattern on monkey cerebellum ( a , d ) and to dner - expressing cells ( b , e ) but not control cells used as internal control ( c , f ) . none of these reactivities were seen with serum from controls ( d f ) . ( c ) preincubation with a lysate of hek293 expressing dner ( a , b ) completely abolished the anti - tr / dner fluorescence ( a , c : monkey cerebellum ; b , d : dner - expressing cells ) , but it was not abolished following incubation with a similar lysate from wild - type hek293 ( c , d ) . the extracellular domain of dner was expressed in hek293 cells essentially as described for the full - length protein . the recombinant protein was purified by immobilized metal affinity chromatography , as described for the extracellular domains of desmoglein 1 and 3 . the fractionation of dner(ec ) was monitored by western blot using a monoclonal antibody against hexahistidine and alkaline phosphatase anti - mouse igg ( jackson immunoresearch laboratories , west grove , pa ) , as in reference 14 . the purified antigen was characterized by sds - page using the nupage system ( invitrogen , darmstadt , germany ) according to the manufacturer 's instructions , western blot , and maldi - tof fingerprinting , and maldi - tof tandem mass spectrometry after sds - page and tryptic cleavage . all reagents were obtained from merck or sigma - aldrich , heidelberg , germany , unless otherwise specified . anonymized sera from 38 patients with pcd were sent under code to euroimmun by centers in the netherlands , france , spain , germany , and italy for confirmatory anti - dner testing . all sera had previously tested positive for anti - tr in the contributing laboratories , either by conventional immunohistochemistry or by tissue - based ifa using cryosections of rat cerebellum , according to published criteria for anti - tr staining . clinical information was obtained from forms filled out by the referring neurologists , telephone interviews , and review of the clinical records . the remaining 5 samples included 1 patient with pcd without a confirmed tumor , 1 with limbic encephalitis and hl , 1 with paraneoplastic encephalomyelitis and hl , 1 with hl only , and 1 with unknown diagnosis . five samples from patients with cerebellar ataxia were originally included as disease controls in a study evaluating the utility of antineuronal antibody testing in neuromyelitis optica ( s.j . and b.w . , unpublished data ) ; these samples were tested in an anonymized fashion . in addition , serum samples from 207 controls were tested . controls included 66 patients with pcd with a variety of tumors but no anti - tr antibodies : 25 with non - hl , 12 with hl , 7 with breast cancer , 6 with non - sclc , 3 with ovarian cancer , and 13 with other tumors . furthermore , 53 patients with hl without neurologic symptoms were enrolled , as were 46 patients with other diseases ( 20 rheumatoid arthritis , 10 systemic lupus erythematosus , 10 sjgren syndrome , 4 encephalitis , 2 neuropathy ) and 42 healthy individuals . the cdnas for human dner ( uniprot q8nft8 ) , its extracellular domain ( dner[ec ] : amino acids 1640 ) , cdr2 ( q01850 ) , or cdr2l ( q86x02 ) were obtained by performing pcr on a commercially available cdna ( biosource , solingen , germany , genbank bc035009 ; bc017503 ; bc047534 ) . the amplification products were ligated with ptriex-1 ( merck biosciences , darmstadt , germany ) . dner , dner(ec ) , cdr2/yo , or cdr2l ( each with c - terminal octahistidine - tags ) was expressed in human hek293 cells after exgen500-mediated transfection ( fermentas , st . leon - rot , germany ) according to the manufacturer 's instructions . to prepare substrates for the ifa , hek293 cells were grown on sterile cover glasses , transfected , and allowed to express the recombinant protein for 48 hours . cover glasses were washed with phosphate - buffered saline ( pbs ) , fixed with either acetone or 1% ( w / v ) formalin for 10 minutes at room temperature , air dried , cut into millimeter - sized fragments ( biochips ) , and used as substrates in ifa ( see below ) . for selected experiments , cells were transfected in standard t - flasks and the cell culture medium was harvested after 5 days . the medium was centrifuged at 20,000 g at 4c for 20 minutes , and the resulting supernatant was stored in aliquots at 80c until further use . ifa was conducted using slides with a mosaic of 9 biochip substrates : hek293-dner , hek293-cdr2/yo , hek293-cdr2l , and mock - transfected hek293 , each acetone- and formalin - fixed , as well as 4 m unfixed cryosections of monkey cerebellum ( figure 1a ) . each biochip mosaic was incubated with 70 l of pbs - diluted sample at room temperature for 30 minutes , washed with pbs - tween , and immersed in pbs - tween for 5 minutes . in the second step , fluorescein isothiocyanate labeled goat anti - human igg ( euroimmun , lbeck , germany ) was applied and incubated at room temperature for 30 minutes . slides were washed again with a flush of pbs - tween and then immersed in pbs - tween for 5 minutes . slides were embedded in pbs - buffered dabco containing glycerol ( approximately 20 l per field ) and examined by fluorescence microscopy with 460490 nm led excitation ( eurostar , euroimmun ) . results were evaluated by 2 independent observers with more than 10 years of professional experience . samples were classified according to the fluorescence intensity of the transfected cells in direct comparison with cdr2/yo , crd2l , and mock - transfected cells and control samples . a specific fluorescence of the transfected cells at a dilution of 1:10 or higher was considered positive . a staining of monkey cerebellum was scored as having an anti - tr pattern when a distinct punctate immunoreactivity was detected in purkinje cell dendritic tree and soma , as previously described . endpoint titers refer to the last dilution showing visible fluorescence . in competitive inhibition experiments , recombinant antigens were mixed with diluted serum sample 30 minutes prior to the ifa , as described in stcker et al . each reaction field contained 9 biochips with hek293 cells expressing delta / notch - like epidermal growth factor - related receptor ( dner ) , cdr2/yo , or cdr2l , and mock - transfected control cells fixed with either acetone or formalin as well as cryosections of monkey cerebellum . ( b ) binding of serum igg of a patient with paraneoplastic cerebellar degeneration and hodgkin lymphoma ( a c ) to purkinje cell somata and dendritic trees ( anti - tr ) pattern on monkey cerebellum ( a , d ) and to dner - expressing cells ( b , e ) but not control cells used as internal control ( c , f ) . none of these reactivities were seen with serum from controls ( d f ) . ( c ) preincubation with a lysate of hek293 expressing dner ( a , b ) completely abolished the anti - tr / dner fluorescence ( a , c : monkey cerebellum ; b , d : dner - expressing cells ) , but it was not abolished following incubation with a similar lysate from wild - type hek293 ( c , d ) . the extracellular domain of dner was expressed in hek293 cells essentially as described for the full - length protein . the recombinant protein was purified by immobilized metal affinity chromatography , as described for the extracellular domains of desmoglein 1 and 3 . the fractionation of dner(ec ) was monitored by western blot using a monoclonal antibody against hexahistidine and alkaline phosphatase anti - mouse igg ( jackson immunoresearch laboratories , west grove , pa ) , as in reference 14 . the purified antigen was characterized by sds - page using the nupage system ( invitrogen , darmstadt , germany ) according to the manufacturer 's instructions , western blot , and maldi - tof fingerprinting , and maldi - tof tandem mass spectrometry after sds - page and tryptic cleavage . all reagents were obtained from merck or sigma - aldrich , heidelberg , germany , unless otherwise specified . prior to the testing of all sera , fixation experiments were conducted with representative , decoded , anti - tr positive control sera from erasmus mc previously shown to bind to dner . these sera reacted with both acetone- and formalin - fixed dner - transfected hek293 cells . however , formalin - fixed cells showed lower background staining , which allowed for more reliable evaluation of low - titer sera . accordingly , only data obtained with the formalin - fixed dner substrate were included in the study . in contrast to dner , the control substrates cdr2/yo and cdr2l were expressed intracellularly and , as expected , were only detected by anti - yo positive control sera after acetone fixation but not after treatment with nonpermeabilizing formalin . accordingly , acetone - fixed cdr2/yo and cdr2l cells were used for the study . storing experiments revealed that dust - free storing of desiccated slides containing the dner , cdr2/yo , and cdr2l substrates and mock - transfected control cells at 20c for up to 12 months did not affect the outcome of individual tests . all 38 sera with anti - tr antibodies ( as previously identified by the senders according to conventional or immunofluorescence - based immunohistochemistry ) were positive for anti - dner in the dner rc - ifa but did not react with the control substrates ( figure 1b ) . moreover , all 6 anti - yo positive samples reacted with cdr2/yo- and cdr2l - expressing cells but not with those expressing dner or with the mock - transfected hek293 cells . of the other 201 control sera , this corresponds to a sensitivity of 100% ( 95% confidence interval [ ci ] 92.8%100% ) and a specificity of 100% ( 95% ci 98.7%100% ) of the recombinant cell - based assay for anti - dner . when testing the same sera using the commercial non - recombinant ifa using monkey cerebellar tissue sections , only 34 of the 38 sera produced the typical anti - tr pattern on tissue ( table ) , corresponding to a sensitivity of 89.5% ( 95% ci 80.6%89.5% ) . all control sera were also negative for anti - tr in the monkey tissue based ifa ( specificity 100% , 95% ci 98.7%100% ) . one control serum showed a weak cytoplasmic purkinje cell staining pattern but no anti - tr specific fluorescence at a 1:10 dilution . correlation of indirect immunofluorescence using cryosections of monkey cerebellum or hek293 expressing dner endpoint titers were determined in all 38 anti - dner positive sera on the recombinant cells and monkey cerebellum . all but 3 sera showed higher titers when the recombinant cells were used in comparison to monkey tissue ( p < 0.001 , fisher exact test ) ( figure 2 ) , which yielded low - titer or negative results in 20 of 38 cases . full reproducibility was demonstrated with representative samples on the basis of 3 subsequent slide lots , and all criteria for the ce - labeling of in vitro diagnostics in norm iso 13485 were fulfilled . each dot represents the endpoint titer of a single serum ( n = 38 ) . dner = delta / notch - like epidermal growth factor - related receptor . in neutralization experiments , lysates from hek293-dner and purified dner(ec ) abolished anti - dner reactivities of human sera with recombinant cells and the corresponding anti - tr reactivity on monkey cerebellum in a dose - dependent manner ( figure 1c ) . in contrast , similar fractions of hek293 cells expressing cdr2/yo , mock - transfected hek293 , and the purified extracellular domain of desmoglein 3 did not affect anti - dner or the anti - tr reactivities . prior to the testing of all sera , fixation experiments were conducted with representative , decoded , anti - tr positive control sera from erasmus mc previously shown to bind to dner . these sera reacted with both acetone- and formalin - fixed dner - transfected hek293 cells . however , formalin - fixed cells showed lower background staining , which allowed for more reliable evaluation of low - titer sera . accordingly , only data obtained with the formalin - fixed dner substrate were included in the study . in contrast to dner , the control substrates cdr2/yo and cdr2l were expressed intracellularly and , as expected , were only detected by anti - yo positive control sera after acetone fixation but not after treatment with nonpermeabilizing formalin . accordingly , acetone - fixed cdr2/yo and cdr2l cells were used for the study . storing experiments revealed that dust - free storing of desiccated slides containing the dner , cdr2/yo , and cdr2l substrates and mock - transfected control cells at 20c for up to 12 months did not affect the outcome of individual tests . all 38 sera with anti - tr antibodies ( as previously identified by the senders according to conventional or immunofluorescence - based immunohistochemistry ) were positive for anti - dner in the dner rc - ifa but did not react with the control substrates ( figure 1b ) . moreover , all 6 anti - yo positive samples reacted with cdr2/yo- and cdr2l - expressing cells but not with those expressing dner or with the mock - transfected hek293 cells . of the other 201 control sera , this corresponds to a sensitivity of 100% ( 95% confidence interval [ ci ] 92.8%100% ) and a specificity of 100% ( 95% ci 98.7%100% ) of the recombinant cell - based assay for anti - dner . when testing the same sera using the commercial non - recombinant ifa using monkey cerebellar tissue sections , only 34 of the 38 sera produced the typical anti - tr pattern on tissue ( table ) , corresponding to a sensitivity of 89.5% ( 95% ci 80.6%89.5% ) . all control sera were also negative for anti - tr in the monkey tissue based ifa ( specificity 100% , 95% ci 98.7%100% ) . one control serum showed a weak cytoplasmic purkinje cell staining pattern but no anti - tr specific fluorescence at a 1:10 dilution . correlation of indirect immunofluorescence using cryosections of monkey cerebellum or hek293 expressing dner endpoint titers were determined in all 38 anti - dner positive sera on the recombinant cells and monkey cerebellum . all but 3 sera showed higher titers when the recombinant cells were used in comparison to monkey tissue ( p < 0.001 , fisher exact test ) ( figure 2 ) , which yielded low - titer or negative results in 20 of 38 cases . full reproducibility was demonstrated with representative samples on the basis of 3 subsequent slide lots , and all criteria for the ce - labeling of in vitro diagnostics in norm iso 13485 were fulfilled . each dot represents the endpoint titer of a single serum ( n = 38 ) . dner = delta / notch - like epidermal growth factor - related receptor . in neutralization experiments , lysates from hek293-dner and purified dner(ec ) abolished anti - dner reactivities of human sera with recombinant cells and the corresponding anti - tr reactivity on monkey cerebellum in a dose - dependent manner ( figure 1c ) . in contrast , similar fractions of hek293 cells expressing cdr2/yo , mock - transfected hek293 , and the purified extracellular domain of desmoglein 3 did not affect anti - dner or the anti - tr reactivities . we report on the first well - standardized rc - ifa , which was developed to determine autoantibodies against tr / dner and then evaluated using samples collected in several european centers . in a blinded study of 245 sera , including a large panel of 38 sera with anti - tr autoantibodies , the new assay yielded 100% sensitivity and specificity with respect to the serologic precharacterization that was performed at the contributing centers . in comparison to the reference procedure , which had to use the elaborate epitope blocking step as a confirmatory measure in many cases our results are in agreement with the initial report on the identification of dner as the target antigen of anti - tr antibodies and a subsequent independent confirmation study that used in - house rc - ifas and significantly smaller sample cohorts . rca - ifa has already been shown to be a suitable method to sensitively and specifically detect other anti - neural surface autoantibodies such as anti - nmda receptor or anti - aquaporin-4 . likewise , although intracellularly expressed , hek293 cells expressing cdr2/yo or cdr2l are apparently competent substrates for detecting autoantibodies against cdr2/yo . all in all , recombinant cell substrates complement or even improve indirect immunofluorescence for the detection of paraneoplastic anti - neural autoantibodies for which cryosections of mammalian cerebellum are classically used . by specific neutralization of the autoantibody reactions with recombinant full - length dner expressed in hek293 , on the one hand , and the anti - tr pattern on cerebellum using dner(ec ) , we confirmed the earlier report on the exclusive presence of relevant epitopes in the extracellular domain of dner . we also demonstrated that the recombinant dner(ec ) construct is a fully competent antigen that may help in the development of a microplate elisa or dot / lineblot for integration with other paraneoplastic neurologic syndrome markers such as hu , yo , ri , and cv2 . as a rule , adult patients with subacute - onset cerebellar ataxia should be tested for anti - dner and the other well - characterized onconeural antibodies . first , an immunohistochemical screening can be performed using cryosections of cerebellum and then be confirmed in anti - dner rc - ifa when the anti - tr staining pattern is observed or suspected . alternatively , direct screening can be performed using the anti - dner rc - ifa ; if the results are equivocal or the clinical data are highly suggestive , an additional immunohistochemistry could be performed as well . it is most preferable , however , to determine the autoantibodies by using a multiparametric approach using mosaics of tissue and recombinant cells , similar to the approach used in this study . symptomatology - driven sets of test substrates can be assembled , and a broad spectrum of anti - neural autoantibodies can be tested for simultaneously . , w. schlumberger , w. stcker , and p.a.e.s.s . were involved in the conception and organization of the research project and in the writing of the manuscript . m. van coevorden - hameete , v. rogemond , j. honnorat , and l. sabater report no disclosure . f. graus is on the editorial board for lancet neurology and has received research support from fondo investigaciones santitarias . b. wildemann has received travel funding and speaker honoraria from bayer healthcare , biogen idec , merck serono , teva , and sanofi - aventis / genzyme and has received research support from biogen idec , merck serono , teva , novartis , biotest , dietmar - hopp - foundation , and german ministry for education and research . w. stcker holds patents for alibrationsstreifen fr einen immunoblot , vorrichtung und verfahren zur automatischen fokussierung fr die mikroskopie schwach leuchtender substrate , verfahren zum spezifischen und quantitativen nachweis von antikrpern in einer probe , zum nachweis von anti - nmda - rezeptor - autoantikrpern zum einsatz in diagnoseverfahren , verfahren und vorrichtung zur bearbeitung von proben biologischen materials , diagnosekit sowie verfahren zur untersuchung einer menschlichen patientenprobe auf das vorhandensein neuromyelitis - optica - spezifischen antikrpern , testkit fr die labordiagnostik , vorrichtung zur steuerung einer pipettiervorrichtung , verfahren und analysevorrichtung zur mikroskopischen untersuchung eines gewebeschnittes oder eines zellausstriches , and verfahren sowie vorrichtung zur inkubation von patentientenproben ; is the head of the board for euroimmun medizinische labordiagnostika ag ; is employed by klinisch - immunologisches ; and holds stock in euroimmun medizinische labordiagnostika ag .

objective : to determine sensitivity and specificity of a standardized recombinant cell - based indirect immunofluorescence assay ( rc - ifa ) for anti - tr antibodies in comparison to a reference procedure.methods:delta/notch-like epidermal growth factor - related receptor ( dner ) was expressed in hek293 and used as a substrate for rc - ifa . hek293 control cells expressing cdr2/yo and cdr2l as well as mock - transfected hek293 cells were used as controls . serum samples from 38 patients with anti - tr antibodies ( 33 with paraneoplastic cerebellar degeneration [ pcd ] and hodgkin lymphoma ) , 66 patients with anti - tr negative pcd , 53 patients with hodgkin lymphoma without neurologic symptoms , 40 patients with rheumatic diseases , and 42 healthy blood donors were tested for anti - dner reactivity in the rc - ifa . in addition , rc - ifa results were compared to those from a commercial tissue - based ifa using monkey cerebellum.results:using the rc - ifa , anti - dner was detected in all anti - tr positive patients but in none of the controls ( sensitivity 100% , 95% confidence interval [ ci ] 92.8%100% ; specificity 100% , 95% ci 98.7%100% ) . in comparison , anti - tr was not detected in 4 samples with low - titer autoantibodies using the commercial tissue - based assay . preadsorption of sera with either recombinant full - length dner or its extracellular domain selectively abolished anti - tr reactivity.conclusion:anti-tr antibodies bind to the extracellular domain of dner and can be detected by rc - ifa using hek293 cells expressing the recombinant receptor . the new method performs better than a frequently used commercial tissue - based indirect immunofluorescence assay ( ifa ) in samples with low - titer antibodies.classification of evidence : this study provides class ii evidence that rc - ifa accurately detects anti - tr as compared to conventional ifa .

Classification of evidence: METHODS Human sera. Standard protocol approvals, registrations, and patient consents. Cloning and expression of DNER in HEK293. Detection of anti-DNER antibodies by indirect immunofluorescence. Purification of recombinant human DNER(ec) from HEK293 cells. Miscellaneous. RESULTS Determination of anti-DNER by indirect immunofluorescence. DISCUSSION AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

this study provides class ii evidence that rc - ifa accurately detects anti - tr as compared to conventional ifa . anonymized sera from 38 patients with pcd were sent under code to euroimmun by centers in the netherlands , france , spain , germany , and italy for confirmatory anti - dner testing . all sera had previously tested positive for anti - tr in the contributing laboratories , either by conventional immunohistochemistry or by tissue - based ifa using cryosections of rat cerebellum , according to published criteria for anti - tr staining . in addition , serum samples from 207 controls were tested . controls included 66 patients with pcd with a variety of tumors but no anti - tr antibodies : 25 with non - hl , 12 with hl , 7 with breast cancer , 6 with non - sclc , 3 with ovarian cancer , and 13 with other tumors . furthermore , 53 patients with hl without neurologic symptoms were enrolled , as were 46 patients with other diseases ( 20 rheumatoid arthritis , 10 systemic lupus erythematosus , 10 sjgren syndrome , 4 encephalitis , 2 neuropathy ) and 42 healthy individuals . the cdnas for human dner ( uniprot q8nft8 ) , its extracellular domain ( dner[ec ] : amino acids 1640 ) , cdr2 ( q01850 ) , or cdr2l ( q86x02 ) were obtained by performing pcr on a commercially available cdna ( biosource , solingen , germany , genbank bc035009 ; bc017503 ; bc047534 ) . dner , dner(ec ) , cdr2/yo , or cdr2l ( each with c - terminal octahistidine - tags ) was expressed in human hek293 cells after exgen500-mediated transfection ( fermentas , st . to prepare substrates for the ifa , hek293 cells were grown on sterile cover glasses , transfected , and allowed to express the recombinant protein for 48 hours . cover glasses were washed with phosphate - buffered saline ( pbs ) , fixed with either acetone or 1% ( w / v ) formalin for 10 minutes at room temperature , air dried , cut into millimeter - sized fragments ( biochips ) , and used as substrates in ifa ( see below ) . ifa was conducted using slides with a mosaic of 9 biochip substrates : hek293-dner , hek293-cdr2/yo , hek293-cdr2l , and mock - transfected hek293 , each acetone- and formalin - fixed , as well as 4 m unfixed cryosections of monkey cerebellum ( figure 1a ) . in the second step , fluorescein isothiocyanate labeled goat anti - human igg ( euroimmun , lbeck , germany ) was applied and incubated at room temperature for 30 minutes . samples were classified according to the fluorescence intensity of the transfected cells in direct comparison with cdr2/yo , crd2l , and mock - transfected cells and control samples . a staining of monkey cerebellum was scored as having an anti - tr pattern when a distinct punctate immunoreactivity was detected in purkinje cell dendritic tree and soma , as previously described . each reaction field contained 9 biochips with hek293 cells expressing delta / notch - like epidermal growth factor - related receptor ( dner ) , cdr2/yo , or cdr2l , and mock - transfected control cells fixed with either acetone or formalin as well as cryosections of monkey cerebellum . ( b ) binding of serum igg of a patient with paraneoplastic cerebellar degeneration and hodgkin lymphoma ( a c ) to purkinje cell somata and dendritic trees ( anti - tr ) pattern on monkey cerebellum ( a , d ) and to dner - expressing cells ( b , e ) but not control cells used as internal control ( c , f ) . ( c ) preincubation with a lysate of hek293 expressing dner ( a , b ) completely abolished the anti - tr / dner fluorescence ( a , c : monkey cerebellum ; b , d : dner - expressing cells ) , but it was not abolished following incubation with a similar lysate from wild - type hek293 ( c , d ) . the extracellular domain of dner was expressed in hek293 cells essentially as described for the full - length protein . the fractionation of dner(ec ) was monitored by western blot using a monoclonal antibody against hexahistidine and alkaline phosphatase anti - mouse igg ( jackson immunoresearch laboratories , west grove , pa ) , as in reference 14 . the purified antigen was characterized by sds - page using the nupage system ( invitrogen , darmstadt , germany ) according to the manufacturer 's instructions , western blot , and maldi - tof fingerprinting , and maldi - tof tandem mass spectrometry after sds - page and tryptic cleavage . anonymized sera from 38 patients with pcd were sent under code to euroimmun by centers in the netherlands , france , spain , germany , and italy for confirmatory anti - dner testing . all sera had previously tested positive for anti - tr in the contributing laboratories , either by conventional immunohistochemistry or by tissue - based ifa using cryosections of rat cerebellum , according to published criteria for anti - tr staining . in addition , serum samples from 207 controls were tested . controls included 66 patients with pcd with a variety of tumors but no anti - tr antibodies : 25 with non - hl , 12 with hl , 7 with breast cancer , 6 with non - sclc , 3 with ovarian cancer , and 13 with other tumors . furthermore , 53 patients with hl without neurologic symptoms were enrolled , as were 46 patients with other diseases ( 20 rheumatoid arthritis , 10 systemic lupus erythematosus , 10 sjgren syndrome , 4 encephalitis , 2 neuropathy ) and 42 healthy individuals . dner , dner(ec ) , cdr2/yo , or cdr2l ( each with c - terminal octahistidine - tags ) was expressed in human hek293 cells after exgen500-mediated transfection ( fermentas , st . to prepare substrates for the ifa , hek293 cells were grown on sterile cover glasses , transfected , and allowed to express the recombinant protein for 48 hours . cover glasses were washed with phosphate - buffered saline ( pbs ) , fixed with either acetone or 1% ( w / v ) formalin for 10 minutes at room temperature , air dried , cut into millimeter - sized fragments ( biochips ) , and used as substrates in ifa ( see below ) . ifa was conducted using slides with a mosaic of 9 biochip substrates : hek293-dner , hek293-cdr2/yo , hek293-cdr2l , and mock - transfected hek293 , each acetone- and formalin - fixed , as well as 4 m unfixed cryosections of monkey cerebellum ( figure 1a ) . in the second step , fluorescein isothiocyanate labeled goat anti - human igg ( euroimmun , lbeck , germany ) was applied and incubated at room temperature for 30 minutes . samples were classified according to the fluorescence intensity of the transfected cells in direct comparison with cdr2/yo , crd2l , and mock - transfected cells and control samples . a staining of monkey cerebellum was scored as having an anti - tr pattern when a distinct punctate immunoreactivity was detected in purkinje cell dendritic tree and soma , as previously described . each reaction field contained 9 biochips with hek293 cells expressing delta / notch - like epidermal growth factor - related receptor ( dner ) , cdr2/yo , or cdr2l , and mock - transfected control cells fixed with either acetone or formalin as well as cryosections of monkey cerebellum . ( b ) binding of serum igg of a patient with paraneoplastic cerebellar degeneration and hodgkin lymphoma ( a c ) to purkinje cell somata and dendritic trees ( anti - tr ) pattern on monkey cerebellum ( a , d ) and to dner - expressing cells ( b , e ) but not control cells used as internal control ( c , f ) . ( c ) preincubation with a lysate of hek293 expressing dner ( a , b ) completely abolished the anti - tr / dner fluorescence ( a , c : monkey cerebellum ; b , d : dner - expressing cells ) , but it was not abolished following incubation with a similar lysate from wild - type hek293 ( c , d ) . the extracellular domain of dner was expressed in hek293 cells essentially as described for the full - length protein . the fractionation of dner(ec ) was monitored by western blot using a monoclonal antibody against hexahistidine and alkaline phosphatase anti - mouse igg ( jackson immunoresearch laboratories , west grove , pa ) , as in reference 14 . the purified antigen was characterized by sds - page using the nupage system ( invitrogen , darmstadt , germany ) according to the manufacturer 's instructions , western blot , and maldi - tof fingerprinting , and maldi - tof tandem mass spectrometry after sds - page and tryptic cleavage . prior to the testing of all sera , fixation experiments were conducted with representative , decoded , anti - tr positive control sera from erasmus mc previously shown to bind to dner . these sera reacted with both acetone- and formalin - fixed dner - transfected hek293 cells . in contrast to dner , the control substrates cdr2/yo and cdr2l were expressed intracellularly and , as expected , were only detected by anti - yo positive control sera after acetone fixation but not after treatment with nonpermeabilizing formalin . accordingly , acetone - fixed cdr2/yo and cdr2l cells were used for the study . storing experiments revealed that dust - free storing of desiccated slides containing the dner , cdr2/yo , and cdr2l substrates and mock - transfected control cells at 20c for up to 12 months did not affect the outcome of individual tests . all 38 sera with anti - tr antibodies ( as previously identified by the senders according to conventional or immunofluorescence - based immunohistochemistry ) were positive for anti - dner in the dner rc - ifa but did not react with the control substrates ( figure 1b ) . moreover , all 6 anti - yo positive samples reacted with cdr2/yo- and cdr2l - expressing cells but not with those expressing dner or with the mock - transfected hek293 cells . of the other 201 control sera , this corresponds to a sensitivity of 100% ( 95% confidence interval [ ci ] 92.8%100% ) and a specificity of 100% ( 95% ci 98.7%100% ) of the recombinant cell - based assay for anti - dner . when testing the same sera using the commercial non - recombinant ifa using monkey cerebellar tissue sections , only 34 of the 38 sera produced the typical anti - tr pattern on tissue ( table ) , corresponding to a sensitivity of 89.5% ( 95% ci 80.6%89.5% ) . all control sera were also negative for anti - tr in the monkey tissue based ifa ( specificity 100% , 95% ci 98.7%100% ) . correlation of indirect immunofluorescence using cryosections of monkey cerebellum or hek293 expressing dner endpoint titers were determined in all 38 anti - dner positive sera on the recombinant cells and monkey cerebellum . all but 3 sera showed higher titers when the recombinant cells were used in comparison to monkey tissue ( p < 0.001 , fisher exact test ) ( figure 2 ) , which yielded low - titer or negative results in 20 of 38 cases . dner = delta / notch - like epidermal growth factor - related receptor . in neutralization experiments , lysates from hek293-dner and purified dner(ec ) abolished anti - dner reactivities of human sera with recombinant cells and the corresponding anti - tr reactivity on monkey cerebellum in a dose - dependent manner ( figure 1c ) . in contrast , similar fractions of hek293 cells expressing cdr2/yo , mock - transfected hek293 , and the purified extracellular domain of desmoglein 3 did not affect anti - dner or the anti - tr reactivities . prior to the testing of all sera , fixation experiments were conducted with representative , decoded , anti - tr positive control sera from erasmus mc previously shown to bind to dner . these sera reacted with both acetone- and formalin - fixed dner - transfected hek293 cells . in contrast to dner , the control substrates cdr2/yo and cdr2l were expressed intracellularly and , as expected , were only detected by anti - yo positive control sera after acetone fixation but not after treatment with nonpermeabilizing formalin . accordingly , acetone - fixed cdr2/yo and cdr2l cells were used for the study . storing experiments revealed that dust - free storing of desiccated slides containing the dner , cdr2/yo , and cdr2l substrates and mock - transfected control cells at 20c for up to 12 months did not affect the outcome of individual tests . all 38 sera with anti - tr antibodies ( as previously identified by the senders according to conventional or immunofluorescence - based immunohistochemistry ) were positive for anti - dner in the dner rc - ifa but did not react with the control substrates ( figure 1b ) . moreover , all 6 anti - yo positive samples reacted with cdr2/yo- and cdr2l - expressing cells but not with those expressing dner or with the mock - transfected hek293 cells . of the other 201 control sera , this corresponds to a sensitivity of 100% ( 95% confidence interval [ ci ] 92.8%100% ) and a specificity of 100% ( 95% ci 98.7%100% ) of the recombinant cell - based assay for anti - dner . when testing the same sera using the commercial non - recombinant ifa using monkey cerebellar tissue sections , only 34 of the 38 sera produced the typical anti - tr pattern on tissue ( table ) , corresponding to a sensitivity of 89.5% ( 95% ci 80.6%89.5% ) . all control sera were also negative for anti - tr in the monkey tissue based ifa ( specificity 100% , 95% ci 98.7%100% ) . correlation of indirect immunofluorescence using cryosections of monkey cerebellum or hek293 expressing dner endpoint titers were determined in all 38 anti - dner positive sera on the recombinant cells and monkey cerebellum . all but 3 sera showed higher titers when the recombinant cells were used in comparison to monkey tissue ( p < 0.001 , fisher exact test ) ( figure 2 ) , which yielded low - titer or negative results in 20 of 38 cases . dner = delta / notch - like epidermal growth factor - related receptor . in neutralization experiments , lysates from hek293-dner and purified dner(ec ) abolished anti - dner reactivities of human sera with recombinant cells and the corresponding anti - tr reactivity on monkey cerebellum in a dose - dependent manner ( figure 1c ) . in contrast , similar fractions of hek293 cells expressing cdr2/yo , mock - transfected hek293 , and the purified extracellular domain of desmoglein 3 did not affect anti - dner or the anti - tr reactivities . we report on the first well - standardized rc - ifa , which was developed to determine autoantibodies against tr / dner and then evaluated using samples collected in several european centers . in a blinded study of 245 sera , including a large panel of 38 sera with anti - tr autoantibodies , the new assay yielded 100% sensitivity and specificity with respect to the serologic precharacterization that was performed at the contributing centers . in comparison to the reference procedure , which had to use the elaborate epitope blocking step as a confirmatory measure in many cases our results are in agreement with the initial report on the identification of dner as the target antigen of anti - tr antibodies and a subsequent independent confirmation study that used in - house rc - ifas and significantly smaller sample cohorts . all in all , recombinant cell substrates complement or even improve indirect immunofluorescence for the detection of paraneoplastic anti - neural autoantibodies for which cryosections of mammalian cerebellum are classically used . by specific neutralization of the autoantibody reactions with recombinant full - length dner expressed in hek293 , on the one hand , and the anti - tr pattern on cerebellum using dner(ec ) , we confirmed the earlier report on the exclusive presence of relevant epitopes in the extracellular domain of dner . we also demonstrated that the recombinant dner(ec ) construct is a fully competent antigen that may help in the development of a microplate elisa or dot / lineblot for integration with other paraneoplastic neurologic syndrome markers such as hu , yo , ri , and cv2 . as a rule , adult patients with subacute - onset cerebellar ataxia should be tested for anti - dner and the other well - characterized onconeural antibodies . first , an immunohistochemical screening can be performed using cryosections of cerebellum and then be confirmed in anti - dner rc - ifa when the anti - tr staining pattern is observed or suspected . alternatively , direct screening can be performed using the anti - dner rc - ifa ; if the results are equivocal or the clinical data are highly suggestive , an additional immunohistochemistry could be performed as well . symptomatology - driven sets of test substrates can be assembled , and a broad spectrum of anti - neural autoantibodies can be tested for simultaneously . w. stcker holds patents for alibrationsstreifen fr einen immunoblot , vorrichtung und verfahren zur automatischen fokussierung fr die mikroskopie schwach leuchtender substrate , verfahren zum spezifischen und quantitativen nachweis von antikrpern in einer probe , zum nachweis von anti - nmda - rezeptor - autoantikrpern zum einsatz in diagnoseverfahren , verfahren und vorrichtung zur bearbeitung von proben biologischen materials , diagnosekit sowie verfahren zur untersuchung einer menschlichen patientenprobe auf das vorhandensein neuromyelitis - optica - spezifischen antikrpern , testkit fr die labordiagnostik , vorrichtung zur steuerung einer pipettiervorrichtung , verfahren und analysevorrichtung zur mikroskopischen untersuchung eines gewebeschnittes oder eines zellausstriches , and verfahren sowie vorrichtung zur inkubation von patentientenproben ; is the head of the board for euroimmun medizinische labordiagnostika ag ; is employed by klinisch - immunologisches ; and holds stock in euroimmun medizinische labordiagnostika ag .

statin therapy has been well established as a cornerstone of cardiovascular prevention , and yet despite potent therapies for lowering lowdensity lipoprotein cholesterol ( ldlc ) , substantial residual risk remains.1 , 2 , 3 clinical and epidemiological studies have demonstrated that triglyceride ( tg ) elevation is an independent risk factor for increased cardiovascular ( cv ) events , and therefore may represent one contributive factor of residual cv risk beyond statin therapy.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 more recently , elegant mendelian randomization studies have supported a causal role for tg in the pathogenesis of cardiovascular disease ( cvd ) , showing that elevated tg are not merely a risk marker , but rather a risk factor and thus potentially modifiable.3 despite the available data , an important question that remains is whether treatment of modest degrees of tg elevation would decrease cv events , in particular in patients already receiving ldlc lowering therapy with statins . prior cv outcome studies that administered therapies with tglowering effects ( niacin or fenofibrate ) on top of statin therapy did not reach their primary endpoints . nonetheless , these studies also did not prospectively enroll patients with elevated tg levels despite statin therapy,6 , 12 , 13 , 14 , 15 and subgroup analyses suggested possible benefits to tg lowering in patients with dyslipidemia.5 , 6 outcome studies of relatively low doses of prescription omega3 therapies in japan ( the japan epa lipid intervention study [ jelis])16 and italy ( gruppo italiano per lo studio della sopravvivenza nell'infarto miocardico [ gissi])17 , 18 have suggested that omega3 therapy may provide cv protection . however , these studies were performed in single countries prior to current treatment guidelines , and therefore provide supportive but not conclusive evidence of cv benefit . other more recent omega3 therapy outcome studies conducted in the presence of statins have been less encouraging , but these studies were characterized by evaluating nonhypertriglyceridemic patient populations ( eg , tg < 200 mg / dl ) and administering low doses of longchain omega3 fatty acids ( eg , eicosapentaenoic acid [ epa ] and/or docosoahexaenoic acid [ dha]).19 , 20 , 21 , 22 , 23 , 24 omega3 therapies , including epa , have been postulated to have cardioprotective effects such as beneficial changes to tg and other lipid and lipoprotein parameters ( eg , non highdensity lipoprotein cholesterol [ nonhdlc ] , apolipoprotein ciii ) , as well as other potential benefits beyond plasma lipid modification.19 , 25 , 26 , 27 , 28 , 29 , 30 icosapent ethyl ( vascepa ; amarin pharma inc . , bedminster , nj ) is a highly purified ethyl ester of epa , which has been reported to improve atherogenic dyslipidemia characterized by reductions in tg , tgrich lipoproteins , and factors involved in their metabolism , without raising ldlc.25 , 26 , 27 , 28 , 29 based on trials with tg lowering as the primary endpoint , this prescription therapy is currently approved for use in the united states by the us food and drug administration ( fda ) as an adjunct to diet to reduce tg levels in adult patients with severe hypertriglyceridemia ( 500 mg / dl).25 , 26 in this range of very elevated tg levels , reduction is considered to be clinically necessary to decrease the risk of pancreatitis . in addition to beneficial changes to tgrich lipoproteins and other plasma lipid markers , some clinical studies with higherdose epa also suggest beneficial effects on markers of oxidation and inflammation , coronary plaque characteristics , and major cv events.16 , 25 , 26 , 29 , 31 , 32 , 33 for example , in contrast to the fenofibrate and niacin studies , jelis found a 19% relative risk reduction in cv events in statintreated patients with relatively normal tg but a more pronounced 53% reduction in the subgroup with mixed dyslipidemia , specifically tg 150 mg / dl and hdlc < 40 mg / dl.4 , 16 although confirmation of these results is needed in western populations , the reduction of cv events with epa therapy in a patient population with relatively normal tg levels suggests that epa may have pleiotropic effects beyond plasmalipid modification . it is worth noting that the promising results from jelis occurred with a highpurity epa preparation dosed at 1.8 g / d in a japanese population , for whom the baseline epa levels are higher than in western populations due to greater dietary intake of marine omega3 fatty acids . icosapent ethyl 12week dosing at 4 g / d in a highrisk population similar to that within the reduction of cardiovascular events with icosapent ethyl intervention trial ( reduceit ) who had persistent elevations of tg after treatment with statins resulted in significant reductions in tg and atherogenic lipoproteins,26 , 27 , 28 as well as comparable plasma epa levels as the 1.8 g / d dosing group in jelis.34 therefore , a dose of 4 g / d was selected as the dose for further study . in this context , reduceit was designed to determine if treatment with icosapent ethyl 4 g / d vs placebo would reduce ischemic events in patients at increased cv risk already being treated with statins . reduceit ( nct01492361 ) is a phase 3b , international , multicenter , prospective , randomized , doubleblinded , placebocontrolled , parallelgroup trial of icosapent ethyl 4 g / d ( 2 g twice daily with food ) vs placebo ( figure ) . the main objective is to evaluate whether treatment with icosapent ethyl reduces ischemic events in patients at elevated cv risk concurrently treated with statins . inclusion and exclusion criteria are listed in table 1 and table 2 , respectively . men or women age 45 years with established cvd ( cv risk stratum 1 , table 1 ) or age 50 years with diabetes mellitus in combination with 1 additional risk factor for cvd ( cv risk stratum 2 , table 1 ) were eligible for inclusion . fasting tg levels 150 mg / dl and < 500 mg / dl were required . a study amendment was made during the early part of the trial , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl , to increase enrollment of patients with more significant tg elevations . ldlc levels needed to be > 40 mg / dl and 100 mg / dl , with patients on stable statin therapy ( ezetimibe ) for 4 weeks prior to the ldlc and tg qualifying measurements for randomization . if patients met the inclusion criteria at visit 1 , they were asked to return for the randomization visit ( visit 2 ) and entered the treatment / followup period . patients who were not eligible at visit 1 but who became eligible in the next 28 days ( such as patients whose statin dose changed at visit 1 and/or needed to wash out prohibited medications ) may have returned for an optional second screening visit ( visit 1.1 ) . such patients entered a statin stabilization / medication washout period of 28 days prior to rescreening . patients who were eligible following screening / rescreening entered the treatment / followup period , with followup visits occurring at 4 months , 12 months , and annually thereafter . * a study amendment ( may 2013 ) was made , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl to increase enrollment of patients with tg 200 mg / dl ; it is anticipated that mean and median qualifying tg levels will be > 200 mg / dl . eventdriven design : approximately 1612 primary efficacy events will be required during the study ; study duration will vary accordingly . abbreviations : cv , cardiovascular ; cvd , cardiovascular disease ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; reduceit , reduction of cardiovascular events with icosapent ethyl intervention trial ; t2 dm , type 2 diabetes mellitus ; tg , triglycerides . abbreviations : abi , anklebrachial index ; bp , blood pressure ; cad , coronary artery disease ; cr , creatinine ; crcl , creatinine clearance ; cv , cardiovascular ; cvd , cardiovascular disease ; dm , diabetes mellitus ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highsensitivity creactive protein ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; nsteacs , non stsegment elevation acute coronary syndrome ; pad , peripheral arterial disease ; tg , triglycerides . note : patients with dm and cvd as defined above are eligible based on the cvd requirements and will be counted under cv risk stratum 1 . only patients with dm and no documented cvd as defined above need 1 additional risk factor as listed , and they will be counted under cv risk stratum 2 . a study amendment ( may 2013 ) was made , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl to increase enrollment of patients with tg 200 mg / dl ; it is anticipated that mean and median qualifying tg levels will be > 200 mg / dl . abbreviations : aids , acquired immunodeficiency syndrome ; apocii , apolipoprotein cii ; ck , creatine kinase ; crcl , creatinine clearance ; cvd , cardiovascular disease ; dbp , diastolic blood pressure ; hba1c , glycated hemoglobin ; hf , heart failure ; hiv , human immunodeficiency virus ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; nyha , new york heart association ; om3 , omega3 ; pcsk9 , proprotein convertase subtilisin / kexin type 9 ; sbp , systolic blood pressure ; tg , triglyceride ; uln , upper limit of normal . the primary endpoint is a composite of cv death , nonfatal myocardial infarction ( mi ) , nonfatal stroke , coronary revascularization , or unstable angina . the key secondary endpoint is the composite of cv death , nonfatal mi , or nonfatal stroke . several other secondary , tertiary , and exploratory endpoints are being assessed ( table 3 ) , which were designed to provide additional insights into the potential effects of epa therapy on various outcomes and in distinct highrisk patient populations . abbreviations : apob , apolipoprotein b ; chf , coronary heart failure ; cv , cardiovascular ; dm , diabetes mellitus ; ecg , electrocardiography ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highsensitivity creactive protein ; hstnt , highsensitivity troponin t ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; non hdlc , non highdensity lipoprotein cholesterol ; pvd , peripheral vascular disease ; rlpc , remnant lipoprotein cholesterol ; tc , total cholesterol ; tg , triglycerides ; tia , transient ischemic attack ; ua , unstable angina ; vldlc , very lowdensity lipoprotein cholesterol . the first occurrence of any of these major adverse vascular events during the followup period of the study will be included in the incidence . for the secondary and tertiary endpoints that count a single event , the time from randomization to the first occurrence of this type of event will be counted for each patient . for secondary and tertiary endpoints that are composites of 2 types of events , the time from randomization to the first occurrence of any of the event types included in the composite the time from randomization to occurrence of the first and all recurrent major cv events defined as cv death , nonfatal mi ( including silent mi ) , nonfatal stroke , coronary revascularization , or ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization . including silent mi ; ecg will be performed annually for the detection of silent mi . composite endpoints include : composite of cv death , nonfatal mi ( including silent mi ) , nonfatal stroke , cardiac arrhythmia requiring hospitalization of 24 hours , or cardiac arrest ; composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , or ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization ; composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization , nonfatal stroke , or pvd requiring intervention such as angioplasty , bypass surgery , or aneurysm repair ; and composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization , pvd requiring intervention , or cardiac arrhythmia requiring hospitalization of 24 hours . assessment of the relationship between baseline biomarker values and treatment effects within the primary and key secondary composite endpoints ; assessment of the effect of study drug on each marker ; and assessment of the relationship between postbaseline biomarker values and treatment effects within the primary and key secondary composite endpoints by including postbaseline biomarker values ( for example , at 4 months , or at 1 year ) as a covariate . therefore , 1612 events would be required to have approximately 90% power with a 1sided level of 2.5% and with 2 interim analyses . approximately 70% of randomized patients were to be in cv risk stratum 1 ( established cvd ) and approximately 30% of randomized patients were to be in cv risk stratum 2 ( highrisk primary prevention defined by diabetes mellitus and other risk factors ) . randomization was stratified by cv risk strata , ezetimibe use , and by geographical region . protocol amendment 1 ( may 2013 ) changed the lower limit of tg levels for entry into the trial from 150 mg / dl to 200 mg / dl , as a majority of the steering committee members felt that those were the patients most likely to benefit from tg lowering . protocol amendment 2 ( july 2016 ) designated the composite of hard major adverse cardiovascular events ( cv death , nonfatal mi , nonfatal stroke ) as the key secondary endpoint per suggestions from the fda with steering committee concordance . approximately 8000 patients have been randomized at approximately 470 centers worldwide ( see supporting information , appendix , in the online version of this article ) . follow up will continue in this eventdriven trial until approximately 1612 adjudicated primary efficacy endpoint events have occurred . this study is being conducted in accordance with a special protocol assessment agreement with the fda . despite cv risk reduction through potent ldlc lowering therapies such as statins , substantial residual cv risk remains . epidemiological , biological , and genetic studies have provided robust evidence of a strong association between elevated tg levels and higher rates of cv events.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 furthermore , tg reduction lowers several inflammatory markers associated with cv risk , and subgroup and post hoc analyses of outcome studies suggest possible reductions in major cv events with tglowering therapy.3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 finally , studies administering higherdose epa suggest additional beneficial effects beyond lipidlowering that may be unique to epa relative to other tglowering therapies , such as beneficial changes in coronary plaque characteristics , which may lead to reductions in major cv events.4 , 16 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 however , randomized data from large outcome studies across broad populations regarding pharmacological tg lowering and effect on cv outcomes have been mixed ( table 4).1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 part of the reason may involve differences between the classes of drugs studied , such as fibrates , niacin , and omega3 fatty acids . even among omega3 fatty acid studies , there are marked differences with respect to the relatively low doses of omega3 administered and the ratio of epa to dha.16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 in addition , different tglowering therapies may exert differential effects across lipid profiles . for example , fibrates and dhacontaining omega3 fatty acid mixtures have been shown to increase ldlc , which in turn might adversely influence trial results . among outcome studies administering tglowering agents beyond statin therapy , only the jelis trial using pure epa demonstrated a significant reduction in cv events in patients with relatively normal tg levels.16 the subgroup data ( table 5 ) from the action to control cardiovascular risk in diabetes ( accord ) lipid,6 atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides and impact on global health outcomes ( aimhigh),5 and jelis4 trials further support a prospective study of epa in a broader patient population with hypertriglyceridemia , as exemplified in reduceit , as a potential addon to statin therapy to reduce residual cv risk . that the lipid changes in jelis were relatively modest ( ie , approximately a 5% tg reduction ) raises the possibility that other pleiotropic effects beyond lipid lowering may have also contributed to the reduction in cv risk.4 , 16 finally , any benefits to tglowering therapies may be most pronounced among statintreated patients in the higher range of tg elevation ( ie , 200 mg / dl),4 , 5 , 6 for whom randomized prospectively designed outcome studies have not been previously conducted prior to the reduceit study . cv outcome trials administering therapies that can be used for tg lowering abbreviations : accord , action to control cardiovascular risk in diabetes ; aimhigh , atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides : impact on global health outcomes ; ala , alphalinolenic acid ; alphaomega , study of omega3 fatty acids and coronary mortality ; bip , bezafibrate infarction prevention ; bl , baseline ; chd , coronary heart disease ; ci , confidence interval ; cv , cardiovascular ; cvd , cardiovascular disease ; dha , docosahexaenoic acid ; eos , end of study ; epa , eicosapentaenoic acid ; er , extended release ; feno , fenofibrate ; field , fenofibrate intervention and event lowering in diabetes ; gissip , gruppo italiano per lo studio della sopravvivenza nell'infarto miocardicoprevenzione ; gissihf , gruppo italiano per lo studio della sopravvivenza nell'infarto miocardicoheart failure ; hdlc , highdensity lipoprotein cholesterol ; hf , heart failure ; hps2thrive , heart protection study 2treatment of hdl to reduce the incidence of vascular events ; hr , hazard ratio ; jelis , japan epa lipid intervention study ; mace , major adverse cardiac events ; mi , myocardial infarction ; nyha , new york heart association ; oae , omega acid esters ; omega , effect of omega 3fatty acids on the reduction of sudden cardiac death after myocardial infarction ; or , odds ratio ; origin , outcome reduction with an initial glargine intervention ; pbo , placebo ; rrr , relative risk reduction ; scd , sudden cardiac death ; su.fol.om3 , supplmentation en folates et omega3 ; t2 dm , type 2 diabetes mellitus ; tc , total cholesterol ; tg , triglycerides ; vahit , veterans affairs cooperative studies program highdensity lipoprotein cholesterol intervention trial ; vit e , vitamin e. mean or median values are presented . subgroup analyses of patients with dyslipidemia from cv outcome trials administering tglowering therapies added to statin therapy vs statin monotherapy abbreviations : accord , action to control cardiovascular risk in diabetes ; aimhigh , atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides : impact on global health outcomes ; bl , baseline ; cv , cardiovascular ; epa , eicosapentaenoic acid ; er , extended release ; hdlc , highdensity lipoprotein cholesterol ; hps2thrive , heart protection study 2treatment of hdl to reduce the incidence of vascular events ; jelis , japan epa lipid intervention study ; mace , major adverse cardiac events ; tg , triglycerides . bolded values approached / achieved statistical significance . reduceit is designed to evaluate whether treating atrisk patients with highdose epa will lower the rates of important ischemic events beyond statin therapy . however , this trial alone will not validate whether lowering tg specifically in patients with elevated tg levels will result in lower rates of important ischemic events , because the effects of epa may be broader than tg reduction alone . several trials , including reduceit , are ongoing or planned to determine if different tglowering therapies in patients with elevated tg levels lower the rate of important ischemic events.35 , 36 , 37 , 38 the use of different therapeutic agents across these trials may in aggregate help us better understand the relative importance of tg lowering alone and may also help define which potential effects observed in reduceit might be uniquely attributable to epa therapy . several lines of data , including comparison of the jelis study results to those of fibrate and niacin outcome studies , suggest that epa may be differentiated from other tglowering agents as statin addon therapy , by potentially providing unique pleiotropic cardioprotective benefits in addition to tg lowering . other changes in the lipidlowering field may also affect the interpretation of the ongoing reduceit trial . for example , the proprotein convertase subtilisin / kexin type 9 inhibitors are being tested in large cv outcome trials of patients for whom ldlc control from statin ( ezetimibe ) therapy may be insufficient or poorly tolerated . if these ldlc lowering agents are found to be beneficial , epa therapy could potentially serve as a complementary approach to reduce residual cv risk even further , though this specific combination would not have been studied well in terms of incremental effects on cv events . importantly , residual cv risk remains high in patients with ldlc well controlled by statins , and many of these patients will likely need to be treated from multiple angles . the growing body of tgrelated evidence suggests that tgrich lipoproteins may be a causal factor in such residual risk . consequently , tg lowering represents a target of great interest to optimize further cv risk reduction beyond the ldlc lowering benefits attained with statin use . epaspecific studies suggest that epa may provide unique cv benefits through favorable effects on plasma lipid parameters , as well as on other pleiotropic pathways . a major remaining question is how to achieve cv risk reduction beyond the benefits realized from effective management of ldlc . for patients with persistently high tg levels despite statin therapy , an agent that improves atherogenic dyslipidemia without raising ldlc and provides other potentially pleiotropic benefits may improve cv outcomes . the addition of epa to statin therapy may thus provide additional cv benefit . the reduceit trial with highdose epa is designed to address this longstanding scientific gap and to provide physicians with this muchneeded information to guide clinical care of patients at high cv risk . dr . bhatt has served on advisory boards for cardax , elsevier practice update cardiology , medscape cardiology , and regado biosciences ; has served on the board of directors for boston va research institute and society of cardiovascular patient care ; has been chair of the american heart association quality oversight committee ; has served on data monitoring committees for duke clinical research institute , harvard clinical research institute , mayo clinic , and the population health research institute ; has received honoraria from the american college of cardiology ( senior associate editor , clinical trials and news , for acc.org ) , belvoir publications ( editor in chief , harvard heart letter ) , duke clinical research institute ( clinical trial steering committees ) , harvard clinical research institute ( clinical trial steering committee ) , hmp communications ( editor in chief , journal of invasive cardiology ) , journal of the american college of cardiology ( guest editor , associate editor ) , population health research institute ( clinical trial steering committee ) , slack publications ( chief medical editor , cardiology today 's intervention ) , society of cardiovascular patient care ( secretary / treasurer ) , and webmd ( cme steering committees ) ; has served as deputy editor of clinical cardiology , as chair of the ncdraction registry steering committee , and as chair of the va cart research and publications committee ; has received research funding from amarin ( for his role as chair of the steering committee and principal investigator of reduceit ) , amgen , astrazeneca , bristolmyers squibb , eisai , ethicon , forest laboratories , ischemix , lilly , medtronic , pfizer , roche , sanofiaventis , and the medicines company ; has received royalties from elsevier ( editor , cardiovascular intervention : a companion to braunwald 's heart disease ) ; has served as site coinvestigator for biotronik , boston scientific , and st . jude medical ; has served as a trustee of the american college of cardiology ; and reports unfunded research with flowco , plx pharma , and takeda . dr . steg has received research grants from merck , sanofi , and servier and has received speaking or consulting fees from amarin , amgen , astrazeneca , bayer , boehringer ingelheim , bristolmyers squibb , cslbehring , daiichisankyo , glaxosmithkline , janssen , lilly , merck , novartis , pfizer , regeneron , sanofi , servier , and the medicines company . dr . brinton has received speaking and/or consulting honoraria from alexion , amarin , amgen , aralez , arisaph , astrazeneca , janssen , kastle , kowa , merck , pts diagnostics , regeneron , and sanofiaventis and has received research funding from amarin ( for his role as steering committee member of reduceit ) and kowa ( for his role as steering committee member of prominent ) . jacobson has served as a consultant for amarin , amgen , astrazeneca , merck , regeneron , and sanofi and has done research for amgen and regeneron / sanofi . miller has served as a consultant for amarin , akcea , gemphire , and pfizer . dr . tardif has received research grants from amarin , astrazeneca , dalcor , eli lilly , esperion , merck , pfizer , sanofi , and servier ; has received honoraria from amarin , astrazeneca , dalcor , sanofi , and servier ; and holds equity ( modest position ) in dalcor . ballantyne discloses grant / research support ( all paid to the institution , not individual ) from amarin , amgen , eli lilly , esperion , ionis , novartis , pfizer , regeneron , sanofisynthelabo , the national institutes of health , the american heart association , and the american diabetes association , and has served as a consultant for amarin , amgen , astrazeneca , boehringer ingelheim , eli lilly , esperion , ionis , matinas biopharma inc , merck , novartis , pfizer , regeneron , and sanofisynthelabo .

residual cardiovascular risk persists despite statins , yet outcome studies of lipidtargeted therapies beyond lowdensity lipoprotein cholesterol ( ldlc ) have not demonstrated added benefit . triglyceride elevation is an independent risk factor for cardiovascular events . highdose eicosapentaenoic acid ( epa ) reduces triglyceriderich lipoproteins without raising ldlc . omega3s have postulated pleiotropic cardioprotective benefits beyond triglyceridelowering . to date , no large , multinational , randomized clinical trial has proved that lowering triglycerides on top of statin therapy improves cardiovascular outcomes . the reduction of cardiovascular events with icosapent ethyl intervention trial ( reduceit ; nct01492361 ) is a phase 3b randomized , doubleblinded , placebocontrolled trial of icosapent ethyl , a highly purified ethyl ester of epa , vs placebo . the main objective is to evaluate whether treatment with icosapent ethyl reduces ischemic events in statintreated patients with high triglycerides at elevated cardiovascular risk . reduceit enrolled men or women age 45 years with established cardiovascular disease or age 50 years with diabetes mellitus and 1 additional risk factor . randomization required fasting triglycerides 150 mg / dl and < 500 mg / dl and ldlc > 40 mg / dl and 100 mg / dl with stable statin ( ezetimibe ) 4 weeks prior to qualifying measurements . the primary endpoint is a composite of cardiovascular death , nonfatal myocardial infarction , nonfatal stroke , coronary revascularization , or unstable angina . the key secondary endpoint is the composite of cardiovascular death , nonfatal myocardial infarction , or nonfatal stroke . several secondary , tertiary , and exploratory endpoints will be assessed . approximately 8000 patients have been randomized at approximately 470 centers worldwide . followup will continue in this eventdriven trial until approximately 1612 adjudicated primaryefficacy endpoint events have occurred .

INTRODUCTION METHODS DISCUSSION CONCLUSION Supporting information Conflicts of Interest

statin therapy has been well established as a cornerstone of cardiovascular prevention , and yet despite potent therapies for lowering lowdensity lipoprotein cholesterol ( ldlc ) , substantial residual risk remains.1 , 2 , 3 clinical and epidemiological studies have demonstrated that triglyceride ( tg ) elevation is an independent risk factor for increased cardiovascular ( cv ) events , and therefore may represent one contributive factor of residual cv risk beyond statin therapy.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 more recently , elegant mendelian randomization studies have supported a causal role for tg in the pathogenesis of cardiovascular disease ( cvd ) , showing that elevated tg are not merely a risk marker , but rather a risk factor and thus potentially modifiable.3 despite the available data , an important question that remains is whether treatment of modest degrees of tg elevation would decrease cv events , in particular in patients already receiving ldlc lowering therapy with statins . prior cv outcome studies that administered therapies with tglowering effects ( niacin or fenofibrate ) on top of statin therapy did not reach their primary endpoints . other more recent omega3 therapy outcome studies conducted in the presence of statins have been less encouraging , but these studies were characterized by evaluating nonhypertriglyceridemic patient populations ( eg , tg < 200 mg / dl ) and administering low doses of longchain omega3 fatty acids ( eg , eicosapentaenoic acid [ epa ] and/or docosoahexaenoic acid [ dha]).19 , 20 , 21 , 22 , 23 , 24 omega3 therapies , including epa , have been postulated to have cardioprotective effects such as beneficial changes to tg and other lipid and lipoprotein parameters ( eg , non highdensity lipoprotein cholesterol [ nonhdlc ] , apolipoprotein ciii ) , as well as other potential benefits beyond plasma lipid modification.19 , 25 , 26 , 27 , 28 , 29 , 30 icosapent ethyl ( vascepa ; amarin pharma inc . , bedminster , nj ) is a highly purified ethyl ester of epa , which has been reported to improve atherogenic dyslipidemia characterized by reductions in tg , tgrich lipoproteins , and factors involved in their metabolism , without raising ldlc.25 , 26 , 27 , 28 , 29 based on trials with tg lowering as the primary endpoint , this prescription therapy is currently approved for use in the united states by the us food and drug administration ( fda ) as an adjunct to diet to reduce tg levels in adult patients with severe hypertriglyceridemia ( 500 mg / dl).25 , 26 in this range of very elevated tg levels , reduction is considered to be clinically necessary to decrease the risk of pancreatitis . in addition to beneficial changes to tgrich lipoproteins and other plasma lipid markers , some clinical studies with higherdose epa also suggest beneficial effects on markers of oxidation and inflammation , coronary plaque characteristics , and major cv events.16 , 25 , 26 , 29 , 31 , 32 , 33 for example , in contrast to the fenofibrate and niacin studies , jelis found a 19% relative risk reduction in cv events in statintreated patients with relatively normal tg but a more pronounced 53% reduction in the subgroup with mixed dyslipidemia , specifically tg 150 mg / dl and hdlc < 40 mg / dl.4 , 16 although confirmation of these results is needed in western populations , the reduction of cv events with epa therapy in a patient population with relatively normal tg levels suggests that epa may have pleiotropic effects beyond plasmalipid modification . icosapent ethyl 12week dosing at 4 g / d in a highrisk population similar to that within the reduction of cardiovascular events with icosapent ethyl intervention trial ( reduceit ) who had persistent elevations of tg after treatment with statins resulted in significant reductions in tg and atherogenic lipoproteins,26 , 27 , 28 as well as comparable plasma epa levels as the 1.8 g / d dosing group in jelis.34 therefore , a dose of 4 g / d was selected as the dose for further study . in this context , reduceit was designed to determine if treatment with icosapent ethyl 4 g / d vs placebo would reduce ischemic events in patients at increased cv risk already being treated with statins . reduceit ( nct01492361 ) is a phase 3b , international , multicenter , prospective , randomized , doubleblinded , placebocontrolled , parallelgroup trial of icosapent ethyl 4 g / d ( 2 g twice daily with food ) vs placebo ( figure ) . the main objective is to evaluate whether treatment with icosapent ethyl reduces ischemic events in patients at elevated cv risk concurrently treated with statins . men or women age 45 years with established cvd ( cv risk stratum 1 , table 1 ) or age 50 years with diabetes mellitus in combination with 1 additional risk factor for cvd ( cv risk stratum 2 , table 1 ) were eligible for inclusion . fasting tg levels 150 mg / dl and < 500 mg / dl were required . a study amendment was made during the early part of the trial , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl , to increase enrollment of patients with more significant tg elevations . ldlc levels needed to be > 40 mg / dl and 100 mg / dl , with patients on stable statin therapy ( ezetimibe ) for 4 weeks prior to the ldlc and tg qualifying measurements for randomization . * a study amendment ( may 2013 ) was made , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl to increase enrollment of patients with tg 200 mg / dl ; it is anticipated that mean and median qualifying tg levels will be > 200 mg / dl . abbreviations : cv , cardiovascular ; cvd , cardiovascular disease ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; reduceit , reduction of cardiovascular events with icosapent ethyl intervention trial ; t2 dm , type 2 diabetes mellitus ; tg , triglycerides . abbreviations : abi , anklebrachial index ; bp , blood pressure ; cad , coronary artery disease ; cr , creatinine ; crcl , creatinine clearance ; cv , cardiovascular ; cvd , cardiovascular disease ; dm , diabetes mellitus ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highsensitivity creactive protein ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; nsteacs , non stsegment elevation acute coronary syndrome ; pad , peripheral arterial disease ; tg , triglycerides . only patients with dm and no documented cvd as defined above need 1 additional risk factor as listed , and they will be counted under cv risk stratum 2 . a study amendment ( may 2013 ) was made , increasing the lower end of the fasting tg level from 150 mg / dl to 200 mg / dl to increase enrollment of patients with tg 200 mg / dl ; it is anticipated that mean and median qualifying tg levels will be > 200 mg / dl . the primary endpoint is a composite of cv death , nonfatal myocardial infarction ( mi ) , nonfatal stroke , coronary revascularization , or unstable angina . the key secondary endpoint is the composite of cv death , nonfatal mi , or nonfatal stroke . several other secondary , tertiary , and exploratory endpoints are being assessed ( table 3 ) , which were designed to provide additional insights into the potential effects of epa therapy on various outcomes and in distinct highrisk patient populations . abbreviations : apob , apolipoprotein b ; chf , coronary heart failure ; cv , cardiovascular ; dm , diabetes mellitus ; ecg , electrocardiography ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highsensitivity creactive protein ; hstnt , highsensitivity troponin t ; htn , hypertension ; ldlc , lowdensity lipoprotein cholesterol ; mi , myocardial infarction ; non hdlc , non highdensity lipoprotein cholesterol ; pvd , peripheral vascular disease ; rlpc , remnant lipoprotein cholesterol ; tc , total cholesterol ; tg , triglycerides ; tia , transient ischemic attack ; ua , unstable angina ; vldlc , very lowdensity lipoprotein cholesterol . for secondary and tertiary endpoints that are composites of 2 types of events , the time from randomization to the first occurrence of any of the event types included in the composite the time from randomization to occurrence of the first and all recurrent major cv events defined as cv death , nonfatal mi ( including silent mi ) , nonfatal stroke , coronary revascularization , or ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization . composite endpoints include : composite of cv death , nonfatal mi ( including silent mi ) , nonfatal stroke , cardiac arrhythmia requiring hospitalization of 24 hours , or cardiac arrest ; composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , or ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization ; composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization , nonfatal stroke , or pvd requiring intervention such as angioplasty , bypass surgery , or aneurysm repair ; and composite of cv death , nonfatal mi ( including silent mi ) , nonelective coronary revascularizations ( defined as emergent or urgent classifications ) , ua determined to be caused by myocardial ischemia by invasive / noninvasive testing and requiring emergent hospitalization , pvd requiring intervention , or cardiac arrhythmia requiring hospitalization of 24 hours . protocol amendment 2 ( july 2016 ) designated the composite of hard major adverse cardiovascular events ( cv death , nonfatal mi , nonfatal stroke ) as the key secondary endpoint per suggestions from the fda with steering committee concordance . approximately 8000 patients have been randomized at approximately 470 centers worldwide ( see supporting information , appendix , in the online version of this article ) . follow up will continue in this eventdriven trial until approximately 1612 adjudicated primary efficacy endpoint events have occurred . epidemiological , biological , and genetic studies have provided robust evidence of a strong association between elevated tg levels and higher rates of cv events.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 furthermore , tg reduction lowers several inflammatory markers associated with cv risk , and subgroup and post hoc analyses of outcome studies suggest possible reductions in major cv events with tglowering therapy.3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 finally , studies administering higherdose epa suggest additional beneficial effects beyond lipidlowering that may be unique to epa relative to other tglowering therapies , such as beneficial changes in coronary plaque characteristics , which may lead to reductions in major cv events.4 , 16 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 however , randomized data from large outcome studies across broad populations regarding pharmacological tg lowering and effect on cv outcomes have been mixed ( table 4).1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 part of the reason may involve differences between the classes of drugs studied , such as fibrates , niacin , and omega3 fatty acids . among outcome studies administering tglowering agents beyond statin therapy , only the jelis trial using pure epa demonstrated a significant reduction in cv events in patients with relatively normal tg levels.16 the subgroup data ( table 5 ) from the action to control cardiovascular risk in diabetes ( accord ) lipid,6 atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides and impact on global health outcomes ( aimhigh),5 and jelis4 trials further support a prospective study of epa in a broader patient population with hypertriglyceridemia , as exemplified in reduceit , as a potential addon to statin therapy to reduce residual cv risk . that the lipid changes in jelis were relatively modest ( ie , approximately a 5% tg reduction ) raises the possibility that other pleiotropic effects beyond lipid lowering may have also contributed to the reduction in cv risk.4 , 16 finally , any benefits to tglowering therapies may be most pronounced among statintreated patients in the higher range of tg elevation ( ie , 200 mg / dl),4 , 5 , 6 for whom randomized prospectively designed outcome studies have not been previously conducted prior to the reduceit study . cv outcome trials administering therapies that can be used for tg lowering abbreviations : accord , action to control cardiovascular risk in diabetes ; aimhigh , atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides : impact on global health outcomes ; ala , alphalinolenic acid ; alphaomega , study of omega3 fatty acids and coronary mortality ; bip , bezafibrate infarction prevention ; bl , baseline ; chd , coronary heart disease ; ci , confidence interval ; cv , cardiovascular ; cvd , cardiovascular disease ; dha , docosahexaenoic acid ; eos , end of study ; epa , eicosapentaenoic acid ; er , extended release ; feno , fenofibrate ; field , fenofibrate intervention and event lowering in diabetes ; gissip , gruppo italiano per lo studio della sopravvivenza nell'infarto miocardicoprevenzione ; gissihf , gruppo italiano per lo studio della sopravvivenza nell'infarto miocardicoheart failure ; hdlc , highdensity lipoprotein cholesterol ; hf , heart failure ; hps2thrive , heart protection study 2treatment of hdl to reduce the incidence of vascular events ; hr , hazard ratio ; jelis , japan epa lipid intervention study ; mace , major adverse cardiac events ; mi , myocardial infarction ; nyha , new york heart association ; oae , omega acid esters ; omega , effect of omega 3fatty acids on the reduction of sudden cardiac death after myocardial infarction ; or , odds ratio ; origin , outcome reduction with an initial glargine intervention ; pbo , placebo ; rrr , relative risk reduction ; scd , sudden cardiac death ; su.fol.om3 , supplmentation en folates et omega3 ; t2 dm , type 2 diabetes mellitus ; tc , total cholesterol ; tg , triglycerides ; vahit , veterans affairs cooperative studies program highdensity lipoprotein cholesterol intervention trial ; vit e , vitamin e. mean or median values are presented . subgroup analyses of patients with dyslipidemia from cv outcome trials administering tglowering therapies added to statin therapy vs statin monotherapy abbreviations : accord , action to control cardiovascular risk in diabetes ; aimhigh , atherothrombosis intervention in metabolic syndrome with low hdl / high triglycerides : impact on global health outcomes ; bl , baseline ; cv , cardiovascular ; epa , eicosapentaenoic acid ; er , extended release ; hdlc , highdensity lipoprotein cholesterol ; hps2thrive , heart protection study 2treatment of hdl to reduce the incidence of vascular events ; jelis , japan epa lipid intervention study ; mace , major adverse cardiac events ; tg , triglycerides . reduceit is designed to evaluate whether treating atrisk patients with highdose epa will lower the rates of important ischemic events beyond statin therapy . for example , the proprotein convertase subtilisin / kexin type 9 inhibitors are being tested in large cv outcome trials of patients for whom ldlc control from statin ( ezetimibe ) therapy may be insufficient or poorly tolerated . importantly , residual cv risk remains high in patients with ldlc well controlled by statins , and many of these patients will likely need to be treated from multiple angles . for patients with persistently high tg levels despite statin therapy , an agent that improves atherogenic dyslipidemia without raising ldlc and provides other potentially pleiotropic benefits may improve cv outcomes . bhatt has served on advisory boards for cardax , elsevier practice update cardiology , medscape cardiology , and regado biosciences ; has served on the board of directors for boston va research institute and society of cardiovascular patient care ; has been chair of the american heart association quality oversight committee ; has served on data monitoring committees for duke clinical research institute , harvard clinical research institute , mayo clinic , and the population health research institute ; has received honoraria from the american college of cardiology ( senior associate editor , clinical trials and news , for acc.org ) , belvoir publications ( editor in chief , harvard heart letter ) , duke clinical research institute ( clinical trial steering committees ) , harvard clinical research institute ( clinical trial steering committee ) , hmp communications ( editor in chief , journal of invasive cardiology ) , journal of the american college of cardiology ( guest editor , associate editor ) , population health research institute ( clinical trial steering committee ) , slack publications ( chief medical editor , cardiology today 's intervention ) , society of cardiovascular patient care ( secretary / treasurer ) , and webmd ( cme steering committees ) ; has served as deputy editor of clinical cardiology , as chair of the ncdraction registry steering committee , and as chair of the va cart research and publications committee ; has received research funding from amarin ( for his role as chair of the steering committee and principal investigator of reduceit ) , amgen , astrazeneca , bristolmyers squibb , eisai , ethicon , forest laboratories , ischemix , lilly , medtronic , pfizer , roche , sanofiaventis , and the medicines company ; has received royalties from elsevier ( editor , cardiovascular intervention : a companion to braunwald 's heart disease ) ; has served as site coinvestigator for biotronik , boston scientific , and st .

it is well known that it serves as the precursor for serotonin , but what is less well known is that the majority ( 9095% ) of tryptophan besides that utilized for protein synthesis , is shuttled to the kynurenine pathway , the end point of which is the generation of nicotinamide adenine dinucleotide ( nad+ ) . metabolites along the kynurenine pathway both are synthesized in the brain and can travel into the brain . furthermore , upregulation of the kynurenine pathway has been demonstrated in depressive disorders , and the intensity of depressive symptoms has been found to correlate with the upregulation of kynurenine pathway [ 210 ] . the enhancement of this differential metabolic route of tryptophan has been hypothesized to contribute to potential alterations in serotonergic function in depression , such that an increase shuttling of tryptophan to the kynurenine pathway could result in a deficit in serotonin synthesis . the rate - limiting step in this pathway is the conversion of tryptophan to kynurenine by either tryptophan 2,3-dioxygenase ( tdo ) or indoleamine 2,3-dioxygenase 1 ( ido1 ) . one pathway results in the conversion of kynurenine to kynurenic acid ( kyna ) by kynurenine aminotransferase1 ( kat1 ) . kyna is an n - methyl - d - aspartate receptor ( nmdar ) and 7 nicotinic acetylcholine receptor ( 7nachr ) antagonist and is thought to play a neuroprotective role through inhibiting glutamate signaling . the other pathway , which results in the generation of quinolinic acid ( quin ) , has multiple steps , by which kynurenine is converted to intermediates through kynurenine 3-monooxygenase ( kmo ) and kynureninase ( kynu ) and , finally , by 3-hydroxyanthranilic acid 3,4-dioxygenase ( 3-hoa ) to quin ( figure 1 ) . as a highly selective nmdar agonist when taken altogether , the balance of the kynurenine metabolites , particularly kyna and quin , could play an important role in central nervous system functioning , and that disruption of this balance could be important for a number of neurological disorders , including neurodegenerative and affective disorders . it has been well established that inflammation can affect the activity of enzymes regulating the kynurenine pathway . specifically , ido1 is upregulated by proinflammatory cytokines , such as tnf- and ifn , and it has also been shown to be downregulated by anti - inflammatory cytokines , such as il-4 . furthermore , there is evidence that stress , and specifically the major output of the hpa axis , glucocorticoids , can upregulate tdo . taken together , these data suggest that stress and/or inflammation have the ability to upregulate levels of kynurenine . consistent with this , kynurenine levels are elevated in diseases that have elevated glucocorticoid output and inflammatory mediators , such as major depression and coronary heart disease [ 1 , 2 ] . this increased pool of kynurenine may serve as a precursor for the generation of downstream metabolites , and in the context of disease states , it may amplify the production of the neurotoxic quin , which has been hypothesized to contribute to structural alterations and loss of neuropil in disease states such as major depression [ 8 , 12 , 13 ] . it is now well established that acute stress upregulates inflammatory mediators , both in humans , peripherally , and in rodents , both peripherally and in the brain [ 14 , 15 ] . recent work from our group has demonstrated that stress - induced changes in inflammatory mediators within the brain are not uniform and exhibit significant spatiotemporal specificity , with the amygdala appearing to be highly sensitive to induction of proinflammatory molecules following exposure to acute psychological stress . given that stress increases inflammatory cytokines and glucocorticoid levels , both of which have been shown to increase kynurenine levels , it seems highly plausible that stress could exert robust and dynamic effects on the pathway regulating kynurenine synthesis . consistent with this hypothesis , it has been shown that various acute stressors can lead to an upregulation of the kynurenine pathway [ 1719 ] ; however , only one of these studies actually examined expression levels of the enzymes involved in kynurenine metabolism and did so in a whole brain manner without any regional specificity . therefore , the goal of this study was to investigate whether acute stress exposure affected gene expression levels of the enzymes in the kynurenine pathway , particularly investigating the key corticolimbic brain regions involved in regulating the stress response , the amygdala , hippocampus , hypothalamus , and medial prefrontal cortex , in a time - dependent manner . adult ( approximately 70 postnatal days old , 300 g ) , male , sprague dawley rats ( n = 48 ) , obtained from charles river ( st . constant , qc , canada ) , were used in the following experiment . animals were allowed to acclimate for at least one week prior to testing and kept on a 12:12 hour ( h ) light / dark cycle with ad libitum access to food and water . the university of calgary animal care committee and the canadian council for animal care approved all animal use . animals were subjected to 120 minutes ( min ) of restraint stress within the first 2 h of the light cycle , using clear , plexiglas , broome style restrainers ( plas - labs , lansing , mi , united states ) . rats were then decapitated 1 h , 2 h , 4 h , 6 h , or 24 h following stress termination ( n = 6 for all time points ) . a nonstressed basal group ( n = 6 ) , designated as b , was implemented to allow for comparison of relative mrna expression . however , given that circadian variations in inflammatory mediators exist in rats [ 2022 ] , two additional basal groups , referred to as 4b and 6b , were implemented for the 4 h ( n = 6 ) and 6 h ( n = 6 ) time points . hence , there were three control groups : b , 4b , and 6b , which were used as controls for animals in the 1 h , 2 h , and 24 h groups , the 4 h group , and the 6 h group , respectively . following animal sacrifice , trunk blood was collected in vacutainer blood collection tubes containing k2 ethylenediaminetetraacetic acid ( edta ) ( bd , mississauga , on , canada ) and placed on ice for a maximum of 30 min before centrifugation at 10,000 g and 4c for 20 min . brains were extracted and the amygdala , hippocampus , hypothalamus , and medial prefrontal cortex were dissected out as previously described . following dissection , brain regions were immediately flash frozen on dry ice and stored in 2 ml rnase - free microtubes ( diamed , mississauga , on , canada ) at 80c for further processing . tissue collection areas and tools were sterilized with 70% ethanol and rnasezap ( sigma - aldrich , oakville , on , canada ) between dissections to prevent rnase activity . plasma corticosterone levels were measured in duplicate using a commercially available enzyme linked immunosorbent assay ( elisa ) kit ( cayman chemical company , ann arbor , mi , united states ) , according to the manufacturer 's protocol . the limit of detection for this assay at 80% binding / maximal binding is 30 pg / ml , while the standard curve ranges from 8.3 pg / ml to 5,000 pg / ml . intra- and interassay variability are less than 10% at the middle points of the standard curve . all plasma samples were diluted 1 : 1000 so that values fell within the standard curve . throughout the entirety of the mrna isolation procedure , 70% ethanol and rnasezap ( sigma - aldrich , oakville , on , canada ) were used to ensure that contamination by rnases did not occur . mrna was isolated from each sample using the rneasy plus universal mini kit ( qiagen , toronto , on , canada ) and the qiacube ( qiagen ) , according to the manufacturer 's protocols . briefly , samples were placed in 900 l of qiazol lysis reagent ( qiagen ) , containing phenol and guanidine thiocyanate , and homogenized for 2 min at 50 hz using 50 mm steel beads ( qiagen ) with a tissuelyser lt homogenizer ( qiagen ) . after lysis , 100 l of gdna eliminator solution and 180 l of chloroform were added to the samples , followed by centrifugation for 15 min at 4c and 12,000 g. the aqueous phase containing mrna was transferred to new microcentrifuge tubes and placed in the qiacube to carry out the rneasy plus universal program . in this automated protocol , 600 l of 70% ethanol is added to the samples , which are transferred to spin columns and centrifuged for 15 sec at 20c and 8,000 g. the centrifugation process is repeated and followed by buffer rinses , to aid in the removal of non - mrna biomolecules . finally , the spin column is placed in a 1.5 ml collection tube and nuclease - free h2o ( qiagen ) was added to the samples so that they were eluted to a final volume of 100 l . a nanodrop spectrophotometer ( thermo fisher scientific , waltham , ma , united states ) all samples had an a260/a280 ratio value between 1.9 and 2.1 , indicating high mrna purity . in order to synthesize cdna , qscript cdna supermix ( quanta biosciences , gaithersburg , md , united states ) was used , according to manufacturer 's protocols . to summarize , 1 g of mrna was added to 4 l of a reaction buffer ( mgcl2 , dntps , recombinant rnase inhibitor protein , qscript reverse transcriptase , random primers , oligo(dt ) primer , and stabilizers ) and nuclease - free h2o to result in a solution with a final volume of 20 l . samples were then incubated for 5 min at 25c , 30 min at 42c , and 5 min at 85c and held at 4c . as with mrna isolation , cdna samples were read on a nanodrop spectrophotometer to measure cdna content and stored at 20c . prior to quantitative pcr ( qpcr ) analysis , cdna aliquots were diluted in nuclease - free h2o to a final concentration of 25 ng/l . primers were first designed using the idtdna software ( coralville , ia , united states ) , primerquest . although all efforts were taken to check the literature for duplication of previous primers , any replication of previously published primers is purely accidental and likely due to using the same software program . the program was then instructed to construct primers that would result in products of no longer than 200 base pairs ( bp ) and had annealing temperatures ( tm ) of 5255c . primer pairs that met these criteria were then analyzed with the ncbi blast tool to check for specificity . finally , primers were reconstituted and diluted in nuclease - free h2o to obtain a final stock concentration of 10 m . for a list of primer sequences , see table 1 . quantitative pcr ( qpcr ) was performed using the quanta perfecta sybr green fastmix ( quanta biosciences ) on a rotor - gene q ( qiagen ) , according to manufacturer 's instructions . briefly , 50 ng of cdna , primers ( final concentration of 1 m for forward and reverse each ) , and nuclease - free h2o were added to a reaction buffer ( mgcl2 , dntps , accufast taq dna polymerase , sybr green i dye , and stabilizers ) . first , each primer was analyzed in a serially diluted standard curve to assess proper reaction conditions ( number of cycles and annealing temperature ) and to check for primer reaction efficiency ( must be between 80 and 120% ) . all primers were assayed using the following specifications : 1 min at 90c , 40 cycles of 95c for 5 sec and 60c for 30 sec . this was followed by a melt step ( 72c to 95c ) to assess for single primer products . each gene of interest 's primer pair was compared with all reference primer pairs to ascertain which ones have the most similar reaction efficiencies . for each gene of interest , since all samples per brain region could not fit in one qpcr assay , they were divided as follows : assays contained at least one sample from the b , 1 h , 2 h , and 24 h groups , from the 4b and 4 h group , or from the 6b and 6 h group . for each run , likewise , for each sample , control primers and primers of interest were assayed in triplicate , while no template controls ( containing nuclease - free h2o ) were assayed singularly . the ct value , or the point at which fluorescent detection crosses the threshold , was assessed for each well . the three ct values for each sample were averaged for both the reference primer and primer of interest . the difference between the reference primer and primer of interest , ct , was calculated for each sample . subsequently , the difference between the average ct value for the basal groups ( b , 4b , and 6b ) and each sample ( from the 1 h , 2 h , 24 h , 4 h , and 6 h groups ) , ct , was calculated . finally , the 2 values for each sample were calculated and the data was normalized so that the averages 2 of the basal groups were 1 . all data were analyzed using graphpad prism software ( graphpad , la jolla , ca , united states ) . before any analyses were performed , outliers were removed using the robust regression and outlier removal ( rout ) method in the software . it involves using nonlinear regression to fit a curve that is not influenced by outliers . this is followed by the identification of outliers using the residuals of the robust fit . one - way analyses of variance ( anova ) , and if applicable post hoc analyses with bonferroni 's correction for multiple comparisons , was used to compare the means of the 1 h , 2 h , and 24 h groups to the b group , while two - tailed student 's t - tests were used to compare the relative mrna expression levels of the 4 h and 6 h groups to the 4b and 6b group , respectively . f- or t - values , p - values , and eta square ( r ) values are reported for all data . all data are reported as means standard error of the mean ( sem ) and p < 0.05 was considered the threshold for statistical significance . two hours of restraint stress reliably elevates plasma corticosterone in our hands , but to determine if stress had residual effects on plasma corticosterone at the time points we examined gene expression , we measured plasma corticosterone levels at all time points . plasma corticosterone levels were not altered 1 h , 2 h , or 24 h post - stress termination ( f(3,19 ) = 1.75 , p > 0.05 , and r = 0.22 ) , compared to their time - matched basal ( b ) group ( table 2 ) . similarly , corticosterone levels remained the same at both the 4 h ( t(9 ) = 2.15 , p > 0.05 , and r = 0.34 ) and 6 h time points post - stress termination ( t(9 ) = 0.17 , p > 0.05 , and r = 0.003 ) , relative to their time - matched controls ( table 2 ) . finally , two - tailed t - tests showed a significant increase in the 6b group compared to the b group ( t(10 ) = 2.60 , p = 0.03 , and r = 0.40 ) but not between the 4b and b ( t(10 ) = 0.72 , p > 0.05 , and r = 0.05 ) , which is indicative of circadian changes in basal levels of corticosterone ( table 2 ) . this suggests that stress - induced corticosterone levels had recovered to basal levels at all time points examined in this study . 120 min of restraint stress altered amygdalar mrna expression of ido1 , kmo , and kynu but not of tdo , kat1 , or haao ( 3-hoa ) ( figure 2 ) . there was a significant effect of stress on amygdalar ido1 mrna expression ( f(3,17 ) = 5.71 , p = 0.007 ) , r = 0.50 , and subsequent post hoc analysis demonstrated that a significant difference between the b group and the 1 h group existed ( p < 0.05 ) but not between the b and the 2 h or 24 h groups ( figure 2 ) . there were no significant increases or decreases at the 4 h ( t(10 ) = 0.19 , p = 0.86 , and r = 0.003 ) or 6 h ( t(10 ) = 0.61 , p = 0.56 , and r = 0.04 ) time point with regard to ido1 mrna expression ( figure 2 ) . interestingly , acute restraint stress also produced a trending increase in kmo ( t(7 ) = 2.01 , p = 0.09 , and r = 0.36 ) and a significant increase in kynu ( t(7 ) = 3.03 , p = 0.02 , and r = 0.57 ) mrna expression at the 4 h time point ( figure 2 ) . there were no differences in kmo mrna expression at the 1 h , 2 h , 24 h ( f(3,17 ) = 1.80 , p = 0.19 , and r = 0.24 ) , or 6 h time point ( t(10 ) = 0.01 , p = 0.99 , and r = 0.000009 ) , compared to their time - matched basal controls . likewise , kynu mrna expression at the 1 h , 2 h , 24 h ( f(3,16 ) = 2.63 , p = 0.09 , and r = 0.33 ) , and 6 h time points ( t(7 ) = 0.17 , p = 0.87 , and r = 0.004 ) remained the same as time - matched controls ( figure 2 ) . at all time points examined ( 1 h , 2 h , and 24 h ; 4 h ; 6 h ) , there were no significant changes compared to time - matched basal controls ( b , 4b , and 6b , resp . ) with tdo ( f(3,19 ) = 0.94 , p = 0.44 , and r = 0.13 ; t(10 ) = 0.41 , p = 0.69 , and r = 0.02 ; t(10 ) = 0.78 , p = 0.45 , and r = 0.06 ) , kat1 ( f(3,18 ) = 0.43 , p = 0.74 , and r = 0.07 ; t(10 ) = 0.44 , p = 0.67 , and r = 0.02 ; t(10 ) = 0.87 , p = 0.41 , and r = 0.07 ) , and haao ( f(3,17 ) = 0.89 , p = 0.47 , and r = 0.14 ; t(10 ) = 0.85 , p = 0.41 , and r = 0.07 ; t(10 ) = 1.03 , p = 0.33 , and r = 0.10 ) gene expression ( figure 2 ) . there were no significant alterations of mrna expression in any of the kynurenine pathway enzymes ; however , hippocampal tdo expression was trending towards a decrease at the 4 h time point ( t(9 ) = 1.95 , p = 0.08 , and r = 0.30 ) , when compared to its time - matched control ( figure 3 ) . there were no significant differences between the 1 h , 2 h , and 24 h time points and the b group ( f(3,12 ) = 0.85 , p = 0.49 , and r = 0.18 ) or the 6 h time point and the 6b group ( t(9 ) = 0.50 , p = 0.623 , and r = 0.03 ) for tdo ( figure 3 ) . as mentioned above , at all time points examined ( 1 h , 2 h , and 24 h ; 4 h ; 6 h ) , there were no significant changes compared to time - matched basal controls ( b , 4b , and 6b , resp . ) in gene expression of ido1 ( f(3,14 ) = 1.40 , p = 0.29 , and r = 0.23 ; t(9 ) = 1.24 , p = 0.25 ; t(9 ) = 0.70 , p = 0.50 , and r = 0.05 ) , kat1 ( f(3,11 ) = 0.72 , p = 0.56 , and r = 0.16 ; t(9 ) = 0.30 , p = 0.77 , and r = 0.01 ; t(10 ) = 1.61 , p = 0.14 , and r = 0.21 ) , kmo ( f(3,15 ) = 1.13 , p = 0.37 , and r = 0.18 ; t(7 ) = 0.65 , p = 0.54 , and r = 0.06 ; t(8 ) = 0.43 , p = 0.68 , and r = 0.02 ) , kynu ( f(3,9 ) = 1.07 , p = 0.41 , and r = 0.26 ; t(7 ) = 0.45 , p = 0.67 , and r = 0.03 ; t(8 ) = 0.23 , p = 0.83 , and r = 0.006 ) , and haao ( f(3,11 ) = 1.20 , p = 0.35 , and r = 0.25 ; t(9 ) = 0.13 , p = 0.90 , and r = 0.002 ; t(8 ) = 0.27 , p = 0.79 , and r = 0.009 ) ( figure 3 ) . 120 min of restraint stress significantly altered hypothalamic mrna expression of tdo and haao but not of ido1 , kat1 , kynm , or kynu ( figure 4 ) . tdo mrna levels were significantly elevated in the hypothalamus at the 4 h time point ( t(6 ) = 2.48 , p = 0.05 , and r = 0.51 ) but not at the 1 h , 2 h , and 24 h ( f(3,17 ) = 0.79 , p = 0.52 , and r = 0.12 ) or 6 h time point ( t(8 ) = 1.81 , p = 0.11 , and r = 0.29 ) ( figure 4 ) . furthermore , even though haao mrna expression was not significantly altered at the 1 h , 2 h , or 24 h time point ( f(3,17 ) = 0.07 , p = 0.97 , and r = 0.01 ) or the 4 h time point ( t(9 ) = 0.10 , p = 0.93 , and r = 0.001 ) , it was significantly downregulated 6 h post - stress termination ( t(8 ) = 2.41 , p = 0.04 , and r = 0.42 ) ( figure 4 ) . alterations in kat1 mrna expression trended toward significance between the 1 h , 2 h , and 24 h and its time - matched basal control ( f(3,17 ) = 2.51 , p = 0.09 , and r = 0.31 ) , but post hoc analysis showed that this was not between the basal groups and any of the stress groups but between the stress groups ; furthermore , there were no changes at the 4 h or 6 h post - stress termination time point with kat1 mrna expression compared to their time - matched basal controls ( t(8 ) = 0.66 , p = 0.53 , and r = 0.05 ; t(8 ) = 0.25 , p = 0.81 , and r = 0.008 ) . there were also no significant differences with regard to ido1 ( f(3,17 ) = 1.61 , p = 0.22 , and r = 0.22 ; t(7 ) = 0.31 , p = 0.77 , and r = 0.01;t(8 ) = 0.65 , p = 0.54 , and r = 0.05 ) , kmo ( f(3,20 ) = 1.10 , p = 0.37 , and r = 0.14 ; t(10 ) = 0.09 , p = 0.93 , and r = 0.0009 ; t(10 ) = 0.11 , p = 0.91 , and r = 0.001 ) , or kynu ( f(3,19 ) = 0.92 , p = 0.45 , and r = 0.13 ) ; 4 h ( t(10 ) = 0.86 , p = 0.41 , and r = 0.07 ) ; 6 h ( t(9 ) = 1.03 , p = 0.33 , and r = 0.10 ) hypothalamic mrna expression at any time point examined compared to time - matched basal controls ( figure 4 ) . 120 min of acute restraint stress significantly altered mrna expression of kynu but not of tdo , ido1 , kat1 , kmo , or haao in the medial prefrontal cortex ( figure 5 ) . restraint stress altered kynu mrna expression between the 1 h , 2 h , and 24 h groups and their time - matched control b ( f(3,16 ) = 5.70 , p = 0.008 , and r = 0.52 ) , and post hoc analysis revealed that kynu mrna was downregulated at 1 h ( p < 0.01 ) , 2 h ( p < 0.01 ) , and 24 h ( p < 0.05 ) , in comparison to the b group . this decrease was not present at the 4 h ( t(7 ) = 0.62 , p = 0.55 , and r = 0.05 ) or 6 h post - stress ( t(10 ) = 0.60 , p = 0.56 , and r = 0.03 ) time point , when compared to their basal controls ( figure 5 ) . despite trending differences in tdo ( f(3,19 ) = 2.88 , p = 0.06 , and r = 0.31 ) expression between the b , 1 h , 2 h , and 24 h groups , post hoc analysis revealed that tdo expression was not significantly different between the 1 h , 2 h , and 24 h post - stress termination groups when compared to their time - matched basal control . furthermore , there were no differences between the 4 h ( t(10 ) = 1.03 , p = 0.33 , and r = 0.10 ) and 6 h ( t(10 ) = 0.74 , p = 0.48 , and r = 0.05 ) termination groups when compared to their time - matched basal controls . for the other enzymes in the kynurenine pathway , at all time points examined ( 1 h , 2 h , and 24 h ; 4 h ; 6 h ) , there were no significant changes compared to time - matched basal controls ( b , 4b , and 6b , resp . ) in gene expression of ido1 ( f(3,18 ) = 2.33 , p = 0.11 , and r = 0.28 ; t(10 ) = 0.25 , p = 0.81 , and r = 0.006 ; t(10 ) = 0.33 , p = 0.75 , and r = 0.01 ) , kat1 ( f(3,19 ) = 1.79 , p = 0.18 , and r = 0.22 ; t(9 ) = 1.42 , p = 0.19 , and r = 0.18 ; t(10 ) = 1.32 , p = 0.22 , and r = 0.15 ) , kmo ( f(3,15 ) = 0.88 , p = 0.48 , and r = 0.15 ; t(7 ) = 1.06 , p = 0.33 , and r = 0.14 ; t(10 ) = 0.17 , p = 0.87 , and r = 0.003 ) , and haao ( f(3,16 ) = 0.81 , p = 0.51 , and r = 0.13 ; t(7 ) = 0.12 , p = 0.91 , and r = 0.002 ; t(8 ) = 1.34 , p = 0.22 , and r = 0.18 ) ( figure 5 ) . our results are the first to show that acute stress alters gene expression of enzymes that regulate the kynurenine pathway in a regionally dependent manner ( figure 1 ) . specifically , we showed that in the amygdala there is an increase in ido1 , kmo , and kynu mrna expression following acute stress exposure ( figure 2 ) . interestingly , these increases occur along the pathway in a temporal manner , with ido1 mrna expression coming up 1 h post - stress termination followed by kmo and kynu mrna expression increases at 4 h post - stress termination ( figure 1 ) . the only other enzyme that was increased by acute stress exposure was tdo mrna expression in the hypothalamus ( figure 4 ) . in addition to these increases , it was also found that within the hypothalamus there was a delayed decrease in haao mrna expression ( figure 4 ) . decreases in the gene expression of enzymes in the kynurenine pathway were also found in the medial prefrontal cortex , specifically in kynu mrna expression , which was found to decrease at 1 h , 2 h , and 24 h post - stress termination time points ( figure 5 ) . finally , there were essentially no alterations from stress exposure on kynurenine pathway enzymes in the hippocampus , aside from a trending decrease in tdo mrna expression ( figure 3 ) . taken altogether this work showed that acute stress induces temporal and regional alterations in mrna levels of enzymes along the kynurenine pathway ; specifically , enzymes that promote the excitotoxic nmdar agonist quinolinic acid are upregulated in the amygdala following acute stress exposure , and quinolinic pathway enzymes are decreased in the medial prefrontal cortex ( figure 1 ) . these regional differences have been highlighted before , both in terms of acute stress - induced inflammation and also in kynurenine pathway metabolites in response to chronic stress [ 26 , 27 ] . furthermore , given the ability of quinolinic acid to act as a nmdar agonist and thus potentially increase excitability , these regional differences in the kynurenine pathway may somewhat underlie the regional changes in neuronal activity observed as a result of chronic stress or psychiatric illness , such as depression , specifically , hyperexcitable amygdala , and hypoexcitable cortex [ 1 , 410 ] . it should be noted that there is some variability in the mrna expression data . in spite of this , there were significant changes in enzymes , such that along the quin neurotoxic arm of the kynurenine pathway several enzymes are increased within the amygdala , while a downregulation of mrna expression of these enzymes was seen in the medial prefrontal cortex , following exposure to acute stress ( figure 1 ) . it is unclear what could be cause of this variability ; however , it has been previously reported that genes that are regulated by stress or inflammation exhibit a high degree of expression variability [ 26 , 28 ] . it is possible that this variability of gene expression is caused by differences in gene expression regulators ( e.g. , transcription factors , upstream activators ) across individuals [ 27 , 28 ] . as inflammation is known to induce enzymes along the kynurenine pathway , specifically , upregulation of ido1 and enzymes that increase quin , a neurotoxic kynurenine metabolite [ 1 , 13 , 29 ] , this regional specificity in response to acute restraint stress is aligned with recent work from our group demonstrating that acute stress produces regional effects on inflammatory mediators in the brain . specifically , we found that acute restraint stress leads to an increase of inflammatory mediators in circulation and the amygdala but a decrease in the medial prefrontal cortex . these regionally specific changes in inflammatory cytokine levels following acute stress parallel , to some degree , the region - specific changes we found herein . given the ability of inflammatory cytokines to modulate tryptophan metabolism along the kynurenine pathway , it seems quite plausible that stress modulates the expression of these enzymes in a regional manner through the regulation of inflammatory mediators . consistent with this , previous reports have demonstrated that glucocorticoids and inflammation can modulate the kynurenine pathway [ 12 , 29 , 30 ] and these data suggest that this may occur , in part , through alterations in the expression of the enzymes involved in the kynurenine metabolism . whole brain alterations of kynurenine metabolites and enzymes following stress exposure require tnf- and ifn . as we have recently shown that acute psychological stress reliably increases tnf- levels in the amygdala , it is quite likely that stress - induced tnf- signalling mediates the documented changes here in enzymes involved in kynurenine metabolism . having established this pattern , future work will seek to determine the discrete role of both tnf- and glucocorticoids in this process . given that the hippocampus has traditionally been studied as a region highly sensitive to the effects of stress , it is interesting to note that this was the only brain region examined in which we did not find any significant changes in the expression of enzymes involved in kynurenine metabolism . this is consistent with the apparent lack of effect we previously reported of acute stress on inflammatory mediators in the hippocampus and parallels studies demonstrating a lack of effect of chronic stress on downstream by - products of kynurenine metabolism within the hippocampus [ 31 , 32 ] . as our analysis did not subdivide the hippocampus into distinct regions ( e.g. , ca1 , ca3 , and dentate gyrus ) , these data do not preclude the possibility of discrete subregion changes in these enzymes within the hippocampus , a hypothesis which is consistent with other data indicating that there are differences in cytokine responses within hippocampal subregions . with regard to cellular sources of kynurenine metabolites , there are numerous reports that have shown that kyna producing enzymes are primarily localized to astrocytes in the brain , while quin producing enzymes are primarily localized to microglia [ 1 , 24 ] . as it has been reported that stress can increase microglial activation [ 34 , 35 ] , it is possible that these observed effects of stress on quin producing enzymes are due to increased microglial activation and the subsequent upregulation of gene transcription within these cells . future work should investigate the cell - type specific origins of kynurenine pathway metabolites and enzymes in response to stress . these regional differences in enzymes regulating the kynurenine pathway , in response to acute stress , could underlie changes that others have observed with chronic stress paradigms . specifically , previous reports have documented an upregulation of quin pathway metabolites in subcortical regions ( i.e. , the amygdala ) and downregulation of these metabolites in the cortex ( cingulate cortex ) [ 31 , 32 ] , which parallels the increase and decrease we documented of enzymes in these cascades , respectively . the functional relevance of these changes has yet to be established , but given that quin can activate nmda channels and promote excitotoxicity , it is interesting to note that biochemical indications of hyperexcitability and structural damage have been found within the amygdala following exposure to chronic stress and in depression . to summarize , these data present the first investigation of the effects of acute stress on regional changes in mrna expression of enzymes regulating the kynurenine pathway . specifically , we found changes in enzymes along the quin neurotoxic arm of the kynurenine pathway are increased within the amygdala , while a downregulation of mrna expression of these enzymes was seen in the medial prefrontal cortex , following exposure to acute stress ( figure 1 ) . more so , it seems quite plausible that these changes are a consequence of increased inflammatory mediators in discrete brain regions and could provide a mechanistic pathway linking stress - induced inflammation to alterations in cellular integrity within the brain . the data herein , demonstrating acute stress - induced changes in the gene expression of enzymes leading to kynurenine metabolites that alter excitatory signalling , may may provide insight into processes that can become dysregulated during chronic stress conditions , which could , in turn , contribute to some of the neuropathological effects documented following stress or inflammatory - related illnesses , particularly major depression .

tryptophan is an essential dietary amino acid that is necessary for protein synthesis , but also serves as the precursor for serotonin . however , in addition to these biological functions , tryptophan also serves as a precursor for the kynurenine pathway , which has neurotoxic ( quinolinic acid ) and neuroprotective ( kynurenic acid ) metabolites . glucocorticoid hormones and inflammatory mediators , both of which are increased by stress , have been shown to bias tryptophan along the kynurenine pathway and away from serotonin synthesis ; however , to date , there is no published data regarding the effects of stress on enzymes regulating the kynurenine pathway in a regional manner throughout the brain . herein , we examined the effects of an acute psychological stress ( 120 min restraint ) on gene expression patterns of enzymes along the kynurenine pathway over a protracted time - course ( 124 h post - stress termination ) within the amygdala , hippocampus , hypothalamus , and medial prefrontal cortex . time - dependent changes in differential enzymes along the kynurenine metabolism pathway , particularly those involved in the production of quinolinic acid , were found within the amygdala , hypothalamus , and medial prefrontal cortex , with no changes seen in the hippocampus . these regional differences acutely may provide mechanistic insight into processes that become dysregulated chronically in stress - associated disorders .

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

it is well known that it serves as the precursor for serotonin , but what is less well known is that the majority ( 9095% ) of tryptophan besides that utilized for protein synthesis , is shuttled to the kynurenine pathway , the end point of which is the generation of nicotinamide adenine dinucleotide ( nad+ ) . metabolites along the kynurenine pathway both are synthesized in the brain and can travel into the brain . furthermore , upregulation of the kynurenine pathway has been demonstrated in depressive disorders , and the intensity of depressive symptoms has been found to correlate with the upregulation of kynurenine pathway [ 210 ] . the enhancement of this differential metabolic route of tryptophan has been hypothesized to contribute to potential alterations in serotonergic function in depression , such that an increase shuttling of tryptophan to the kynurenine pathway could result in a deficit in serotonin synthesis . the other pathway , which results in the generation of quinolinic acid ( quin ) , has multiple steps , by which kynurenine is converted to intermediates through kynurenine 3-monooxygenase ( kmo ) and kynureninase ( kynu ) and , finally , by 3-hydroxyanthranilic acid 3,4-dioxygenase ( 3-hoa ) to quin ( figure 1 ) . as a highly selective nmdar agonist when taken altogether , the balance of the kynurenine metabolites , particularly kyna and quin , could play an important role in central nervous system functioning , and that disruption of this balance could be important for a number of neurological disorders , including neurodegenerative and affective disorders . it has been well established that inflammation can affect the activity of enzymes regulating the kynurenine pathway . specifically , ido1 is upregulated by proinflammatory cytokines , such as tnf- and ifn , and it has also been shown to be downregulated by anti - inflammatory cytokines , such as il-4 . furthermore , there is evidence that stress , and specifically the major output of the hpa axis , glucocorticoids , can upregulate tdo . consistent with this , kynurenine levels are elevated in diseases that have elevated glucocorticoid output and inflammatory mediators , such as major depression and coronary heart disease [ 1 , 2 ] . this increased pool of kynurenine may serve as a precursor for the generation of downstream metabolites , and in the context of disease states , it may amplify the production of the neurotoxic quin , which has been hypothesized to contribute to structural alterations and loss of neuropil in disease states such as major depression [ 8 , 12 , 13 ] . it is now well established that acute stress upregulates inflammatory mediators , both in humans , peripherally , and in rodents , both peripherally and in the brain [ 14 , 15 ] . recent work from our group has demonstrated that stress - induced changes in inflammatory mediators within the brain are not uniform and exhibit significant spatiotemporal specificity , with the amygdala appearing to be highly sensitive to induction of proinflammatory molecules following exposure to acute psychological stress . given that stress increases inflammatory cytokines and glucocorticoid levels , both of which have been shown to increase kynurenine levels , it seems highly plausible that stress could exert robust and dynamic effects on the pathway regulating kynurenine synthesis . consistent with this hypothesis , it has been shown that various acute stressors can lead to an upregulation of the kynurenine pathway [ 1719 ] ; however , only one of these studies actually examined expression levels of the enzymes involved in kynurenine metabolism and did so in a whole brain manner without any regional specificity . therefore , the goal of this study was to investigate whether acute stress exposure affected gene expression levels of the enzymes in the kynurenine pathway , particularly investigating the key corticolimbic brain regions involved in regulating the stress response , the amygdala , hippocampus , hypothalamus , and medial prefrontal cortex , in a time - dependent manner . however , given that circadian variations in inflammatory mediators exist in rats [ 2022 ] , two additional basal groups , referred to as 4b and 6b , were implemented for the 4 h ( n = 6 ) and 6 h ( n = 6 ) time points . hence , there were three control groups : b , 4b , and 6b , which were used as controls for animals in the 1 h , 2 h , and 24 h groups , the 4 h group , and the 6 h group , respectively . brains were extracted and the amygdala , hippocampus , hypothalamus , and medial prefrontal cortex were dissected out as previously described . in this automated protocol , 600 l of 70% ethanol is added to the samples , which are transferred to spin columns and centrifuged for 15 sec at 20c and 8,000 g. the centrifugation process is repeated and followed by buffer rinses , to aid in the removal of non - mrna biomolecules . to summarize , 1 g of mrna was added to 4 l of a reaction buffer ( mgcl2 , dntps , recombinant rnase inhibitor protein , qscript reverse transcriptase , random primers , oligo(dt ) primer , and stabilizers ) and nuclease - free h2o to result in a solution with a final volume of 20 l . subsequently , the difference between the average ct value for the basal groups ( b , 4b , and 6b ) and each sample ( from the 1 h , 2 h , 24 h , 4 h , and 6 h groups ) , ct , was calculated . two hours of restraint stress reliably elevates plasma corticosterone in our hands , but to determine if stress had residual effects on plasma corticosterone at the time points we examined gene expression , we measured plasma corticosterone levels at all time points . plasma corticosterone levels were not altered 1 h , 2 h , or 24 h post - stress termination ( f(3,19 ) = 1.75 , p > 0.05 , and r = 0.22 ) , compared to their time - matched basal ( b ) group ( table 2 ) . similarly , corticosterone levels remained the same at both the 4 h ( t(9 ) = 2.15 , p > 0.05 , and r = 0.34 ) and 6 h time points post - stress termination ( t(9 ) = 0.17 , p > 0.05 , and r = 0.003 ) , relative to their time - matched controls ( table 2 ) . finally , two - tailed t - tests showed a significant increase in the 6b group compared to the b group ( t(10 ) = 2.60 , p = 0.03 , and r = 0.40 ) but not between the 4b and b ( t(10 ) = 0.72 , p > 0.05 , and r = 0.05 ) , which is indicative of circadian changes in basal levels of corticosterone ( table 2 ) . there was a significant effect of stress on amygdalar ido1 mrna expression ( f(3,17 ) = 5.71 , p = 0.007 ) , r = 0.50 , and subsequent post hoc analysis demonstrated that a significant difference between the b group and the 1 h group existed ( p < 0.05 ) but not between the b and the 2 h or 24 h groups ( figure 2 ) . at all time points examined ( 1 h , 2 h , and 24 h ; 4 h ; 6 h ) , there were no significant changes compared to time - matched basal controls ( b , 4b , and 6b , resp . ) there were no significant alterations of mrna expression in any of the kynurenine pathway enzymes ; however , hippocampal tdo expression was trending towards a decrease at the 4 h time point ( t(9 ) = 1.95 , p = 0.08 , and r = 0.30 ) , when compared to its time - matched control ( figure 3 ) . as mentioned above , at all time points examined ( 1 h , 2 h , and 24 h ; 4 h ; 6 h ) , there were no significant changes compared to time - matched basal controls ( b , 4b , and 6b , resp . ) furthermore , even though haao mrna expression was not significantly altered at the 1 h , 2 h , or 24 h time point ( f(3,17 ) = 0.07 , p = 0.97 , and r = 0.01 ) or the 4 h time point ( t(9 ) = 0.10 , p = 0.93 , and r = 0.001 ) , it was significantly downregulated 6 h post - stress termination ( t(8 ) = 2.41 , p = 0.04 , and r = 0.42 ) ( figure 4 ) . alterations in kat1 mrna expression trended toward significance between the 1 h , 2 h , and 24 h and its time - matched basal control ( f(3,17 ) = 2.51 , p = 0.09 , and r = 0.31 ) , but post hoc analysis showed that this was not between the basal groups and any of the stress groups but between the stress groups ; furthermore , there were no changes at the 4 h or 6 h post - stress termination time point with kat1 mrna expression compared to their time - matched basal controls ( t(8 ) = 0.66 , p = 0.53 , and r = 0.05 ; t(8 ) = 0.25 , p = 0.81 , and r = 0.008 ) . 120 min of acute restraint stress significantly altered mrna expression of kynu but not of tdo , ido1 , kat1 , kmo , or haao in the medial prefrontal cortex ( figure 5 ) . restraint stress altered kynu mrna expression between the 1 h , 2 h , and 24 h groups and their time - matched control b ( f(3,16 ) = 5.70 , p = 0.008 , and r = 0.52 ) , and post hoc analysis revealed that kynu mrna was downregulated at 1 h ( p < 0.01 ) , 2 h ( p < 0.01 ) , and 24 h ( p < 0.05 ) , in comparison to the b group . this decrease was not present at the 4 h ( t(7 ) = 0.62 , p = 0.55 , and r = 0.05 ) or 6 h post - stress ( t(10 ) = 0.60 , p = 0.56 , and r = 0.03 ) time point , when compared to their basal controls ( figure 5 ) . despite trending differences in tdo ( f(3,19 ) = 2.88 , p = 0.06 , and r = 0.31 ) expression between the b , 1 h , 2 h , and 24 h groups , post hoc analysis revealed that tdo expression was not significantly different between the 1 h , 2 h , and 24 h post - stress termination groups when compared to their time - matched basal control . furthermore , there were no differences between the 4 h ( t(10 ) = 1.03 , p = 0.33 , and r = 0.10 ) and 6 h ( t(10 ) = 0.74 , p = 0.48 , and r = 0.05 ) termination groups when compared to their time - matched basal controls . for the other enzymes in the kynurenine pathway , at all time points examined ( 1 h , 2 h , and 24 h ; 4 h ; 6 h ) , there were no significant changes compared to time - matched basal controls ( b , 4b , and 6b , resp . ) in gene expression of ido1 ( f(3,18 ) = 2.33 , p = 0.11 , and r = 0.28 ; t(10 ) = 0.25 , p = 0.81 , and r = 0.006 ; t(10 ) = 0.33 , p = 0.75 , and r = 0.01 ) , kat1 ( f(3,19 ) = 1.79 , p = 0.18 , and r = 0.22 ; t(9 ) = 1.42 , p = 0.19 , and r = 0.18 ; t(10 ) = 1.32 , p = 0.22 , and r = 0.15 ) , kmo ( f(3,15 ) = 0.88 , p = 0.48 , and r = 0.15 ; t(7 ) = 1.06 , p = 0.33 , and r = 0.14 ; t(10 ) = 0.17 , p = 0.87 , and r = 0.003 ) , and haao ( f(3,16 ) = 0.81 , p = 0.51 , and r = 0.13 ; t(7 ) = 0.12 , p = 0.91 , and r = 0.002 ; t(8 ) = 1.34 , p = 0.22 , and r = 0.18 ) ( figure 5 ) . our results are the first to show that acute stress alters gene expression of enzymes that regulate the kynurenine pathway in a regionally dependent manner ( figure 1 ) . specifically , we showed that in the amygdala there is an increase in ido1 , kmo , and kynu mrna expression following acute stress exposure ( figure 2 ) . interestingly , these increases occur along the pathway in a temporal manner , with ido1 mrna expression coming up 1 h post - stress termination followed by kmo and kynu mrna expression increases at 4 h post - stress termination ( figure 1 ) . in addition to these increases , it was also found that within the hypothalamus there was a delayed decrease in haao mrna expression ( figure 4 ) . decreases in the gene expression of enzymes in the kynurenine pathway were also found in the medial prefrontal cortex , specifically in kynu mrna expression , which was found to decrease at 1 h , 2 h , and 24 h post - stress termination time points ( figure 5 ) . finally , there were essentially no alterations from stress exposure on kynurenine pathway enzymes in the hippocampus , aside from a trending decrease in tdo mrna expression ( figure 3 ) . taken altogether this work showed that acute stress induces temporal and regional alterations in mrna levels of enzymes along the kynurenine pathway ; specifically , enzymes that promote the excitotoxic nmdar agonist quinolinic acid are upregulated in the amygdala following acute stress exposure , and quinolinic pathway enzymes are decreased in the medial prefrontal cortex ( figure 1 ) . these regional differences have been highlighted before , both in terms of acute stress - induced inflammation and also in kynurenine pathway metabolites in response to chronic stress [ 26 , 27 ] . furthermore , given the ability of quinolinic acid to act as a nmdar agonist and thus potentially increase excitability , these regional differences in the kynurenine pathway may somewhat underlie the regional changes in neuronal activity observed as a result of chronic stress or psychiatric illness , such as depression , specifically , hyperexcitable amygdala , and hypoexcitable cortex [ 1 , 410 ] . in spite of this , there were significant changes in enzymes , such that along the quin neurotoxic arm of the kynurenine pathway several enzymes are increased within the amygdala , while a downregulation of mrna expression of these enzymes was seen in the medial prefrontal cortex , following exposure to acute stress ( figure 1 ) . it is unclear what could be cause of this variability ; however , it has been previously reported that genes that are regulated by stress or inflammation exhibit a high degree of expression variability [ 26 , 28 ] . as inflammation is known to induce enzymes along the kynurenine pathway , specifically , upregulation of ido1 and enzymes that increase quin , a neurotoxic kynurenine metabolite [ 1 , 13 , 29 ] , this regional specificity in response to acute restraint stress is aligned with recent work from our group demonstrating that acute stress produces regional effects on inflammatory mediators in the brain . specifically , we found that acute restraint stress leads to an increase of inflammatory mediators in circulation and the amygdala but a decrease in the medial prefrontal cortex . given the ability of inflammatory cytokines to modulate tryptophan metabolism along the kynurenine pathway , it seems quite plausible that stress modulates the expression of these enzymes in a regional manner through the regulation of inflammatory mediators . consistent with this , previous reports have demonstrated that glucocorticoids and inflammation can modulate the kynurenine pathway [ 12 , 29 , 30 ] and these data suggest that this may occur , in part , through alterations in the expression of the enzymes involved in the kynurenine metabolism . as we have recently shown that acute psychological stress reliably increases tnf- levels in the amygdala , it is quite likely that stress - induced tnf- signalling mediates the documented changes here in enzymes involved in kynurenine metabolism . given that the hippocampus has traditionally been studied as a region highly sensitive to the effects of stress , it is interesting to note that this was the only brain region examined in which we did not find any significant changes in the expression of enzymes involved in kynurenine metabolism . this is consistent with the apparent lack of effect we previously reported of acute stress on inflammatory mediators in the hippocampus and parallels studies demonstrating a lack of effect of chronic stress on downstream by - products of kynurenine metabolism within the hippocampus [ 31 , 32 ] . , ca1 , ca3 , and dentate gyrus ) , these data do not preclude the possibility of discrete subregion changes in these enzymes within the hippocampus , a hypothesis which is consistent with other data indicating that there are differences in cytokine responses within hippocampal subregions . with regard to cellular sources of kynurenine metabolites , there are numerous reports that have shown that kyna producing enzymes are primarily localized to astrocytes in the brain , while quin producing enzymes are primarily localized to microglia [ 1 , 24 ] . as it has been reported that stress can increase microglial activation [ 34 , 35 ] , it is possible that these observed effects of stress on quin producing enzymes are due to increased microglial activation and the subsequent upregulation of gene transcription within these cells . these regional differences in enzymes regulating the kynurenine pathway , in response to acute stress , could underlie changes that others have observed with chronic stress paradigms . , the amygdala ) and downregulation of these metabolites in the cortex ( cingulate cortex ) [ 31 , 32 ] , which parallels the increase and decrease we documented of enzymes in these cascades , respectively . the functional relevance of these changes has yet to be established , but given that quin can activate nmda channels and promote excitotoxicity , it is interesting to note that biochemical indications of hyperexcitability and structural damage have been found within the amygdala following exposure to chronic stress and in depression . to summarize , these data present the first investigation of the effects of acute stress on regional changes in mrna expression of enzymes regulating the kynurenine pathway . specifically , we found changes in enzymes along the quin neurotoxic arm of the kynurenine pathway are increased within the amygdala , while a downregulation of mrna expression of these enzymes was seen in the medial prefrontal cortex , following exposure to acute stress ( figure 1 ) . more so , it seems quite plausible that these changes are a consequence of increased inflammatory mediators in discrete brain regions and could provide a mechanistic pathway linking stress - induced inflammation to alterations in cellular integrity within the brain . the data herein , demonstrating acute stress - induced changes in the gene expression of enzymes leading to kynurenine metabolites that alter excitatory signalling , may may provide insight into processes that can become dysregulated during chronic stress conditions , which could , in turn , contribute to some of the neuropathological effects documented following stress or inflammatory - related illnesses , particularly major depression .

interpersonal conflicts are emotionally difficult and require regulation of negative affect and behavior for successful resolution ( arriaga and rusbult , 1998 ; lopes et al . , 2011 ) . these self - regulatory mechanisms are reliant on cognitive control processes mediated by the lateral prefrontal cortex ( lpfc ; heatherton and wagner , 2011 ; ochsner et al . , lpfc dysfunction in cognitive control is a well - established neural impairment in schizophrenia ( barch , 2005 ; manoach , 2003 ; minzenberg et al . , 2009 ) , 2001 ; nishimura et al . , 2011 ; perlstein et al . , 2001 ; van veelen et al . , 2010 ) and global functioning deficits ( sanz et al . , 2009 ; yoon et al . , 2008 ) . however , there is a paucity of research examining how lpfc dysfunction contributes to illness severity . consistent with the diathesis stress model , one proposal is that lpfc dysfunction is a biological vulnerability that , in the presence of an interpersonal stressor , contributes to symptom exacerbation via impaired cognitive control of emotion ( hooker et al . , 2010 ; cognitive control of emotion comprises the dual processes of explicit and effortful control of the experience and expression of internal emotional states ( emotion regulation ) and the implicit and more automatic control of how external emotional information influences behavior ( gyurak et al . , 2011 ; gross and thompson , 2007 , ochsner et al . , 2005 ; quirk et al . , 2006 ) . the lpfc , comprising both dorsolateral ( dlpfc ) and ventrolateral ( vlpfc ) regions , is consistently implicated in laboratory assessed cognitive control of emotion ( ochsner and gross , 2005 ; ochsner et al . , 2012 ; pessoa , 2008 ) , including paradigms assessing explicit emotion regulation ( e.g. reappraisal paradigms ) as well as paradigms assessing implicit cognitive control of external and/or irrelevant emotional information ( e.g. emotional stroop , emotional flanker tasks ) ( gyurak et al . , lower vlpfc activity during social exclusion predicts higher self - reported distress ( eisenberger et al . , 2003 ) . similarly , lower vlpfc activity when viewing negative facial expressions predicts increased negative mood and maladaptive behavior following interpersonal conflicts ( hooker et al . , 2010 ) . in schizophrenia , interpersonal conflicts , especially conflicts characterized by criticism , predict symptom exacerbation and higher relapse rates ( hooley , 2007 ) , and symptom exacerbation in response to interpersonal criticism is related to poor working memory and cognitive control ( rosenfarb et al . , 2000 ) neurocognitive processes known to be mediated by the lpfc ( aron et al . , 2004 ; curtis and d'esposito , 2003 ) and to predict functional outcome ( milev et al . , 2005 ) . collectively , these data suggest that compromised lpfc function is a vulnerability for symptom exacerbation and functional difficulties in response to interpersonal conflict . however , research attempting to connect lpfc activity , interpersonal conflict , and schizophrenia symptomatology is sparse . to date , studies have primarily focused on the direct relationship between the lpfc and symptoms ( goghari et al . , 2010 ; macdonald et al . , 2005 ; menon et al . , 2001 ; nishimura et al . , 2011 ; perlstein et al . , 2001 ; van veelen et al . , , only one study has examined the interaction between lpfc activation , symptoms , and real - world social interactions . in a sample of healthy individuals characterized along the schizophrenia - risk dimension social anhedonia , individuals at high - risk for schizophrenia ( i.e. those with high social anhedonia ) with low lpfc activation had worse symptoms of paranoia on days with distressing interpersonal conflicts compared to days without distressing interpersonal conflicts ( hooker et al . , 2014 ) . to date , no study has examined this relationship in individuals with schizophrenia . the scarcity of studies directly linking lpfc function to social interactions and subsequent symptoms in schizophrenia may result from limitations inherent in the currently available and commonly used methods . first , the tasks traditionally used to assess lpfc function , such as response inhibition or working memory tasks ( barch , 2005 ) , although robust activators , may not be the most sensitive measures for assessing how lpfc activity relates to real - world social functioning because they do not directly capture cognitive control of emotional information . given the inherently affective nature of social interactions and the accompanying need for self - regulation ( arriaga and rusbult , 1998 ) , tasks assessing the interaction between lpfc mediated cognitive control and emotional information may provide a more accurate reflection of the inhibitory demands of real - world social contexts . by using cold cognitive tasks and not assessing lpfc mediated cognitive control in relation to emotional information , previous studies may have lacked the sensitivity necessary to identify the role of the lpfc in individuals ' response to social conflict . second , prior research has primarily assessed social interactions using laboratory - based one - time retrospective measures of functioning . although these provide an overview of an individual 's general level of functioning , they are not well suited for capturing the multidimensional nature of social interactions in daily life , and rarely provide the context in which they occur ( trull and ebner - priemer , 2009 ) . social interactions do not occur in a vacuum ; day - to - day changes in social behavior may be prompted at a specific time , in a specific setting , or in the context of a particular interpersonal relationship . one - time retrospective evaluations of social functioning miss these nuances , calling into question their ecological validity ( yager and ehmann , 2006 ) . experience sampling methods ( esm ) , a technique in which assessments are collected in the person 's natural environment and repeated over time , have revealed a nuanced relationship between changes in the social environment , particularly social stressors , and symptoms ( myin - germeys et al . , 2009 ; oorschot et al . , 2009 ) , thus , using esm in conjunction with neuroimaging techniques may provide a more sensitive and ecologically valid approach to understanding the contribution of lpfc dysfunction to social deficits in schizophrenia . the present study addressed these prior limitations by combining fmri and esm to test whether people with schizophrenia have lpfc deficits in cognitive control of negative emotional information , and , if so , whether these lpfc deficits are related to changes in mood and symptoms following interpersonal conflict . individuals with schizophrenia and demographically - matched healthy controls completed an adapted version of the multi - source interference task ( msit ) , a cognitive control task specifically designed to activate the cingulo frontal parietal cognitive control network ( bush and shin , 2006 ) . in our adapted version , the msit - emotion , msit stimuli are superimposed on a negative emotional scene that is irrelevant to the central task demand . thus , rather than requiring explicit manipulation of emotional material and/or the explicit regulation of internal emotional states ( i.e. emotion regulation through reappraisal / suppression ) , the task requires participants to engage cognitive control mechanisms to inhibit the effect of irrelevant emotional information in the external environment on task performance . this process is thought to more accurately reflect the interaction of emotion and cognitive control in real - world social contexts ( silbersweig et al . , 2007 ) , and considered to be a form of implicit cognitive control of emotion ( gyurak et al . , 2011 ) . our measure of cognitive control of emotional information was lpfc activity when inhibiting the effect of irrelevant emotional information during high interference trials ( when cognitive control skills are most challenged ) . stimuli were also superimposed on neutral pictures , included to test the specificity of schizophrenia participants ' lpfc deficits for controlling emotional information . following the scan , schizophrenia participants completed an online , structured daily - diary questionnaire of mood and symptoms every evening for 3 weeks . end - of - the - day reports provide data on daily events and day - to - day symptom fluctuations whilst minimizing interference with participants ' daily experience . participants rated the extent to which they experienced mood and schizophrenia - spectrum symptoms , as well as the occurrence of interpersonal conflict and associated distress . we hypothesized that : 1 ) schizophrenia participants would show reduced lpfc activity during cognitive control of emotional information compared to healthy participants ; and 2 ) among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . specifically , we expect that schizophrenia participants with low lpfc activity will have an increase in negative mood and psychotic symptoms the day after highly distressing interpersonal conflict . 23 individuals with schizophrenia or schizoaffective disorder and 24 healthy controls were recruited from the greater boston area . groups were matched for gender , age , education , and iq ( table 1 ) . inclusion criteria for all participants were : age 1865 , primary english speaker , no neurological or major medical illness , no head trauma history , no substance abuse within 6 months , and no current / past substance dependence . inclusion criteria for schizophrenia participants were : diagnosis of schizophrenia or schizoaffective disorder , no comorbid axis i disorders , and no history of electroconvulsive therapy . inclusion criteria for healthy participants were : no current / past axis i disorders , no first - degree relative with a psychotic disorder , scores within 1.5 standard deviations of the population mean on five measures of schizotypal personality ( perceptual aberration scale ( chapman et al . , 1978 ) , magical ideation scale ( eckblad & chapman , 1983 ) , referential thinking scale ( lenzenweger et al . , 1997 ) , physical anhedonia scale ( chapman et al . , 1976 ) , and revised social anhedonia scale ( eckblad et al . , 1982 ) ) . psychopathology was assessed with the structured clinical interview for dsm - iv axis i disorders ( first et al . , 2002 ) ; symptoms were assessed using the positive and negative syndrome scale ( panss ; kay et al . , 1987 ) ; social functioning was assessed with the social adjustment scale self - report ( sas - sr ; weissman et al . , 1978 ) and the global functioning : social scale ( gf : s ; auther et al . , 2006 ) ; positive and negative mood were assessed using the positive and negative affect schedule ( panas ; watson et al . , 1988 ) . clinical assessments were conducted by trained phd - level clinical psychologists ( lmt , shl ) and supervised by a licensed clinical psychologist ( cih ) . participants completed behavioral assessments , returned a separate day for the scan , and were subsequently oriented to the daily - diary . 1 ) , an adaptation of the msit , a standard cognitive control paradigm that combines the flanker ( eriksen and eriksen , 1974 ) and simon ( simon and berbaum , 1990 ) effects to robustly activate the cingulo frontal parietal cognitive control network within individual subjects ( bush and shin , 2006 ) . in the msit , subjects see sets of three numbers ( 1 , 2 , or 3 ) and report the identity of the number that differs from the other two . during control trials , position of the target number is always congruent with its position on the button box and the other two distracter numbers are zeros ; thus there is no spatial or semantic interference . during interference trials , position of the target number is incongruent with its position on the button box and the distracters are other numbers . this creates two sources of interference : spatial incongruence between the target and response ( simon effect ) and semantic incongruence between the target and distracter numbers ( flanker effect ) . in the msit - emotion , task stimuli ( i.e. the numbers for interference and control trials ) are shown on a background of either a neutral or negative picture , resulting in four conditions : neutral control ( neucon ) , neutral interference ( neuint ) , negative control ( negcon ) , and negative interference ( negint ) . 48 neutral pictures and 48 negative pictures were selected from the international affective picture system ( iaps ; lang et al . , 2005 ; see supplemental methods ) . trials were presented in an event - related design so that correct and incorrect trials could be analyzed separately . each trial was presented for 1.75 s followed by a variable inter - trial interval ( central fixation - cross ) for 410 s. pictures were randomly assigned to conditions ; trial types were presented in a fixed , pseudo - random order . before entering the scanner , participants completed a practice version of the task ( 10 trials for each of the 4 conditions ) . participants were required to achieve 80% accuracy or higher before entering the scanner to ensure task competency ; thus any group differences would not be due to task difficulty / performance differences . after the scan , participants rated the valence of each neutral and negative picture ( see supplemental methods ) . images were acquired on a siemens 3 t timtrio scanner and analyzed using spm8 within the general linear model ( glm ) framework . for single - subject glms , vectors of onset times with 0 s duration were defined for each event in each condition ( neucon , neuint , negcon , negint ) and convolved with the canonical hrf . the data were high - pass filtered ( cut - off period : 128 s ) . artifact detection and movement correction were conducted using the artifact detection tools software package ( art ; whitfield - gabrieli , 2009 ) . regressors were created to exclude volumes with gross motion ( > 3 mm relative to previous time frame ) or spiking artifacts ( global mean image intensity greater than 3sd from the mean of the entire time series within a scan ) from analysis . there were no group differences in number of outliers identified ( sz max = 30 ; hc max = 28 ) . contrasts were calculated for each of the four conditions relative to fixation periods , and for interference versus control for neutral and negative pictures ( i.e. neuint > neucon ; negint > negcon ) . we tested our central hypothesis that , compared to healthy controls ( hc ) , schizophrenia ( sz ) participants would show reduced lpfc activity during cognitive control of emotional information using a 2 2 2 full factorial anova with group ( hc / sz ) , emotion ( negative / neutral ) and condition ( interference / control ) as factors . the predicted group emotion condition interaction was that hc would have greater cognitive control related activity ( interference control ) than sz when inhibiting irrelevant negative emotional information on interference trials versus control trials compared to inhibiting irrelevant neutral information on interference versus control trials . clusters showing the predicted group emotion condition interaction within the lpfc were identified and contrast values for each condition for each subject were extracted from the peak voxel . the difference between cognitive control activation on negative trials and cognitive control activation on neutral trials ( i.e. ( negint neg con ) ( neuint neucon ) ) was calculated and used in the analysis with the daily - diary data . group maps were thresholded at t = 3.18 , p < 0.001 ( uncorrected ) with 10 voxels/270 mm cluster size . , 2004 ; maldjian et al . , 2003 ) to create our lpfc region of interest mask , comprising brodmann 's areas ( ba ) , 9 and 46 ( dorsal lpfc ; middle frontal gyri extending into portions of the superior frontal gyri ) and ba 44 , 45 , and 47 ( ventral lpfc ; inferior frontal gyri ) . activation clusters surviving small volume corrections within this mask ( fwe , p < 0.05 ) are reported . the daily - diary consisted of a structured questionnaire completed online at the end of each day ( i.e. right before bed ) for 21 consecutive days . participants rated the extent to which they experienced mood and psychotic symptoms , including positive and negative mood , paranoia , hallucinations / odd experiences , negative symptoms , and disorganized symptoms . questions were rated on a 1-to-5 scale ( 1 = not at all ; 5 = extremely ) . participants also reported whether they had an interpersonal conflict ( yes / no ) , and if so , the extent to which the conflict caused distress and was resolved . mood items were selected from the profile of mood states - short form ( poms - sf ; curran et al . , 1995 ) . diary questions for psychotic symptoms were adapted from previous literature using esm in schizophrenia ( myin - germeys et al . , 2002 ; myin - germeys and van os , 2007 ; thewissen et al . , 2008 ) as well as commonly used diagnostic interviews and questionnaires ( e.g. schizotypal personality questionnaire ; psychotic - like experiences scale ) . consistent with prior esm literature on stress , we used the subjective rating of conflict distress as the measure of social stress . if more than one conflict occurred , distress ratings were summed . if no conflict occurred that day , distress was coded as 0 . research staff monitored diary entries daily and sent a reminder email if a day was missed . participants could not miss more than 6 days.hypothesis 2among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . analysis of daily - diary responses and lpfc activity used a hierarchical linear modeling ( hlm ) approach with the mixed procedure in sas ( sas institute , cary , nc ) . the daily diary has a hierarchical structure in which the 21 daily assessments are nested within participant . we expected that the relationship between conflict distress and symptom severity within each participant would vary as a function of the between - subject variable , lpfc activity . thus , we conducted two - level models with the mixed procedure in sas , which allows for the simultaneous analysis of within and between subject variables . the repeated assessments over 21 days allowed us to examine the longitudinal effect of conflict distress on the change in mood and symptoms the following day . the main analyses tested whether daily symptom - severity is predicted by the interaction of lpfc activity and the previous day 's conflict distress . thus , by accounting for previous day symptom - severity , we are able to test the effect of lpfc activity on the change in symptom - severity the day after conflict . we expected that the interaction of lpfc activity and previous day conflict distress would significantly predict symptom - severity , such that sz participants with low lpfc activity would have an increase in symptoms the day after high conflict distress . all variables were grand - mean centered . if the interaction between yesterday 's conflict distress and lpfc activity was significant , the effects of the interaction were further examined using simple slope analysis as described by aiken and west ( 1991 ) . simple slope analyses for high and low groups were tested at 1sd above and below each centered mean . for completeness , we also report the relationship between symptom severity and : 1 ) lpfc activity ; 2 ) same day conflict distress ; and 3 ) the interaction of same day conflict distress and lpfc activity . test retest reliability ( i.e. stability of the diary ratings ) was examined by correlating average diary ratings for the first and second halves of the diary period . internal consistency of diary items in each symptom construct was measured with standardized alpha coefficients . construct validity was examined by correlating diary variables with corresponding mood and symptom constructs assessed with standard laboratory - based instruments . one sample t - tests of interference versus control conditions ( i.e. neuint > neucon and negint > negcon ) confirmed that the msit - emotion task activates the expected cognitive control network . both hc and sz participants demonstrated greater activity for interference versus control conditions in cognitive control regions , including inferior , middle and superior frontal gyri ( lpfc ) , and anterior cingulate cortex ( acc ) ( fig . 2 ; supplementary tables 1 and 2 ) . all participants responded slower and were less accurate on interference trials compared to control trials ; participants also responded slower on negative picture trials compared to neutral picture trials . a significant condition emotion interaction in the sz group indicated that individuals with schizophrenia were slower and less accurate on negative interference trials relative to neutral interference trials and negative and neutral control trials . these data indicate that the negative interference trials , posited to be the most taxing on cognitive control resources , are particularly challenging for sz participants . compliance was high ; average number of diary days completed was 20.0 ( sd 1.8 ) . on average , participants had 11.6 conflicts over the diary period and conflicts occurred on 28.8% of diary days . the consistent daily responses and number of conflicts indicate sufficient data to analyze the relationship between conflict and symptoms . retest reliability shows that diary estimates are stable ( i.e. correlations between the first 10 days and last 11 days had rs > .75 for all variables ) . the items included in each mood and symptom construct were chosen according to existing literature and a priori face validity . after the data were collected , standardized alpha of each construct was examined to verify internal consistency , and items that were not correlated with the construct were removed . this analysis revealed that reverse - coded items were not effective for assessing symptoms and were removed ( i.e. two items were dropped from the negative symptom construct and one item from the disorganized symptom construct ) . alpha was low for the negative symptom construct ( = .58 ) but acceptable for all other mood and symptom constructs ( > .70 ) . analysis of construct validity showed that 21-day average daily - diary ratings for negative mood significantly correlated with laboratory measures of trait negative affect ( panas negative affect , r = .55 , p < .05 ) . daily - diary ratings of positive mood were not correlated with panas positive affect ( r = .01 ) ; however , there is no overlap between the panas and poms positive mood items , so a different lab - based assessment of positive mood may be necessary to test construct validity . daily diary measures of positive , negative , and disorganized symptoms were not correlated with positive and negative symptom scale ( panss ) measures of positive , negative , and disorganized symptoms . this does not necessarily indicate that the diary measures are inaccurate or invalid , but it does suggest that different factors may be influencing symptom assessments from the diary and the panss . research comparing multiple methods of assessment may provide more information regarding the validity of different methods . in addition , there were significant correlations between daily - diary measures of interpersonal conflict and lab - based measure social functioning . for example , participants with worse social impairment as measured by the social adjustment scale had more interpersonal conflicts over the 21 day diary period ( r = .50 , p = .03 ) and worse conflict distress ( r = .50 , p = .03 ) . a similar pattern was observed for the global functioning : social scale but the correlations were not significant . panss measures of interpersonal functioning were also related to daily - diary measures of interpersonal conflict . higher ratings on panss hostility were associated with more interpersonal conflicts ( r = .58 , p = .008 ) and more conflict distress ( r = .66 , p = .002 ) . a higher degree of panss uncooperativeness was also associated with more interpersonal conflicts ( r = .50 , p = .02 ) and more conflict distress ( r = .69 , p = .001 ) . together these data indicate that the daily - diary assessments of mood and interpersonal conflict are measuring the intended construct . since prior studies have found a relationship between social stress and symptoms , we conducted simple regression analysis to look at the relationship between conflict distress and symptom severity . consistent with prior findings , daily ratings of high conflict distress were associated with worse symptom severity , including worse negative mood , paranoia , negative symptoms and disorganized symptoms ( table 3 ) . we also conducted simple regression analysis to examine the relationship between lpfc activity and symptoms regardless of interpersonal conflict : lpfc activity did not relate to any of the symptom measures . we used hlm to investigate the interaction of right dlpfc activity with conflict distress on the same day of the conflict ( i.e. same day conflict distress ) . no significant interactions were found ; that is , on the same day as an interpersonal conflict , individuals ' dlpfc activation during cognitive control of emotion did not interact with conflict distress in predicting symptom severity that day ( table 3 ) . analysis revealed a single cluster of activation in our lpfc region of interest mask , in the right superior frontal gyrus ( ba9 ; dlpfc ) , with a significant group emotion condition interaction(hc > sz ) that survived small volume correction ( fwe p < 0.05 ; see fig . 3 barplots illustrate that hc participants consistently demonstrate the expected pattern of activation in the right dlpfc : increased activation in negint compared to negcon conditions ; sz participants show the opposite pattern , deactivating in negint compared to negcon conditions . activation clusters with significant group emotion condition interaction(hc > sz ) were also present in the right lateral orbital frontal gyrus extending into the insula ( lofg ; ba11 ) and the left dorsal anterior cingulate ( dacc ; ba32 ) , but these did not survive small volume correction in either the lpfc region of interest mask ( i.e. the lofg cluster ) or an anterior cingulate mask comprising ba24 and ba32 ( see supplemental table 3 ) . no regions showed an interaction in the opposite direction ( i.e. sz > hc ) . the cluster in the right superior frontal gyrus ( dlpfc ; ba 9 , peak : x = 27 , y = 53 , z = 40 ) was the only cluster that survived multiple comparison correction within our lpfc region of interest mask . all subsequent analyses investigating lpfc activity and diary data were conducted using the difference score ( negint negcon ) ( neuint neucon ) , i.e. our measure of cognitive control of emotional information , calculated from the contrast values in each condition extracted from the peak of this cluster in the right dlpfc.hypothesis 2among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . we used hlm to examine the interaction of dlpfc activity with conflict distress from the previous day ( i.e. previous day conflict distress ) in predicting symptom severity on days following interpersonal conflicts . dlpfc activity significantly interacted with previous day conflict distress in predicting change in positive mood [ f = 8.03 , b = 0.02 , p = 0.01 ] and negative mood [ f = 4.49 , b = 0.01 , p = 0.047 ] the following day . 4 . the interaction of previous day conflict distress and dlpfc activity did not significantly predict any other symptom ( see table 3 ) . simple slope analyses were conducted to better understand the interactions predicting positive and negative mood . analysis of effects influencing positive mood showed the following : the effect of dlpfc activity on positive mood was examined separately for days when previous day conflict distress was low and days when previous day conflict distress was high . on days when previous day conflict distress was low 0.02 ) , t(19 ) = 1.51 , p = 0.30 ] ( blue line plotted in fig . however , when previous day conflict distress was high , dlpfc activity significantly predicted positive mood the following day [ b = 0.07(s.e . 0.02 ) , t(19 ) = 3.1 , p = 0.006 ] , such that schizophrenia participants with higher dlpfc activity had higher positive mood compared to participants with lower dlpfc activity ( red line plotted in fig . next we examined the effect of conflict distress on positive mood for schizophrenia participants with high dlpfc activity and low dlpfc activity . for schizophrenia participants with low dlpfc activity , previous day conflict distress was not related to positive mood [ b = 0.02(s.e . 0.02 ) , t(19 ) = 1.0 , p = 0.35 ] . however , for schizophrenia participants with high dlpfc activity , previous day conflict distress had a significant effect on positive mood the next day [ b = 0.07(s.e . 0.02 ) , t(19 ) = 3.6 , p = 0.002 ] , such that positive mood was higher on days following high conflict distress . these results indicate that , among schizophrenia participants with high dlpfc activity , positive mood is significantly higher on days preceded by high conflict distress as compared to days preceded by low conflict distress . no relationship to positive mood was found among individuals with low dlpfc activity , regardless of level of previous day conflict distress . analysis of effects influencing negative mood showed the following : on days when previous day conflict distress was low , dlpfc activity had no relationship to negative mood [ b = 0.02(s.e . 0.02 ) , t(19 ) = 1.54 , p = 0.14 ] ( blue line plotted in fig . 4b ) . similarly , on days when previous day conflict distress was high , dlpfc activity had no significant influence on negative mood [ b = 0.02(s.e . 0.01 ) , t(19 ) = 1.52 , p = 0.15 ] ( red line plotted in fig . we then examined the effect of previous day conflict distress for schizophrenia participants with high and low dlpfc activities . for schizophrenia participants with low dlpfc activity , previous day conflict distress was a trend - level significant predictor of the change in negative mood the next day [ b = 0.03(s.e . 0.01 ) , t(19 ) = 2.0 , p = 0.06 ] , such that when previous day conflict distress was high , participants with low dlpfc activity had worse negative mood the next day . however , for schizophrenia participants with high dlpfc activity , previous day conflict distress was not related to the change in negative mood the next day [ b = 0.02(s.e . 0.01 ) , t(19 ) = 1.2 , p = 0.26 ] . these results indicate that , among schizophrenia participants with low dlpfc activity , negative mood is significantly higher on days preceded by high conflict distress as compared to days preceded by low conflict distress . no relationship to negative mood was found among individuals with high dlpfc activity , regardless of level of previous day conflict distress . this study combines fmri and experience sampling methods to investigate whether people with schizophrenia have lpfc deficits in the cognitive control of emotional information , and , if so , whether these lpfc deficits prospectively predict the influence of real - world interpersonal conflict on changes in mood and psychotic symptoms . two key findings emerged . first , analysis of the msit - emotion task revealed a group emotion condition interaction in the right dlpfc ( superior frontal gyrus , ba9 ) . inspection of activity in this region indicates that , compared to healthy participants , individuals with schizophrenia had lower dlpfc activity during cognitive control of task - irrelevant negative emotional information . this is consistent with prior literature demonstrating that schizophrenia is associated with lpfc deficits in cognitive control of emotion ( ursu et al . , 2011 ; vercammen et al . , 2012 , schizophrenia participants showed deficient neural activity in the right dlpfc when controlling emotional information on trials with the greatest cognitive demand ( i.e. negative interference trials ) suggesting that schizophrenia participants ' ability to control the influence of irrelevant negative emotional information on task performance is particularly impacted during high cognitive load tasks . this is consistent with research demonstrating a relationship between dlpfc dysfunction and cognitive load in non - emotional cognitive control tasks ( callicott et al . , 2003 ) , and further indicates that tasks probing cognition emotion interactions may be a more sensitive tool for understanding the role of emotion processing in the pathophysiology of schizophrenia . second , among schizophrenia participants , the interaction of right dlpfc activity during cognitive control of emotional information and the previous day 's conflict distress predicted changes in positive and negative mood the following day . specifically , analysis of positive mood shows that schizophrenia participants with higher dlpfc activity had better positive mood the day after high conflict distress . however , dlpfc activity had no influence on days following low conflict distress , and for schizophrenia participants with low dlpfc activity , positive mood was no different on days following high or low conflict distress . thus , higher dlpfc activity appears to convey a boost to positive mood following an interpersonal conflict , but only when that conflict is experienced as highly distressing . conversely , analysis of negative mood showed that schizophrenia participants with lower dlpfc activity had worse negative mood when the previous day 's conflict distress was high . however , on days when previous day conflict distress was low , dlpfc activation had no relationship to negative mood . thus , lower dlpfc activity appears to contribute to an exacerbation of negative mood following an interpersonal conflict , but only when that conflict is experienced as highly distressing . these data provide the first evidence tying lpfc dysfunction in schizophrenia to daily ratings of mood following real - world social interactions , and have substantive implications for understanding the specific role of lpfc in response to social stress . first , our finding that low dlpfc activity during cognitive control of negative emotional information predicts increased negative mood on days following highly distressing interpersonal conflicts is consistent with our previous finding in healthy individuals ( hooker et al . , 2010 ) , and prior literature demonstrating reduced lpfc engagement in response to social stress predicts higher self - reported social distress ( eisenberger et al . , 2003 ; eisenberger et al . , 2007 ; hooker et al . , 2010 ) together , these data support the well - documented proposal that lpfc control - related mechanisms act to down - regulate negative affect / mood ( kohn et al . , 2014 ; ochsner and gross , 2005 ) , and suggest that adaptive response to social stressors requires lpfc control - related functions to down - regulate negative emotional information . second , our finding that individuals with high dlpfc activity show an increase in their positive mood the day after highly distressing interpersonal conflicts is particularly interesting , especially given that the paradigm we used to probe lpfc - mediated cognitive control focused on control of negative emotional information . this finding supports the proposal that lpfc - mediated control mechanisms are also important in the up - regulation of positive mood ( kim and hamann , 2007 ; mak et al . , 2009 ) even in the context of incidental / automatic cognitive control of emotional information , and replicates parallel findings in our previous study ( hooker et al . , 2010 ) . in the context of explicit emotion regulation , one explanation of this finding is that people with high lpfc control - related activation use more effective cognitive strategies to reevaluate distressing interpersonal conflicts and reframe them in a positive light , possibly by thinking about what they have learned from the interpersonal conflict and how it makes them stronger / better ( i.e. positive reappraisal ) . in line with this interpretation , the use of positive reappraisal is related to increased experience and expression of positive emotions ( gross and john , 2003 ) , and has consistently been shown to predict well - being ( helgeson et al . , 2006 ) and promote an upward - spiral of adaptive responding to negative events ( fredrickson , 2004 ) . interestingly , several studies examining explicit emotion regulation in mood psychopathology ( e.g. depression , bipolar disorder ) have not found strong group differences ( e.g. dillon and pizzagalli , 2013 ; ehring et al . , 2010 ; gruber et al . , 2014 ) . this suggests that , when given clear instructions , individuals with emotion regulation difficulties can effectively engage emotion control mechanisms , but that in the absence of such clear instructions as is typically the case in daily life they struggle to regulate emotional responses . thus , using the msit - emotion to probe incidental / automatic lpfc - mediated emotion control processes may have enhanced the probability of finding group differences and detecting a relationship between lpfc activity and daily social interactions . finally , the results are in line with the diathesis stress model of neural mechanisms of social stress ( e.g. dillon and pizzagalli , 2013 ; ehring et al . , 2010 ; gruber et al . , 2014 ) and have implications for the development of treatment interventions . low or compromised lpfc function may be a vulnerability that contributes to the increased negative impact of highly distressing interpersonal conflicts via impaired cognitive control of emotion . conversely , high or intact lpfc function may be protective against the negative impact of interpersonal conflicts , and appear to convey a benefit in the form of increased positive mood . given that greater dlpfc engagement during our laboratory - based measure of control of emotional information predicted increased positive mood the day after a distressing interpersonal conflict , interventions that aim to improve lpfc function and/or cognitive control of emotional information may also improve real - world forms of emotion control , and consequently , adaptive response to social conflict . consistent with this proposal , normalization / improvement of lpfc function , either in response to medication ( garnefski and kraaij , 2006 ; zlomke and hahn , 2010 ) , cognitive therapy ( heller et al . , 2013 ) , or computerized cognitive training ( edwards et al . , 2010 ) , is associated with improved symptoms in depression , psychosis , and global functioning respectively . this study is also consistent with mounting consensus that ecological measures of daily life can be combined with neuroimaging data to meaningfully connect neural indicators of psychological processes to real - world behavior ( subramaniam et al . , 2014 ) . in healthy individuals , lpfc activity predicts daily levels of social support ( berkman and lieberman , 2011 ) , successful smoking cessation ( eisenberger et al . , 2007 ) , and maladaptive behavior following a conflict with a partner ( berkman et al . , 2011 ) . our findings extend this brain - as - predictor ( hooker et al . , 2010 ) approach to understanding the consequences of well - established lpfc deficits in schizophrenia in the context of social stress ( i.e. interpersonal conflict ) . using a task that specifically assessed the interaction between cognitive control and emotion processing likely increased sensitivity to this brain behavior relationship , and provides further support for the proposal that the consequences of cognitive and emotion processing deficits in schizophrenia may be better understood in the context of cognition emotion interactions ( berkman et al . , 2011 ) . by using these methods together , this study demonstrates the value of the brain - as - predictor approach to research attempting to delineate the mechanisms that contribute to the development and persistence of problematic mood and symptoms in schizophrenia . first , mood and symptom measures on the daily - diary were self - report and thus subject to bias . second , we designed the msit - emotion with the assumption that in order to meet task demands participants must engage cognitive control mechanisms to inhibit the influence of the negative picture on performance . it is possible , however , that participants could use an alternative , non - control related strategy to counteract the influence of the negative picture ( e.g. squint their eyes to blur the picture and focus on the numerical stimuli ) . future investigations should use a paradigm that precludes the use of such alternative strategies by incorporating the emotional stimuli into the response pattern of the task , for example , the face - emotion stroop ( kring and elis , 2013 ; pessoa , 2008 ) . third , based on our previous findings regarding predictive value of lpfc activity ( etkin et al . , 2006 ) , we chose to focus specifically on the role of the lpfc in social behavior . next steps should investigate the contribution of other regions involved in cognitive control of emotional information ( e.g. acc , amygdala ) and cognitive control network connectivity ( e.g. cingulo fronto parietal connectivity ) to mood changes in response to social stress . in sum , this study integrates fmri and experience sampling methods to demonstrate a direct link between lpfc activation during cognitive control of emotional information and response to real - world social conflict in schizophrenia . results indicate that lpfc activity during a laboratory - based measure of cognitive control of emotional information predicts changes in positive and negative mood on days following highly distressing interpersonal conflicts . these findings suggest that treatment interventions targeting lpfc mechanisms of cognitive control of emotion could promote adaptive responding to real - world social conflict in schizophrenia . this work was supported by harvard university research funds to christine i. hooker and a sackler scholar fellowship from the sackler scholar program in psychobiology to laura m. tully .

lpfc dysfunction is a well - established neural impairment in schizophrenia and is associated with worse symptoms . however , how lpfc activation influences symptoms is unclear . previous findings in healthy individuals demonstrate that lateral prefrontal cortex ( lpfc ) activation during cognitive control of emotional information predicts mood and behavior in response to interpersonal conflict , thus impairments in these processes may contribute to symptom exacerbation in schizophrenia . we investigated whether schizophrenia participants show lpfc deficits during cognitive control of emotional information , and whether these lpfc deficits prospectively predict changes in mood and symptoms following real - world interpersonal conflict . during fmri , 23 individuals with schizophrenia or schizoaffective disorder and 24 healthy controls completed the multi - source interference task superimposed on neutral and negative pictures . afterwards , schizophrenia participants completed a 21-day online daily - diary in which they rated the extent to which they experienced mood and schizophrenia - spectrum symptoms , as well as the occurrence and response to interpersonal conflict . schizophrenia participants had lower dorsal lpfc activity ( ba9 ) during cognitive control of task - irrelevant negative emotional information . within schizophrenia participants , dlpfc activity during cognitive control of emotional information predicted changes in positive and negative mood on days following highly distressing interpersonal conflicts . results have implications for understanding the specific role of lpfc in response to social stress in schizophrenia , and suggest that treatments targeting lpfc - mediated cognitive control of emotion could promote adaptive response to social stress in schizophrenia .

Introduction Material and methods Results Discussion Funding Supplementary data

these self - regulatory mechanisms are reliant on cognitive control processes mediated by the lateral prefrontal cortex ( lpfc ; heatherton and wagner , 2011 ; ochsner et al . , lpfc dysfunction in cognitive control is a well - established neural impairment in schizophrenia ( barch , 2005 ; manoach , 2003 ; minzenberg et al . however , there is a paucity of research examining how lpfc dysfunction contributes to illness severity . consistent with the diathesis stress model , one proposal is that lpfc dysfunction is a biological vulnerability that , in the presence of an interpersonal stressor , contributes to symptom exacerbation via impaired cognitive control of emotion ( hooker et al . , 2010 ; cognitive control of emotion comprises the dual processes of explicit and effortful control of the experience and expression of internal emotional states ( emotion regulation ) and the implicit and more automatic control of how external emotional information influences behavior ( gyurak et al . the lpfc , comprising both dorsolateral ( dlpfc ) and ventrolateral ( vlpfc ) regions , is consistently implicated in laboratory assessed cognitive control of emotion ( ochsner and gross , 2005 ; ochsner et al . reappraisal paradigms ) as well as paradigms assessing implicit cognitive control of external and/or irrelevant emotional information ( e.g. similarly , lower vlpfc activity when viewing negative facial expressions predicts increased negative mood and maladaptive behavior following interpersonal conflicts ( hooker et al . in schizophrenia , interpersonal conflicts , especially conflicts characterized by criticism , predict symptom exacerbation and higher relapse rates ( hooley , 2007 ) , and symptom exacerbation in response to interpersonal criticism is related to poor working memory and cognitive control ( rosenfarb et al . collectively , these data suggest that compromised lpfc function is a vulnerability for symptom exacerbation and functional difficulties in response to interpersonal conflict . however , research attempting to connect lpfc activity , interpersonal conflict , and schizophrenia symptomatology is sparse . , , only one study has examined the interaction between lpfc activation , symptoms , and real - world social interactions . those with high social anhedonia ) with low lpfc activation had worse symptoms of paranoia on days with distressing interpersonal conflicts compared to days without distressing interpersonal conflicts ( hooker et al . first , the tasks traditionally used to assess lpfc function , such as response inhibition or working memory tasks ( barch , 2005 ) , although robust activators , may not be the most sensitive measures for assessing how lpfc activity relates to real - world social functioning because they do not directly capture cognitive control of emotional information . given the inherently affective nature of social interactions and the accompanying need for self - regulation ( arriaga and rusbult , 1998 ) , tasks assessing the interaction between lpfc mediated cognitive control and emotional information may provide a more accurate reflection of the inhibitory demands of real - world social contexts . by using cold cognitive tasks and not assessing lpfc mediated cognitive control in relation to emotional information , previous studies may have lacked the sensitivity necessary to identify the role of the lpfc in individuals ' response to social conflict . although these provide an overview of an individual 's general level of functioning , they are not well suited for capturing the multidimensional nature of social interactions in daily life , and rarely provide the context in which they occur ( trull and ebner - priemer , 2009 ) . experience sampling methods ( esm ) , a technique in which assessments are collected in the person 's natural environment and repeated over time , have revealed a nuanced relationship between changes in the social environment , particularly social stressors , and symptoms ( myin - germeys et al . , 2009 ) , thus , using esm in conjunction with neuroimaging techniques may provide a more sensitive and ecologically valid approach to understanding the contribution of lpfc dysfunction to social deficits in schizophrenia . the present study addressed these prior limitations by combining fmri and esm to test whether people with schizophrenia have lpfc deficits in cognitive control of negative emotional information , and , if so , whether these lpfc deficits are related to changes in mood and symptoms following interpersonal conflict . individuals with schizophrenia and demographically - matched healthy controls completed an adapted version of the multi - source interference task ( msit ) , a cognitive control task specifically designed to activate the cingulo frontal parietal cognitive control network ( bush and shin , 2006 ) . this process is thought to more accurately reflect the interaction of emotion and cognitive control in real - world social contexts ( silbersweig et al . , 2007 ) , and considered to be a form of implicit cognitive control of emotion ( gyurak et al . our measure of cognitive control of emotional information was lpfc activity when inhibiting the effect of irrelevant emotional information during high interference trials ( when cognitive control skills are most challenged ) . stimuli were also superimposed on neutral pictures , included to test the specificity of schizophrenia participants ' lpfc deficits for controlling emotional information . following the scan , schizophrenia participants completed an online , structured daily - diary questionnaire of mood and symptoms every evening for 3 weeks . participants rated the extent to which they experienced mood and schizophrenia - spectrum symptoms , as well as the occurrence of interpersonal conflict and associated distress . we hypothesized that : 1 ) schizophrenia participants would show reduced lpfc activity during cognitive control of emotional information compared to healthy participants ; and 2 ) among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . specifically , we expect that schizophrenia participants with low lpfc activity will have an increase in negative mood and psychotic symptoms the day after highly distressing interpersonal conflict . 23 individuals with schizophrenia or schizoaffective disorder and 24 healthy controls were recruited from the greater boston area . inclusion criteria for schizophrenia participants were : diagnosis of schizophrenia or schizoaffective disorder , no comorbid axis i disorders , and no history of electroconvulsive therapy . , 2002 ) ; symptoms were assessed using the positive and negative syndrome scale ( panss ; kay et al . , 2006 ) ; positive and negative mood were assessed using the positive and negative affect schedule ( panas ; watson et al . participants completed behavioral assessments , returned a separate day for the scan , and were subsequently oriented to the daily - diary . after the scan , participants rated the valence of each neutral and negative picture ( see supplemental methods ) . contrasts were calculated for each of the four conditions relative to fixation periods , and for interference versus control for neutral and negative pictures ( i.e. we tested our central hypothesis that , compared to healthy controls ( hc ) , schizophrenia ( sz ) participants would show reduced lpfc activity during cognitive control of emotional information using a 2 2 2 full factorial anova with group ( hc / sz ) , emotion ( negative / neutral ) and condition ( interference / control ) as factors . the predicted group emotion condition interaction was that hc would have greater cognitive control related activity ( interference control ) than sz when inhibiting irrelevant negative emotional information on interference trials versus control trials compared to inhibiting irrelevant neutral information on interference versus control trials . participants rated the extent to which they experienced mood and psychotic symptoms , including positive and negative mood , paranoia , hallucinations / odd experiences , negative symptoms , and disorganized symptoms . participants also reported whether they had an interpersonal conflict ( yes / no ) , and if so , the extent to which the conflict caused distress and was resolved . participants could not miss more than 6 days.hypothesis 2among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . analysis of daily - diary responses and lpfc activity used a hierarchical linear modeling ( hlm ) approach with the mixed procedure in sas ( sas institute , cary , nc ) . the repeated assessments over 21 days allowed us to examine the longitudinal effect of conflict distress on the change in mood and symptoms the following day . both hc and sz participants demonstrated greater activity for interference versus control conditions in cognitive control regions , including inferior , middle and superior frontal gyri ( lpfc ) , and anterior cingulate cortex ( acc ) ( fig . a significant condition emotion interaction in the sz group indicated that individuals with schizophrenia were slower and less accurate on negative interference trials relative to neutral interference trials and negative and neutral control trials . analysis of construct validity showed that 21-day average daily - diary ratings for negative mood significantly correlated with laboratory measures of trait negative affect ( panas negative affect , r = .55 , p < .05 ) . daily - diary ratings of positive mood were not correlated with panas positive affect ( r = .01 ) ; however , there is no overlap between the panas and poms positive mood items , so a different lab - based assessment of positive mood may be necessary to test construct validity . daily diary measures of positive , negative , and disorganized symptoms were not correlated with positive and negative symptom scale ( panss ) measures of positive , negative , and disorganized symptoms . in addition , there were significant correlations between daily - diary measures of interpersonal conflict and lab - based measure social functioning . panss measures of interpersonal functioning were also related to daily - diary measures of interpersonal conflict . together these data indicate that the daily - diary assessments of mood and interpersonal conflict are measuring the intended construct . since prior studies have found a relationship between social stress and symptoms , we conducted simple regression analysis to look at the relationship between conflict distress and symptom severity . consistent with prior findings , daily ratings of high conflict distress were associated with worse symptom severity , including worse negative mood , paranoia , negative symptoms and disorganized symptoms ( table 3 ) . we also conducted simple regression analysis to examine the relationship between lpfc activity and symptoms regardless of interpersonal conflict : lpfc activity did not relate to any of the symptom measures . no significant interactions were found ; that is , on the same day as an interpersonal conflict , individuals ' dlpfc activation during cognitive control of emotion did not interact with conflict distress in predicting symptom severity that day ( table 3 ) . our measure of cognitive control of emotional information , calculated from the contrast values in each condition extracted from the peak of this cluster in the right dlpfc.hypothesis 2among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . among schizophrenia participants , lpfc activity during cognitive control of emotional information will predict changes in mood and symptoms following interpersonal conflict . previous day conflict distress ) in predicting symptom severity on days following interpersonal conflicts . dlpfc activity significantly interacted with previous day conflict distress in predicting change in positive mood [ f = 8.03 , b = 0.02 , p = 0.01 ] and negative mood [ f = 4.49 , b = 0.01 , p = 0.047 ] the following day . simple slope analyses were conducted to better understand the interactions predicting positive and negative mood . however , when previous day conflict distress was high , dlpfc activity significantly predicted positive mood the following day [ b = 0.07(s.e . 0.02 ) , t(19 ) = 3.1 , p = 0.006 ] , such that schizophrenia participants with higher dlpfc activity had higher positive mood compared to participants with lower dlpfc activity ( red line plotted in fig . for schizophrenia participants with low dlpfc activity , previous day conflict distress was not related to positive mood [ b = 0.02(s.e . however , for schizophrenia participants with high dlpfc activity , previous day conflict distress had a significant effect on positive mood the next day [ b = 0.07(s.e . these results indicate that , among schizophrenia participants with high dlpfc activity , positive mood is significantly higher on days preceded by high conflict distress as compared to days preceded by low conflict distress . analysis of effects influencing negative mood showed the following : on days when previous day conflict distress was low , dlpfc activity had no relationship to negative mood [ b = 0.02(s.e . similarly , on days when previous day conflict distress was high , dlpfc activity had no significant influence on negative mood [ b = 0.02(s.e . for schizophrenia participants with low dlpfc activity , previous day conflict distress was a trend - level significant predictor of the change in negative mood the next day [ b = 0.03(s.e . 0.01 ) , t(19 ) = 2.0 , p = 0.06 ] , such that when previous day conflict distress was high , participants with low dlpfc activity had worse negative mood the next day . however , for schizophrenia participants with high dlpfc activity , previous day conflict distress was not related to the change in negative mood the next day [ b = 0.02(s.e . these results indicate that , among schizophrenia participants with low dlpfc activity , negative mood is significantly higher on days preceded by high conflict distress as compared to days preceded by low conflict distress . no relationship to negative mood was found among individuals with high dlpfc activity , regardless of level of previous day conflict distress . this study combines fmri and experience sampling methods to investigate whether people with schizophrenia have lpfc deficits in the cognitive control of emotional information , and , if so , whether these lpfc deficits prospectively predict the influence of real - world interpersonal conflict on changes in mood and psychotic symptoms . inspection of activity in this region indicates that , compared to healthy participants , individuals with schizophrenia had lower dlpfc activity during cognitive control of task - irrelevant negative emotional information . this is consistent with prior literature demonstrating that schizophrenia is associated with lpfc deficits in cognitive control of emotion ( ursu et al . , 2012 , schizophrenia participants showed deficient neural activity in the right dlpfc when controlling emotional information on trials with the greatest cognitive demand ( i.e. negative interference trials ) suggesting that schizophrenia participants ' ability to control the influence of irrelevant negative emotional information on task performance is particularly impacted during high cognitive load tasks . , 2003 ) , and further indicates that tasks probing cognition emotion interactions may be a more sensitive tool for understanding the role of emotion processing in the pathophysiology of schizophrenia . second , among schizophrenia participants , the interaction of right dlpfc activity during cognitive control of emotional information and the previous day 's conflict distress predicted changes in positive and negative mood the following day . however , dlpfc activity had no influence on days following low conflict distress , and for schizophrenia participants with low dlpfc activity , positive mood was no different on days following high or low conflict distress . thus , higher dlpfc activity appears to convey a boost to positive mood following an interpersonal conflict , but only when that conflict is experienced as highly distressing . conversely , analysis of negative mood showed that schizophrenia participants with lower dlpfc activity had worse negative mood when the previous day 's conflict distress was high . however , on days when previous day conflict distress was low , dlpfc activation had no relationship to negative mood . thus , lower dlpfc activity appears to contribute to an exacerbation of negative mood following an interpersonal conflict , but only when that conflict is experienced as highly distressing . these data provide the first evidence tying lpfc dysfunction in schizophrenia to daily ratings of mood following real - world social interactions , and have substantive implications for understanding the specific role of lpfc in response to social stress . first , our finding that low dlpfc activity during cognitive control of negative emotional information predicts increased negative mood on days following highly distressing interpersonal conflicts is consistent with our previous finding in healthy individuals ( hooker et al . , 2010 ) , and prior literature demonstrating reduced lpfc engagement in response to social stress predicts higher self - reported social distress ( eisenberger et al . , 2014 ; ochsner and gross , 2005 ) , and suggest that adaptive response to social stressors requires lpfc control - related functions to down - regulate negative emotional information . second , our finding that individuals with high dlpfc activity show an increase in their positive mood the day after highly distressing interpersonal conflicts is particularly interesting , especially given that the paradigm we used to probe lpfc - mediated cognitive control focused on control of negative emotional information . , 2009 ) even in the context of incidental / automatic cognitive control of emotional information , and replicates parallel findings in our previous study ( hooker et al . in the context of explicit emotion regulation , one explanation of this finding is that people with high lpfc control - related activation use more effective cognitive strategies to reevaluate distressing interpersonal conflicts and reframe them in a positive light , possibly by thinking about what they have learned from the interpersonal conflict and how it makes them stronger / better ( i.e. thus , using the msit - emotion to probe incidental / automatic lpfc - mediated emotion control processes may have enhanced the probability of finding group differences and detecting a relationship between lpfc activity and daily social interactions . low or compromised lpfc function may be a vulnerability that contributes to the increased negative impact of highly distressing interpersonal conflicts via impaired cognitive control of emotion . given that greater dlpfc engagement during our laboratory - based measure of control of emotional information predicted increased positive mood the day after a distressing interpersonal conflict , interventions that aim to improve lpfc function and/or cognitive control of emotional information may also improve real - world forms of emotion control , and consequently , adaptive response to social conflict . consistent with this proposal , normalization / improvement of lpfc function , either in response to medication ( garnefski and kraaij , 2006 ; zlomke and hahn , 2010 ) , cognitive therapy ( heller et al . , 2010 ) , is associated with improved symptoms in depression , psychosis , and global functioning respectively . in healthy individuals , lpfc activity predicts daily levels of social support ( berkman and lieberman , 2011 ) , successful smoking cessation ( eisenberger et al . , 2010 ) approach to understanding the consequences of well - established lpfc deficits in schizophrenia in the context of social stress ( i.e. using a task that specifically assessed the interaction between cognitive control and emotion processing likely increased sensitivity to this brain behavior relationship , and provides further support for the proposal that the consequences of cognitive and emotion processing deficits in schizophrenia may be better understood in the context of cognition emotion interactions ( berkman et al . by using these methods together , this study demonstrates the value of the brain - as - predictor approach to research attempting to delineate the mechanisms that contribute to the development and persistence of problematic mood and symptoms in schizophrenia . first , mood and symptom measures on the daily - diary were self - report and thus subject to bias . third , based on our previous findings regarding predictive value of lpfc activity ( etkin et al . next steps should investigate the contribution of other regions involved in cognitive control of emotional information ( e.g. cingulo fronto parietal connectivity ) to mood changes in response to social stress . in sum , this study integrates fmri and experience sampling methods to demonstrate a direct link between lpfc activation during cognitive control of emotional information and response to real - world social conflict in schizophrenia . results indicate that lpfc activity during a laboratory - based measure of cognitive control of emotional information predicts changes in positive and negative mood on days following highly distressing interpersonal conflicts . these findings suggest that treatment interventions targeting lpfc mechanisms of cognitive control of emotion could promote adaptive responding to real - world social conflict in schizophrenia .

imagine a 6-year - old boy with autism spectrum disorder ( asd ) standing in the middle of the grocery store and screaming . faced with his behavior and the glances she is receiving from others in the store , his mother tries to interpret his behavior and respond appropriately . is he screaming because the store is full of people using eye contact and social smiles that he does n't understand ? is he screaming because he is hungry , sees a cookie , and does n't know how to ask for something to eat ? is he screaming because this is n't the same grocery store that he usually goes to with his mother ? is he screaming because he is overwhelmed by the bright lights in the store ? or , is he screaming because he was asked to put food in the shopping cart and he does n't want to do so ? in other words , is this behavior problem a result of the social - communication impairments , repetitive behaviors , or sensory interests that characterize individuals with asd , or is this boy being noncompliant ? in this moment , his mother attempts to choose an intervention strategy that fits with the underlying reason for his behavior problem . if his mother believes that he is hungry she might coach him to ask for a snack . if his mother believes that he is upset by the change in routine , she might use a visual schedule to show him what to expect in this new location . if her interpretation is accurate , then she is likely to see a decrease in his challenging behavior and have a more successful shopping trip with her son . indeed , mandell and colleagues reported that 40% of children with asd are referred for treatment of disruptive behaviors including aggression , noncompliance , and hyperactivity . from the above example , it is clear that : ( i ) an understanding of the underlying symptoms of asd is necessary for successful management of challenging behaviors ; and ( ii ) the involvement of caregivers in treating challenging behaviors in children and adolescents with asd aids in generalization to community settings . indeed , the involvement of caregivers has been identified as an essential component of a good treatment program . in fact , the utility of parents as interventionists has spanned over four decades , with schopler and reichler cited as being the first advocates for involving parents as cotherapists in the treatment of behavior problems in children with asd . parents are the child 's natural teachers , and caregiver - implemented interventions have been documented to promote skill generalization and maintenance while also potentially reducing parental stress and increasing quality of life . the nature of caregiver involvement varies across programs , with some programs focusing on providing education to caregivers about the child 's symptoms and other programs focusing on teaching caregivers specific technique that can be used in the natural environment . while both approaches have been successful , in order for treatment effects to be maintained , the intervention program needs to be compatible with the family 's daily routines . traditional caregiver - mediated interventions designed to reduce behavior problems in children without asd are typically geared toward treating noncompliance . in these interventions , caregivers are taught to facilitate appropriate behavior by using rewards , and to decrease noncompliance by using consequences including ignoring and time out . little research has been conducted to determine whether these traditional behavior management strategies are effective at treating problem behaviors in children with asd . because it is often an underlying symptom of asd rather than noncompliance that leads to challenging behaviors in these children , a traditional behavior management approach focused on noncompliance may not be effective . there have been several case studies reporting the effectiveness of using a traditional parent - mediated behavioral intervention , parent child interaction therapy ( pot ) , to decrease challenging behaviors in children with asd . these studies suggest that pot may be effective in reducing challenging behaviors , particularly when used with high - functioning children with asd and when adaptations are made to target social skills . however , the use of ignoring is not recommended when targeting repetitive and self - stimulatory behaviors as the lack of social attention may actually provide increased opportunity to engage in these behaviors . solomon and colleagues conducted the only study to date that compared the effectiveness of a traditional caregiver - mediated behavioral intervention ( pot ) in a group of children with asd and a waitlist control group . they found increased shared positive affect in parent - child dyads but no changes in parent stress or parent report of challenging behaviors compared with the comparison group . they noted the importance of building a parent - child relationship for children with asd . thus , caregiver - mediated interventions targeting challenging behaviors for children with asd have not been consistently found to reduce challenging behaviors and may need to be adapted to meet the specific needs of this population . in a comparison of the caregiver - mediated intervention literature for children with asd and children with disruptive behavior disorders ( dbd ) , brookman - frazee and colleagues reported striking differences in intervention techniques . while 100% of the dbd literature reported targeting parenting strategies and discipline techniques , none of the asd literature targeted these skills . in contrast , 68% of the asd literature targeted communication skills while none of the dbd literature targeted communication . therefore , while mental health professionals may be tempted to treat challenging behaviors in children with asd using traditional caregiver - mediated behavior intervention techniques , different techniques may be needed . in the present article , we describe current caregiver - based intervention approaches geared toward understanding behavior problems within the context of asd symptomatology . further , we review the literature on caregiver - mediated interventions treating the most common causes for behavior problems in this population . when faced with a child 's observable challenging behaviors ( ie , those visible above the waterline ) , caregivers are encouraged to use their understanding of asd to identify possible underlying causes for these behaviors ( ie , those hidden below the waterline ) . this image supports the notion of conducting a functional behavior assessment to identify the communicative function or intent of a challenging behavior . indeed , a functional behavior assessment has been recognized as a necessary component in designing interventions to understand and to modify behavior in children with autism . in the behavior analytic literature , the reason why children exhibit problem behavior is often described as either to obtain an item , escape a task , or to seek attention . however , in children with asd the underlying reasons why children may engage in challenging behaviors may be related to autism - specific symptoms . in our example of the boy screaming in the grocery store , the social and sensory demands of the situation may have caused him to want to escape . in contrast , if his screaming was driven by hunger , then his behavior was a form of request . that is , the hidden explanation for his disruptive behavior may be the social , sensory , or communicative demands of the situation . an accurate functional assessment is vital in building effective and efficient behavioral supports . when working with families to conduct a functional behavior assessment and develop an intervention plan , moes and frea emphasized the importance of considering the family 's own environment , values , and beliefs . they suggested that a contextualized behavior support assessment that examines more than just the child 's behavior is important in increasing the compatibility between the behavioral intervention and family routines . in this approach , the emphasis is placed on the collaborative parent - professional relationship in developing behavior plans . moes and frea found positive outcomes for a contextualized behavior support plan with significant reductions in challenging behavior and an increase in compliance during the contextualized behavior phase . this suggests that developing sustainable plans ( eg , compatible with family resources , constraints , beliefs , values , goals , abilities , and needs ) , may contribute to the generalization and maintenance of treatment gains . in our own clinical work , we have found that the most frequent communicative functions of challenging behavior include frustration over inability to communicate , difficulties with social interaction , anxiety , and atypical sensory sensitivities . we review the literature in each of these areas with a focus on interventions that include caregiver - mediated approaches . because one of the functions of challenging behavior is communication , it is not surprising that a considerable amount of intervention research has focused on developing successful procedures for improving communication . in addition to improvements in verbalizations , mean length of utterance , and spontaneity of language use , successful communication intervention has been associated with decreases in problem behavior and increases in positive affect . as a result , the replacement of challenging behaviors with appropriate and increasingly complex communication skills has the potential to have far - reaching implications for academic achievement , social relationship development , and vocational outcomes . if challenging behavior ( eg , screaming in the grocery store ) is a request ( eg , for food ) , then the most effective interventions are directed at increasing appropriate spontaneous and functional communication . clinically , this means that the child must learn to request using a system that is compatible with his / her mental age ( eg , pictures , sign language , words ) . further , the communication must be functional ( eg , instead of learning to sign the word more it would be more effective for him to learn to sign the word cookie ) . there have been a number of behaviorally based communication intervention approaches designed to increase requesting skills , particularly focused on toddlers and preschool - aged children with asd . traditional applied behavior analysis approaches ( eg , discrete trial training ) , have been criticized for teaching prompt dependence ( eg , the screaming boy in the grocery store would wait for a prompt before using his learned verbal skills to say cookie ) , and for limited generalizability across contexts ( eg , he may learn to say cookie only in the grocery store ) . thus , there has been an increased emphasis on naturalistic or child - directed behavioral intervention approaches . these more naturalistic behavior interventions include incidental teaching , enhanced milieu teaching , and pivotal response training to teach requesting and other communication skills . while there are differences among the approaches , naturalistic behavior models share common characteristics , including child choice ( eg , teaching communication skills that fit with the child 's interests ; eg , the boy in the grocery store would learn cookie as a first word ) , intervention in natural contexts ( eg , learning to communicate during family routines such as eating dinner ) , and the use of intrinsically related rewards ( eg , the child would receive a cookie along with social praise ) . results have indicated that , compared with the traditional structured models , interventions that are less structured and more child - focused result not only in faster learning by children with asd , but also result in children displaying higher levels of affect and engaging in less avoidant and disruptive behavior . as our ability to accurately diagnose autism in the first few years of life has improved , there has been an increased demand for intervention approaches that integrate developmental and behavioral approaches to target social communication skills in toddlers and preschoolaged children with asd . kasari 's jasper ( joint attention , symbolic play , emotion regulation ) program resulted in increased expressive language skills with continued gains 1 year post study completion . this combined developmental and naturalistic behavioral approach was more effective than a more traditional applied behavioral intervention preschool program . similarly , the early start denver model incorporates behavioral and developmental strategies and has been associated with increased cognitive development and adaptive behavior . while not directly targeting requesting behavior , the focus on facilitating early social communication skills is designed to increase spontaneous communication skills and decrease challenging behaviors . the caregiver has an advantage as s / he can optimize learning moments throughout naturally occurring opportunities during daily activities within the home and community . this may be more successful than instruction occurring only during specified times , which may burden the family by requiring extra time and effort . thus , naturalistic approaches are ideally suited for caregivers . recently , a large community - based study found improvements for up to 1 year in children 's expressive and receptive communication and decreases in problem behavior with parents who learned how to implement pivotal response treatment . similarly , minjarez and colleagues found significant increases in children 's functional spontaneous speech for parents who participated in a communication intervention training where feedback was delivered based on the videotapes they brought in . however , more research is needed to determine whether caregiver - mediated interventions that combine developmental and behavioral approaches are effective at decreasing challenging behaviors . clinically , we often see behavior problems in children with asd who are overwhelmed by social situations ( eg , the child in a crowded grocery store ) or who want to engage in social interactions but do not have appropriate initiation skills ( eg , a preschool girl whose only way of initiating social interaction with siblings and peers is to pull their hair ) . while the boy in the grocery store may need to learn to request a break , the girl wanting to interact with her peers may need to learn a more appropriate way of initiating social interactions . indeed , bauminger and colleagues reported that children and adolescents with high - functioning asd may be interested in interacting with peers but do not have the knowledge about how to interact appropriately . in their study , children and adolescents with high - functioning asd initiated and responded to peers about half as often as age- and iq - matched children and adolescents with typical development . however , this decreased rate of social interactions may not be an indicator of a lack of interest in social interaction , as children and adolescents with asd reported higher levels of loneliness than their peers with typical development . a variety of skill - based intervention programs have been developed to teach social skills to children with asd . while early intervention programs have focused on relationship - based approaches that combine behavioral and developmental techniques , approaches for older children and adolescents these skill - building programs have taught social scripts to teach specific nonverbal and verbal behavior for specific situations , social stories to teach social norms and expectations , role plays to provide opportunities for practice , and self - monitoring through videotapes and checklists to increase self - awareness . remarkably , few of the skill - building intervention programs have included caregivers as cotherapists , or even included a caregiver education component . in a review of 66 studies published in peer - reviewed journals between 2001 - 2008 targeting social skills , reichow and volkmar reported that parent training was only measured as a main intervention variable in four studies , all targeted at improving social skills in preschool children . thus , while best practices consider parent and family involvement as an essential element of intervention programs for children with autism , very few studies targeting social skills have included parents beyond the toddler and early childhood years . the inclusion of caregivers is particularly important when social skills difficulties lead to challenging behaviors in the home and community settings . indeed , kasari and lawton cautioned that current social skills intervention approaches using behavioral principles seem to train children to produce specific operationalized behaviors rather than facilitating learning and generalization of the behavior across contexts . they caution that many current interventions may teach form but not function . frankel and colleagues taught conversation skills , peer entry , handling teasing , practicing good sportsmanship , and good host behaviors using children 's friendship training . in concurrent sessions , parents were taught how to facilitate the use of these skills at home by arranging for supervised play dates . compared with a delayed treatment control group , parents of children in the intervention group reported that their children showed increased social skills and other appropriate play date behaviors . further , the children in the intervention group self - reported increased popularity and decreased loneliness compared with those who did not receive the intervention . in a study of the skillstreaming approach , lopata and colleagues targeted social skills , emotion recognition , and understanding of metaphoric language . parents received a concurrent educational session focused on understanding symptoms of asd and techniques for generalization of skills learned in the child intervention group . compared with a delayed treatment control group , the intervention group showed increased parent - reported social skills . further , children in the intervention group showed increases on standardized measures of emotion recognition and non - literal language interpretation . both studies are encouraging and suggest that caregiver - mediated social skills interventions may be more successful at increasing flexibility and generalization of skills to the community setting than traditional social skills intervention programs . many children with autism experience clinical significant levels of anxiety , with 11% to 84% of children and adolescents with asd reported to have a diagnosable comorbid anxiety disorder : anxiety symptoms are varied and can include behavioral outbursts ( yelling , aggression ) , repetitive behaviors ( asking repetitive questions , reciting television quotes , pacing ) , withdrawal , and refusal to engage in activities . for example , we saw a child in the clinic with an irrational fear of black top surfaces , which led to temper tantrums when he was asked to leave the car to walk across a store parking lot . the most common anxiety symptoms in children and adolescents with asd are compulsive / ritualistic behavior and irrational fears and beliefs . leyfer and colleagues reported that 44% of the children and adolescents with asd in their study experienced specific phobias and 37% experienced symptoms consistent with obsessive - compulsive disorder . there is a growing literature on the use of adapted cognitive behavioral treatment ( cbt ) techniques to treat fears and irrational beliefs in asd ; using cbt , children with asd have been taught to identify their own anxiety , rate their anxiety levels , replace irrational thoughts with rational thoughts , and use progressive muscle relaxation routines ( see klinger and williams for a review ) . several recent randomized control studies have been conducted examining the efficacy of cbt for treating anxiety in children with asd . approximately 58% to 64% of children who completed the cbt treatments no longer met diagnostic criteria for an anxiety disorder , compared with 0% to 9% of children in waitlist control groups . further , several studies reported that these reductions were maintained 3 months after the intervention was completed . while most of these interventions do not include caregivers , reaven and colleagues ' face your fears program incorporated a parent component into their treatment protocol . parents were taught to identify their child 's anxiety symptoms and to create graded exposure hierarchies to help their child face parents became their child 's coach throughout the cbt intervention and were able to continue using the strategies outside of group . clinically , we have found that the involvement of caregivers in a cbt program provided a way for caregivers to understand the underlying reason for misbehavior . for example , we worked with a child whose fear of failure prevented him from completing his homework . once his mother understood the underlying reason for his refusal to complete homework , they were able to work together to reduce his fear and help him become more successful at completing his homework without any outbursts . to date , there is no data on whether the incorporation of caregivers as coaches increases the effectiveness of cbt interventions for children with asd compared with programs that only include children in the intervention protocol . in addition to irrational fears and beliefs , ritualistic and compulsive behaviors are often indicators of anxiety in children with asd . because of difficulties with flexible thinking , students with asd may prefer routines and become anxious when routines are altered . children with asd may exhibit anxiety by becoming disruptive when there is a deviation from the typical family routine ( eg , when grandparents visit or when a vacation is scheduled ) . the use of a visual schedule can help to significantly reduce a child 's anxiety and in turn , reduce the behavioral symptoms that accompany a new situation . using this preventive approach , caregivers are encouraged to use a daily schedule that lists the activities planned for each day . schedules can include objects , pictures , or written words depending on each child 's developmental levels . rather than causing children to become more inflexible , a daily schedule provides an opportunity to indicate when a change is planned to reduce anxiety . for example , the child that screams in the grocery store may be bothered by the fluorescent lights or by the loudness in the store . indeed , reese , richman , zarcone , and zarcone reported that attempts to escape uncomfortable sensory situations explained disruptive behavior in 14% of the children with asd in their sample . hyperresponsiveness , hyporesponsiveness , and sensory seeking . however , little research has supported these dimensions . recently , brock and colleagues identified sensory hyporesponsiveness in preschool - aged children with asd . despite the fact that it is commonly recognized that challenging behaviors are often exacerbated by atypical sensory processing in children with asd , very little intervention research has been conducted in this area . lang and colleagues conducted a review of all research on sensory integration therapy ( sit ) . only three of 25 studies included in the review considered sit to be an effective therapy based on posttreatment measures . in contrast , 14 of the studies saw no improvement in children with asd who had received sit . thus , to date the most effective approaches for decreasing behavior problems due to sensory sensitivities may be aimed at reducing the anxiety that usually arises as a result of these sensitivities . it is important to consider family social and cultural factors that may impact the successful use of caregivermediated approaches . the requirements of an intervention approach often conflict with the caregiver 's other time demands including workplace , siblings , spouse , and extended family . further family cultural values must be considered , as any attempt to modify the caregiver 's behavior without attending to cultural factors may be ineffective . further , the chronicity of asd and its impact of caregiver stress should be considered . because behavior problems often arise from the underlying symptoms of asd , caregivers are likely to face a lifetime of behavior management challenges . thus , it is important to consider the impact of long - term caregiver stress on effective intervention implementation . indeed , raising a child with asd is associated with higher levels of caregiver stress and psychological distress than raising a child with typical development or a child with another developmental disability . weiss and colleagues reported that the relationship between child behavior problems and parent mental health is mediated by psychological acceptance . that is , those parents who were able to accept the challenges of living with a child with asd showed fewer negative mental health consequences . they argued that caregiver - mediated interventions need to focus on positive coping strategies in addition to the problem focused strategies discussed in this article . furthermore , researchers have suggested the importance of individualizing treatment by matching the intensity of the intervention to the caregivers ' stress level . another consideration for caregiver - mediated intervention programs is how the program content is presented and taught to the caregivers . in a meta - analytic review of program components associated with parent training effectiveness for children with externalizing disorders , kaminski and colleagues found that larger effect sizes were associated with programs that required parents to practice the skills during therapy sessions , focused on parenting consistency , and focused on increasing positive parent - child interactions . conversely , smaller effect sizes were associated with programs focused on problem - solving techniques and promotion of cognitive , academic , and social skills . further research is needed to identify the specific program elements that are associated with larger effects in caregiver - mediated behavioral interventions for children with asd . finally , while the focus of this article has been on treating underlying behavioral causes ( communication frustrations , social skills difficulties , anxiety , sensory sensitivities ) of challenging behaviors , it is important that caregivers and clinicians also consider possible underlying medical complications including gastrointestinal difficulties , sleep disorders , and seizures that often cooccur with asd . a significant proportion of children with asd are referred to mental health centers due to the presence of challenging behaviors . an understanding of the underlying symptoms of asd is essential in managing behavior problems in this population and the involvement of caregivers in treatment is critical to long - term success . while behavioral intervention approaches have been used extensively to improve the social , communication , and anxiety symptoms that often accompany asd , few randomized control studies have been conducted . in a review of the 68 behavior intervention articles published in 2008 to 2009 , kasari and lawton reported that 63% of the studies used case study or single - subject design approaches , 16% used a group design other than randomized control , and 21% of the studies used randomized control trials ( ie , 14 of 68 studies ) . thus , while the literature on the effectiveness of behavioral intervention is growing , there continues to be a need for randomized , controlled studies . further , while the importance of working with caregivers has been emphasized for the past four decades , more research is needed about the effectiveness of caregiver - implemented interventions and the techniques that are most effective at supporting caregivers use of strategies in the natural environment including family beliefs and culture .

a significant proportion of children with autism spectrum disorder ( asd ) are referred to mental health centers due to the presence of challenging behaviors . because challenging behaviors in children and adolescents with asd often result from underlying social and communication difficulties and comorbid anxiety , traditional caregiver - mediated behavior intervention techniques developed for children with disruptive behavior disorders may need to be adapted for this population . behavioral interventions that target communication skills , social skills , anxiety , and sensory responsiveness in children with asd may be needed . notably , while best practice necessitates the involvement of caregivers in treating children and adolescents with asd , few randomized control studies have examined the effectiveness of caregiver - implemented interventions in reducing challenging behaviors . this review summarizes the current literature with regard to caregiver - mediated behavioral interventions for children with asd , and suggests areas for intervention development and research .

Introduction Working with families to understand challenging behaviors Behavioral approaches to improving communication skills Behavioral approach to improving social skills Cognitive behavioral interventions for anxiety Behavioral challenges as a result of sensory sensitivities Other considerations for utilizing caregiver-mediated behavioral interventions Summary

imagine a 6-year - old boy with autism spectrum disorder ( asd ) standing in the middle of the grocery store and screaming . is he screaming because the store is full of people using eye contact and social smiles that he does n't understand ? is he screaming because he is hungry , sees a cookie , and does n't know how to ask for something to eat ? is he screaming because this is n't the same grocery store that he usually goes to with his mother ? in other words , is this behavior problem a result of the social - communication impairments , repetitive behaviors , or sensory interests that characterize individuals with asd , or is this boy being noncompliant ? if his mother believes that he is upset by the change in routine , she might use a visual schedule to show him what to expect in this new location . indeed , mandell and colleagues reported that 40% of children with asd are referred for treatment of disruptive behaviors including aggression , noncompliance , and hyperactivity . from the above example , it is clear that : ( i ) an understanding of the underlying symptoms of asd is necessary for successful management of challenging behaviors ; and ( ii ) the involvement of caregivers in treating challenging behaviors in children and adolescents with asd aids in generalization to community settings . indeed , the involvement of caregivers has been identified as an essential component of a good treatment program . in fact , the utility of parents as interventionists has spanned over four decades , with schopler and reichler cited as being the first advocates for involving parents as cotherapists in the treatment of behavior problems in children with asd . parents are the child 's natural teachers , and caregiver - implemented interventions have been documented to promote skill generalization and maintenance while also potentially reducing parental stress and increasing quality of life . the nature of caregiver involvement varies across programs , with some programs focusing on providing education to caregivers about the child 's symptoms and other programs focusing on teaching caregivers specific technique that can be used in the natural environment . while both approaches have been successful , in order for treatment effects to be maintained , the intervention program needs to be compatible with the family 's daily routines . traditional caregiver - mediated interventions designed to reduce behavior problems in children without asd are typically geared toward treating noncompliance . in these interventions , caregivers are taught to facilitate appropriate behavior by using rewards , and to decrease noncompliance by using consequences including ignoring and time out . little research has been conducted to determine whether these traditional behavior management strategies are effective at treating problem behaviors in children with asd . because it is often an underlying symptom of asd rather than noncompliance that leads to challenging behaviors in these children , a traditional behavior management approach focused on noncompliance may not be effective . there have been several case studies reporting the effectiveness of using a traditional parent - mediated behavioral intervention , parent child interaction therapy ( pot ) , to decrease challenging behaviors in children with asd . these studies suggest that pot may be effective in reducing challenging behaviors , particularly when used with high - functioning children with asd and when adaptations are made to target social skills . solomon and colleagues conducted the only study to date that compared the effectiveness of a traditional caregiver - mediated behavioral intervention ( pot ) in a group of children with asd and a waitlist control group . they found increased shared positive affect in parent - child dyads but no changes in parent stress or parent report of challenging behaviors compared with the comparison group . they noted the importance of building a parent - child relationship for children with asd . thus , caregiver - mediated interventions targeting challenging behaviors for children with asd have not been consistently found to reduce challenging behaviors and may need to be adapted to meet the specific needs of this population . in a comparison of the caregiver - mediated intervention literature for children with asd and children with disruptive behavior disorders ( dbd ) , brookman - frazee and colleagues reported striking differences in intervention techniques . while 100% of the dbd literature reported targeting parenting strategies and discipline techniques , none of the asd literature targeted these skills . in contrast , 68% of the asd literature targeted communication skills while none of the dbd literature targeted communication . therefore , while mental health professionals may be tempted to treat challenging behaviors in children with asd using traditional caregiver - mediated behavior intervention techniques , different techniques may be needed . in the present article , we describe current caregiver - based intervention approaches geared toward understanding behavior problems within the context of asd symptomatology . further , we review the literature on caregiver - mediated interventions treating the most common causes for behavior problems in this population . when faced with a child 's observable challenging behaviors ( ie , those visible above the waterline ) , caregivers are encouraged to use their understanding of asd to identify possible underlying causes for these behaviors ( ie , those hidden below the waterline ) . indeed , a functional behavior assessment has been recognized as a necessary component in designing interventions to understand and to modify behavior in children with autism . in the behavior analytic literature , the reason why children exhibit problem behavior is often described as either to obtain an item , escape a task , or to seek attention . however , in children with asd the underlying reasons why children may engage in challenging behaviors may be related to autism - specific symptoms . in our example of the boy screaming in the grocery store , the social and sensory demands of the situation may have caused him to want to escape . that is , the hidden explanation for his disruptive behavior may be the social , sensory , or communicative demands of the situation . when working with families to conduct a functional behavior assessment and develop an intervention plan , moes and frea emphasized the importance of considering the family 's own environment , values , and beliefs . they suggested that a contextualized behavior support assessment that examines more than just the child 's behavior is important in increasing the compatibility between the behavioral intervention and family routines . this suggests that developing sustainable plans ( eg , compatible with family resources , constraints , beliefs , values , goals , abilities , and needs ) , may contribute to the generalization and maintenance of treatment gains . in our own clinical work , we have found that the most frequent communicative functions of challenging behavior include frustration over inability to communicate , difficulties with social interaction , anxiety , and atypical sensory sensitivities . we review the literature in each of these areas with a focus on interventions that include caregiver - mediated approaches . because one of the functions of challenging behavior is communication , it is not surprising that a considerable amount of intervention research has focused on developing successful procedures for improving communication . in addition to improvements in verbalizations , mean length of utterance , and spontaneity of language use , successful communication intervention has been associated with decreases in problem behavior and increases in positive affect . as a result , the replacement of challenging behaviors with appropriate and increasingly complex communication skills has the potential to have far - reaching implications for academic achievement , social relationship development , and vocational outcomes . clinically , this means that the child must learn to request using a system that is compatible with his / her mental age ( eg , pictures , sign language , words ) . there have been a number of behaviorally based communication intervention approaches designed to increase requesting skills , particularly focused on toddlers and preschool - aged children with asd . traditional applied behavior analysis approaches ( eg , discrete trial training ) , have been criticized for teaching prompt dependence ( eg , the screaming boy in the grocery store would wait for a prompt before using his learned verbal skills to say cookie ) , and for limited generalizability across contexts ( eg , he may learn to say cookie only in the grocery store ) . thus , there has been an increased emphasis on naturalistic or child - directed behavioral intervention approaches . these more naturalistic behavior interventions include incidental teaching , enhanced milieu teaching , and pivotal response training to teach requesting and other communication skills . while there are differences among the approaches , naturalistic behavior models share common characteristics , including child choice ( eg , teaching communication skills that fit with the child 's interests ; eg , the boy in the grocery store would learn cookie as a first word ) , intervention in natural contexts ( eg , learning to communicate during family routines such as eating dinner ) , and the use of intrinsically related rewards ( eg , the child would receive a cookie along with social praise ) . results have indicated that , compared with the traditional structured models , interventions that are less structured and more child - focused result not only in faster learning by children with asd , but also result in children displaying higher levels of affect and engaging in less avoidant and disruptive behavior . as our ability to accurately diagnose autism in the first few years of life has improved , there has been an increased demand for intervention approaches that integrate developmental and behavioral approaches to target social communication skills in toddlers and preschoolaged children with asd . similarly , the early start denver model incorporates behavioral and developmental strategies and has been associated with increased cognitive development and adaptive behavior . while not directly targeting requesting behavior , the focus on facilitating early social communication skills is designed to increase spontaneous communication skills and decrease challenging behaviors . this may be more successful than instruction occurring only during specified times , which may burden the family by requiring extra time and effort . thus , naturalistic approaches are ideally suited for caregivers . recently , a large community - based study found improvements for up to 1 year in children 's expressive and receptive communication and decreases in problem behavior with parents who learned how to implement pivotal response treatment . similarly , minjarez and colleagues found significant increases in children 's functional spontaneous speech for parents who participated in a communication intervention training where feedback was delivered based on the videotapes they brought in . however , more research is needed to determine whether caregiver - mediated interventions that combine developmental and behavioral approaches are effective at decreasing challenging behaviors . clinically , we often see behavior problems in children with asd who are overwhelmed by social situations ( eg , the child in a crowded grocery store ) or who want to engage in social interactions but do not have appropriate initiation skills ( eg , a preschool girl whose only way of initiating social interaction with siblings and peers is to pull their hair ) . while the boy in the grocery store may need to learn to request a break , the girl wanting to interact with her peers may need to learn a more appropriate way of initiating social interactions . indeed , bauminger and colleagues reported that children and adolescents with high - functioning asd may be interested in interacting with peers but do not have the knowledge about how to interact appropriately . in their study , children and adolescents with high - functioning asd initiated and responded to peers about half as often as age- and iq - matched children and adolescents with typical development . however , this decreased rate of social interactions may not be an indicator of a lack of interest in social interaction , as children and adolescents with asd reported higher levels of loneliness than their peers with typical development . a variety of skill - based intervention programs have been developed to teach social skills to children with asd . while early intervention programs have focused on relationship - based approaches that combine behavioral and developmental techniques , approaches for older children and adolescents these skill - building programs have taught social scripts to teach specific nonverbal and verbal behavior for specific situations , social stories to teach social norms and expectations , role plays to provide opportunities for practice , and self - monitoring through videotapes and checklists to increase self - awareness . remarkably , few of the skill - building intervention programs have included caregivers as cotherapists , or even included a caregiver education component . in a review of 66 studies published in peer - reviewed journals between 2001 - 2008 targeting social skills , reichow and volkmar reported that parent training was only measured as a main intervention variable in four studies , all targeted at improving social skills in preschool children . thus , while best practices consider parent and family involvement as an essential element of intervention programs for children with autism , very few studies targeting social skills have included parents beyond the toddler and early childhood years . the inclusion of caregivers is particularly important when social skills difficulties lead to challenging behaviors in the home and community settings . indeed , kasari and lawton cautioned that current social skills intervention approaches using behavioral principles seem to train children to produce specific operationalized behaviors rather than facilitating learning and generalization of the behavior across contexts . they caution that many current interventions may teach form but not function . frankel and colleagues taught conversation skills , peer entry , handling teasing , practicing good sportsmanship , and good host behaviors using children 's friendship training . in concurrent sessions , parents were taught how to facilitate the use of these skills at home by arranging for supervised play dates . compared with a delayed treatment control group , parents of children in the intervention group reported that their children showed increased social skills and other appropriate play date behaviors . in a study of the skillstreaming approach , lopata and colleagues targeted social skills , emotion recognition , and understanding of metaphoric language . compared with a delayed treatment control group , the intervention group showed increased parent - reported social skills . further , children in the intervention group showed increases on standardized measures of emotion recognition and non - literal language interpretation . both studies are encouraging and suggest that caregiver - mediated social skills interventions may be more successful at increasing flexibility and generalization of skills to the community setting than traditional social skills intervention programs . many children with autism experience clinical significant levels of anxiety , with 11% to 84% of children and adolescents with asd reported to have a diagnosable comorbid anxiety disorder : anxiety symptoms are varied and can include behavioral outbursts ( yelling , aggression ) , repetitive behaviors ( asking repetitive questions , reciting television quotes , pacing ) , withdrawal , and refusal to engage in activities . the most common anxiety symptoms in children and adolescents with asd are compulsive / ritualistic behavior and irrational fears and beliefs . leyfer and colleagues reported that 44% of the children and adolescents with asd in their study experienced specific phobias and 37% experienced symptoms consistent with obsessive - compulsive disorder . there is a growing literature on the use of adapted cognitive behavioral treatment ( cbt ) techniques to treat fears and irrational beliefs in asd ; using cbt , children with asd have been taught to identify their own anxiety , rate their anxiety levels , replace irrational thoughts with rational thoughts , and use progressive muscle relaxation routines ( see klinger and williams for a review ) . several recent randomized control studies have been conducted examining the efficacy of cbt for treating anxiety in children with asd . approximately 58% to 64% of children who completed the cbt treatments no longer met diagnostic criteria for an anxiety disorder , compared with 0% to 9% of children in waitlist control groups . further , several studies reported that these reductions were maintained 3 months after the intervention was completed . clinically , we have found that the involvement of caregivers in a cbt program provided a way for caregivers to understand the underlying reason for misbehavior . to date , there is no data on whether the incorporation of caregivers as coaches increases the effectiveness of cbt interventions for children with asd compared with programs that only include children in the intervention protocol . in addition to irrational fears and beliefs , ritualistic and compulsive behaviors are often indicators of anxiety in children with asd . because of difficulties with flexible thinking , students with asd may prefer routines and become anxious when routines are altered . children with asd may exhibit anxiety by becoming disruptive when there is a deviation from the typical family routine ( eg , when grandparents visit or when a vacation is scheduled ) . using this preventive approach , caregivers are encouraged to use a daily schedule that lists the activities planned for each day . for example , the child that screams in the grocery store may be bothered by the fluorescent lights or by the loudness in the store . indeed , reese , richman , zarcone , and zarcone reported that attempts to escape uncomfortable sensory situations explained disruptive behavior in 14% of the children with asd in their sample . hyperresponsiveness , hyporesponsiveness , and sensory seeking . recently , brock and colleagues identified sensory hyporesponsiveness in preschool - aged children with asd . despite the fact that it is commonly recognized that challenging behaviors are often exacerbated by atypical sensory processing in children with asd , very little intervention research has been conducted in this area . only three of 25 studies included in the review considered sit to be an effective therapy based on posttreatment measures . in contrast , 14 of the studies saw no improvement in children with asd who had received sit . thus , to date the most effective approaches for decreasing behavior problems due to sensory sensitivities may be aimed at reducing the anxiety that usually arises as a result of these sensitivities . it is important to consider family social and cultural factors that may impact the successful use of caregivermediated approaches . the requirements of an intervention approach often conflict with the caregiver 's other time demands including workplace , siblings , spouse , and extended family . further family cultural values must be considered , as any attempt to modify the caregiver 's behavior without attending to cultural factors may be ineffective . further , the chronicity of asd and its impact of caregiver stress should be considered . because behavior problems often arise from the underlying symptoms of asd , caregivers are likely to face a lifetime of behavior management challenges . indeed , raising a child with asd is associated with higher levels of caregiver stress and psychological distress than raising a child with typical development or a child with another developmental disability . weiss and colleagues reported that the relationship between child behavior problems and parent mental health is mediated by psychological acceptance . that is , those parents who were able to accept the challenges of living with a child with asd showed fewer negative mental health consequences . they argued that caregiver - mediated interventions need to focus on positive coping strategies in addition to the problem focused strategies discussed in this article . furthermore , researchers have suggested the importance of individualizing treatment by matching the intensity of the intervention to the caregivers ' stress level . another consideration for caregiver - mediated intervention programs is how the program content is presented and taught to the caregivers . in a meta - analytic review of program components associated with parent training effectiveness for children with externalizing disorders , kaminski and colleagues found that larger effect sizes were associated with programs that required parents to practice the skills during therapy sessions , focused on parenting consistency , and focused on increasing positive parent - child interactions . conversely , smaller effect sizes were associated with programs focused on problem - solving techniques and promotion of cognitive , academic , and social skills . further research is needed to identify the specific program elements that are associated with larger effects in caregiver - mediated behavioral interventions for children with asd . finally , while the focus of this article has been on treating underlying behavioral causes ( communication frustrations , social skills difficulties , anxiety , sensory sensitivities ) of challenging behaviors , it is important that caregivers and clinicians also consider possible underlying medical complications including gastrointestinal difficulties , sleep disorders , and seizures that often cooccur with asd . a significant proportion of children with asd are referred to mental health centers due to the presence of challenging behaviors . an understanding of the underlying symptoms of asd is essential in managing behavior problems in this population and the involvement of caregivers in treatment is critical to long - term success . while behavioral intervention approaches have been used extensively to improve the social , communication , and anxiety symptoms that often accompany asd , few randomized control studies have been conducted . in a review of the 68 behavior intervention articles published in 2008 to 2009 , kasari and lawton reported that 63% of the studies used case study or single - subject design approaches , 16% used a group design other than randomized control , and 21% of the studies used randomized control trials ( ie , 14 of 68 studies ) . thus , while the literature on the effectiveness of behavioral intervention is growing , there continues to be a need for randomized , controlled studies . further , while the importance of working with caregivers has been emphasized for the past four decades , more research is needed about the effectiveness of caregiver - implemented interventions and the techniques that are most effective at supporting caregivers use of strategies in the natural environment including family beliefs and culture .

across the dopps phases , the study design and methodology were structured with the following key elements : 1 ) random selection of dialysis units stratified by type of facility and geographic region in each country ; 2 ) collection of demographic data , diabetes as cause of end - stage renal disease ( esrd ) , and mortality data for all hemodialysis patients in each study unit ; 3 ) collection of detailed patient data from a random selection of 20 to 40 patients within each dialysis unit at study entry and at 4-month intervals ; 4 ) collection of kidney disease quality of life information ; and 5 ) collection of facility practice information , determined from questionnaires completed annually by the dialysis unit s medical director ( medical directors survey ) and by the unit s nurse manager . the methodologies of sample selection and data collection were substantially similar in phases 3 and 4 , with possible differences in operational aspects , such as the proportion of data collected electronically . many of the selected facilities in phase 3 continued participation in phase 4 , and there was overlap in the patient populations but not in the follow - up periods . the dopps 3 ( 20052008 ) and dopps 4 ( 20082011 ) populations included 12,954 patients with type 1 or type 2 diabetes . patients were coded as having diabetes if they had a diagnosis of type 1 or type 2 diabetes , if they had received medications for diabetes ( insulin or oral ) before enrollment , if they had diabetic gastroparesis , or if they were marked as diabetic on the patient census , which is a complete listing of patients dialyzed in the participating facility . there were 28,458 patients in the dopps phases 3 and 4 sample , of whom 12,954 had diabetes . an additional 296 patients were excluded because of missing dates for age , study entry , or study exit . the population for this study consisted of 9,201 patients with type 1 or type 2 diabetes after exclusion of patients with no a1c measurements during the first 8 months of dopps follow - up . the primary analyses were performed with the first available a1c value in those with at least one measurement during the first 8 months of data collection . in our data collection design , laboratory values and medication use were reported at study entry and by interval summary forms collected every 4 months afterward . covariate data were collected at study entry on patient age , sex , race ( black vs. other races ) , bmi , albumin , years of dialysis , creatinine , 12 comorbid conditions , insulin use , oral diabetes medication use , ldl cholesterol , hemoglobin , country , and study phase . missing data for covariates ( potential confounders ) , described in the footnote of table 1 , were accounted for by using multiple imputation on the population of patients with at least one valid a1c measurement , as implemented by the iveware program ( 10 ) , and analyzed with the mianalyze procedure in sas stat 9.2 ( 11 ) . missing a1c levels were not imputed , because patients whose physicians did not order measurement of a1c levels are likely to differ in important ways from patients whose physicians did order the test , invalidating the assumptions behind missing data imputation . analytical results derived from the multiply imputed data were compared with results obtained with other methods for dealing with missing data ( including complete case analyses and with missing data indicators with single - value imputation ) . the differences were trivial ( most of the hazard ratio [ hr ] estimates were within 0.03 ; the largest difference was 0.08 ) and did not affect our conclusions . further sensitivity testing of the effects of missing data for variables missing in more than 10% of the patients ( bmi , weight loss , and cholesterol ) consisted of imputation without these variables and without controlling for these variables and of complete case analyses excluding these variables . in general , the relationships between a1c and mortality were similar among all three methods . clinical and demographic characteristics by number and a1c category certain models evaluated patients with and without indicators of poor nutrition , or with and without recent diabetes treatment . for these models , patients were identified as having poor nutritional status if they had any one or more of the following factors ( n = 2,037 ) : bmi < 19 kg / m , weight loss during the first 8 months of dopps follow - up at a rate equivalent to 10% of body weight per year ; albumin < 3.0 mg / dl , or cachexia . patients were identified as recently treated for diabetes if at the start of follow - up they were receiving oral diabetes medicines ( n = 768 ) , insulin ( n = 2,685 ) , or both ( n = 293 ) . no diabetes medications were recorded for 3,477 patients , and 1,978 patients had insufficient drug information for coding before imputation . standard descriptive statistics were used to characterize the dopps patients with type 1 or type 2 diabetes . follow - up started at a1c measurement and ended at the time of death ( outcome event ) , 7 days after transfer from the facility , or as of the date of most recent data availability ( december 2011 or earlier ) , whichever came first . the effect of a1c on all - cause mortality was examined by cox proportional hazards analyses . all models were adjusted for patient age , sex , race ( black vs. other races ) , bmi , hemoglobin , albumin , years of dialysis , creatinine ( because this is a marker of dietary protein intake and muscle mass and is a predictor of clinical outcome in esrd patients ) , 12 comorbid conditions , insulin use , ldl cholesterol , country , and study phase . tests of the interaction between a1c and either nutritional status or diabetes treatment involved likelihood ratio tests of each covariate multiplied by the six indicator variables of a1c categories , although effect estimates and confidence limits were produced with separate models for each population ( i.e. , with and without diabetes treatment , or with and without indicators of poor nutritional status ) . the assumption of proportional hazards for the a1c categories was evaluated by visual inspection of the log(log[survival ] ) versus log(time ) plot and by testing the interaction between these categories and log(time ) , yielding p = 0.03 . although the relationship was slightly stronger during earlier periods of follow - up for mortality , there was reasonable adherence to proportional hazards . sensitivity analyses were performed by comparing the results obtained through the approach described here with models , with mean a1c during the first 8 months of dopps follow - up as the main predictor and restricting to 6,669 patients with at least two a1c measurements during that baseline period . these analyses were also adjusted for weight loss during the 8-month period . because follow - up for this latter analysis started after the baseline period , the duration of follow - up was on average about 4 months shorter than in the main analysis . serum glucose concentration was also examined , but it had a much weaker relation with mortality , presumably because serum glucose levels are more easily influenced by short - term factors . another sensitivity analysis tested the impact of dialysis information on the adjusted relationship between a1c and mortality . the dialysis information included kt / v ( dialyzer clearance of urea dialysis time / volume of distribution of urea ) , vascular access type , number of sessions per week , and dialysis duration . across the dopps phases , the study design and methodology were structured with the following key elements : 1 ) random selection of dialysis units stratified by type of facility and geographic region in each country ; 2 ) collection of demographic data , diabetes as cause of end - stage renal disease ( esrd ) , and mortality data for all hemodialysis patients in each study unit ; 3 ) collection of detailed patient data from a random selection of 20 to 40 patients within each dialysis unit at study entry and at 4-month intervals ; 4 ) collection of kidney disease quality of life information ; and 5 ) collection of facility practice information , determined from questionnaires completed annually by the dialysis unit s medical director ( medical directors survey ) and by the unit s nurse manager . the methodologies of sample selection and data collection were substantially similar in phases 3 and 4 , with possible differences in operational aspects , such as the proportion of data collected electronically . many of the selected facilities in phase 3 continued participation in phase 4 , and there was overlap in the patient populations but not in the follow - up periods . the dopps 3 ( 20052008 ) and dopps 4 ( 20082011 ) populations included 12,954 patients with type 1 or type 2 diabetes . patients were coded as having diabetes if they had a diagnosis of type 1 or type 2 diabetes , if they had received medications for diabetes ( insulin or oral ) before enrollment , if they had diabetic gastroparesis , or if they were marked as diabetic on the patient census , which is a complete listing of patients dialyzed in the participating facility . there were 28,458 patients in the dopps phases 3 and 4 sample , of whom 12,954 had diabetes . an additional 296 patients were excluded because of missing dates for age , study entry , or study exit . the population for this study consisted of 9,201 patients with type 1 or type 2 diabetes after exclusion of patients with no a1c measurements during the first 8 months of dopps follow - up . the primary analyses were performed with the first available a1c value in those with at least one measurement during the first 8 months of data collection . in our data collection design , laboratory values and medication use were reported at study entry and by interval summary forms collected every 4 months afterward . covariate data were collected at study entry on patient age , sex , race ( black vs. other races ) , bmi , albumin , years of dialysis , creatinine , 12 comorbid conditions , insulin use , oral diabetes medication use , ldl cholesterol , hemoglobin , country , and study phase . missing data for covariates ( potential confounders ) , described in the footnote of table 1 , were accounted for by using multiple imputation on the population of patients with at least one valid a1c measurement , as implemented by the iveware program ( 10 ) , and analyzed with the mianalyze procedure in sas stat 9.2 ( 11 ) . missing a1c levels were not imputed , because patients whose physicians did not order measurement of a1c levels are likely to differ in important ways from patients whose physicians did order the test , invalidating the assumptions behind missing data imputation . analytical results derived from the multiply imputed data were compared with results obtained with other methods for dealing with missing data ( including complete case analyses and with missing data indicators with single - value imputation ) . the differences were trivial ( most of the hazard ratio [ hr ] estimates were within 0.03 ; the largest difference was 0.08 ) and did not affect our conclusions . further sensitivity testing of the effects of missing data for variables missing in more than 10% of the patients ( bmi , weight loss , and cholesterol ) consisted of imputation without these variables and without controlling for these variables and of complete case analyses excluding these variables . in general , the relationships between a1c and mortality were similar among all three methods . clinical and demographic characteristics by number and a1c category certain models evaluated patients with and without indicators of poor nutrition , or with and without recent diabetes treatment . for these models , patients were identified as having poor nutritional status if they had any one or more of the following factors ( n = 2,037 ) : bmi < 19 kg / m , weight loss during the first 8 months of dopps follow - up at a rate equivalent to 10% of body weight per year ; albumin < 3.0 mg / dl , or cachexia . patients were identified as recently treated for diabetes if at the start of follow - up they were receiving oral diabetes medicines ( n = 768 ) , insulin ( n = 2,685 ) , or both ( n = 293 ) . no diabetes medications were recorded for 3,477 patients , and 1,978 patients had insufficient drug information for coding before imputation . standard descriptive statistics were used to characterize the dopps patients with type 1 or type 2 diabetes . follow - up started at a1c measurement and ended at the time of death ( outcome event ) , 7 days after transfer from the facility , or as of the date of most recent data availability ( december 2011 or earlier ) , whichever came first . the effect of a1c on all - cause mortality was examined by cox proportional hazards analyses . all models were adjusted for patient age , sex , race ( black vs. other races ) , bmi , hemoglobin , albumin , years of dialysis , creatinine ( because this is a marker of dietary protein intake and muscle mass and is a predictor of clinical outcome in esrd patients ) , 12 comorbid conditions , insulin use , ldl cholesterol , country , and study phase . tests of the interaction between a1c and either nutritional status or diabetes treatment involved likelihood ratio tests of each covariate multiplied by the six indicator variables of a1c categories , although effect estimates and confidence limits were produced with separate models for each population ( i.e. , with and without diabetes treatment , or with and without indicators of poor nutritional status ) . the assumption of proportional hazards for the a1c categories was evaluated by visual inspection of the log(log[survival ] ) versus log(time ) plot and by testing the interaction between these categories and log(time ) , yielding p = 0.03 . although the relationship was slightly stronger during earlier periods of follow - up for mortality , there was reasonable adherence to proportional hazards . sensitivity analyses were performed by comparing the results obtained through the approach described here with models , with mean a1c during the first 8 months of dopps follow - up as the main predictor and restricting to 6,669 patients with at least two a1c measurements during that baseline period . these analyses were also adjusted for weight loss during the 8-month period . because follow - up for this latter analysis started after the baseline period , the duration of follow - up was on average about 4 months shorter than in the main analysis . serum glucose concentration was also examined , but it had a much weaker relation with mortality , presumably because serum glucose levels are more easily influenced by short - term factors . another sensitivity analysis tested the impact of dialysis information on the adjusted relationship between a1c and mortality . the dialysis information included kt / v ( dialyzer clearance of urea dialysis time / volume of distribution of urea ) , vascular access type , number of sessions per week , and dialysis duration . the first available a1c ranged from 3.1 to 19.2% , whereas the mean a1c across 8 months ranged from 3.4 to 15.0% . table 1 shows clinical and demographic characteristics of the patients with type 1 or type 2 diabetes according to the number of a1c measurements made during the initial 8-month baseline period , as well as the first available a1c level among those with at least one a1c measurement during that period . patients with more a1c measurements during the first 8 months were treated more frequently with insulin or oral medicine than those with fewer a1c measurements and differed with respect to a number of other demographic and comorbid conditions . in addition , patients in whom a1c levels were not measured had a slightly lower prevalence of peripheral vascular disease and were more often black . among patients with at least one measurement of a1c , those with higher initial a1c values were also treated more frequently with insulin than were those with lower values ; on average they were younger , more likely to be black , higher in bmi , and were likely to have initiated dialysis more recently . among patients with at least one measurement of a1c , average follow - up was 1.4 years ( 1st99th percentile , 8 days3.3 years ) . the death rate was 0.16/year ( 1,983 deaths /12,513 patient - years ) . among these patients ( the main analysis ) , the adjusted mortality rate was lowest at a1c levels of 77.9% ( fig . a log likelihood test for nonlinearity showed an overall p value of 0.01 for the combined contribution of the squared and cubic a1c terms to the linear model between a1c and mortality . the relation was similar in shape in an unadjusted analysis , though slightly steeper at lower a1c levels and less steep at higher levels . a : risk of mortality by initial a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . b : risk of mortality by mean a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . ( a high - quality color representation of this figure is available in the online issue . ) our sensitivity analysis used the mean a1c during the first 8 months of dopps follow - up , and this showed a death rate of 0.20 deaths / year ( 1,337 deaths/6,679 patient - years ) . when examined in a cox model , the mortality rates were similar among patients with zero , one , or two measures of a1c during the initial 8 months of follow - up when restricted to patients who survived through all 8 months ( hr for 0 vs. 2 , 1.01 ; 95% ci 0.891.15 ; , 1 vs. 2 , 1.09 ; , 0.951.25 ) , with adjustment for phase and country . the conclusions reached with mean a1c were not different from those based on the initial a1c . most of the hr estimates based on the mean a1c were within 0.12 of the estimates made from the first available a1c in the adjusted model . the results of the adjusted models that used first available a1c level were substantially similar to the results from models that used mean a1c levels ( fig . another sensitivity analysis , excluding bmi , weight loss , and cholesterol and performing a complete case analysis on the remaining variables , yielded similar effect estimates , with the largest difference in the hr being at a1c < 5% ; namely 1.41 ( 1.131.77 ) in the complete case model instead of 1.30 ( 1.051.61 ) in the imputed data . a final sensitivity analysis , adding dialysis information ( kt / v , vascular access type , duration , and sessions per week ) resulted in a model very similar to the one shown in fig . the largest change in an a1c category s hr was only 0.019 , and there were no appreciable differences in statistical significance of any of the categories . in the examination of whether nutritional status modified the relationship between a1c and mortality , markers of poor nutritional status were more common in a1c categories below 6% ( table 2 ) . figure 2 shows the estimated effects of a1c on mortality by nutritional status ( good vs. poor ) . the shapes of the estimated dose - response associations differed noticeably ( p = 0.12 for the likelihood ratio test of the interaction ) between patients with and without indicators of poor nutritional status ( fig . , there was little association with mortality for patients with a1c < 9% , but the rate increased for patients with a1c 9% . in contrast , the association for patients with indicators of poor nutrition demonstrated higher mortality rates for patients with a1c < 7% and 8% . counts of patients with indicators of poor nutrition by mean a1c status risk of mortality by initial a1c among patients with and without indicators of poor nutrition , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . ( a high - quality color representation of this figure is available in the online issue . ) we also examined whether diabetes treatment modified the estimated effect of a1c on mortality . the pattern of dose - response associations between patients who were treated with oral diabetes medicines or insulin and those patients who were not treated with these medicines were similar ; however , the mortality rate among patients with low a1c levels was somewhat higher among patients receiving oral medicine or insulin . the p value for the overall log - likelihood test of interaction between diabetes treatment and a1c category was 0.97 . as expected , the proportion of patients being prescribed oral diabetes medicines or insulin during the baseline period was positively associated with a1c level . the percentage of patients on any diabetes medicine was 22% for patients with a1c levels below 5% and 35% for patients with a1c levels below 6% . this percentage rose to 71% for patients with a1c levels at 9% or above , leaving 29% of these patients untreated . the first available a1c ranged from 3.1 to 19.2% , whereas the mean a1c across 8 months ranged from 3.4 to 15.0% . table 1 shows clinical and demographic characteristics of the patients with type 1 or type 2 diabetes according to the number of a1c measurements made during the initial 8-month baseline period , as well as the first available a1c level among those with at least one a1c measurement during that period . patients with more a1c measurements during the first 8 months were treated more frequently with insulin or oral medicine than those with fewer a1c measurements and differed with respect to a number of other demographic and comorbid conditions . in addition , patients in whom a1c levels were not measured had a slightly lower prevalence of peripheral vascular disease and were more often black . among patients with at least one measurement of a1c , those with higher initial a1c values were also treated more frequently with insulin than were those with lower values ; on average they were younger , more likely to be black , higher in bmi , and were likely to have initiated dialysis more recently . among patients with at least one measurement of a1c , average follow - up was 1.4 years ( 1st99th percentile , 8 days3.3 years ) . the death rate was 0.16/year ( 1,983 deaths /12,513 patient - years ) . among these patients ( the main analysis ) , the adjusted mortality rate was lowest at a1c levels of 77.9% ( fig . a log likelihood test for nonlinearity showed an overall p value of 0.01 for the combined contribution of the squared and cubic a1c terms to the linear model between a1c and mortality . the relation was similar in shape in an unadjusted analysis , though slightly steeper at lower a1c levels and less steep at higher levels . a : risk of mortality by initial a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . b : risk of mortality by mean a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . ( a high - quality color representation of this figure is available in the online issue . ) our sensitivity analysis used the mean a1c during the first 8 months of dopps follow - up , and this showed a death rate of 0.20 deaths / year ( 1,337 deaths/6,679 patient - years ) . when examined in a cox model , the mortality rates were similar among patients with zero , one , or two measures of a1c during the initial 8 months of follow - up when restricted to patients who survived through all 8 months ( hr for 0 vs. 2 , 1.01 ; 95% ci 0.891.15 ; , 1 vs. 2 , 1.09 ; , 0.951.25 ) , with adjustment for phase and country . the conclusions reached with mean a1c were not different from those based on the initial a1c . most of the hr estimates based on the mean a1c were within 0.12 of the estimates made from the first available a1c in the adjusted model . the results of the adjusted models that used first available a1c level were substantially similar to the results from models that used mean a1c levels ( fig . another sensitivity analysis , excluding bmi , weight loss , and cholesterol and performing a complete case analysis on the remaining variables , yielded similar effect estimates , with the largest difference in the hr being at a1c < 5% ; namely 1.41 ( 1.131.77 ) in the complete case model instead of 1.30 ( 1.051.61 ) in the imputed data . a final sensitivity analysis , adding dialysis information ( kt / v , vascular access type , duration , and sessions per week ) resulted in a model very similar to the one shown in fig . 1a . the largest change in an a1c category s hr was only 0.019 , and there were no appreciable differences in statistical significance of any of the categories . in the examination of whether nutritional status modified the relationship between a1c and mortality , markers of poor nutritional status were more common in a1c categories below 6% ( table 2 ) . figure 2 shows the estimated effects of a1c on mortality by nutritional status ( good vs. poor ) . the shapes of the estimated dose - response associations differed noticeably ( p = 0.12 for the likelihood ratio test of the interaction ) between patients with and without indicators of poor nutritional status ( fig . there was little association with mortality for patients with a1c < 9% , but the rate increased for patients with a1c 9% . in contrast , the association for patients with indicators of poor nutrition demonstrated higher mortality rates for patients with a1c < 7% and 8% . counts of patients with indicators of poor nutrition by mean a1c status risk of mortality by initial a1c among patients with and without indicators of poor nutrition , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . ( a high - quality color representation of this figure is available in the online issue . ) the pattern of dose - response associations between patients who were treated with oral diabetes medicines or insulin and those patients who were not treated with these medicines were similar ; however , the mortality rate among patients with low a1c levels was somewhat higher among patients receiving oral medicine or insulin . the p value for the overall log - likelihood test of interaction between diabetes treatment and a1c category was 0.97 . as expected , the proportion of patients being prescribed oral diabetes medicines or insulin during the baseline period was positively associated with a1c level . the percentage of patients on any diabetes medicine was 22% for patients with a1c levels below 5% and 35% for patients with a1c levels below 6% . this percentage rose to 71% for patients with a1c levels at 9% or above , leaving 29% of these patients untreated . a1c levels strongly predict all - cause mortality in hemodialysis patients with type 1 or type 2 diabetes . in the current study , mortality was lowest at a1c levels of 77.9% and increased progressively for either lower or higher a1c levels . the relationship between low a1c and mortality appeared to be even stronger in patients with indicators of poor nutritional status , including low serum albumin , low bmi , or presence of cachexia . these findings suggest that optimal a1c levels among hemodialysis patients with diabetes may need to be less stringent than levels recommended for patients with diabetes who do not have advanced chronic kidney disease ( ckd ) . careful attention to the use of diabetes medicines , which our data indicate are frequently prescribed to hemodialysis patients with a1c < 6% and frequently not prescribed to those with a1c levels 9% , is a readily modifiable practice that may improve clinical outcomes . our findings differ from previous studies that did not show a relationship between a1c levels and mortality . a recent study did not show a relationship between a1c level and patient survival , whereas glycated albumin levels were more predictive of patient outcomes ( 12 ) . that study was limited by a relatively small sample size ( 444 subjects ) , however , and thus by a low power for detecting a1c effects on clinical outcomes . elevated a1c levels were also found not to be associated with mortality in a retrospective cohort study of maintenance hemodialysis patients in canada ( 13 ) . differences in findings between these studies and ours may relate to variations in patient case mix , other confounding factors , or differences in duration of follow - up . indeed , although a study based on data from a large dialysis organization did not identify an association between a1c level and survival during a 12-month follow - up period ( 6 ) , updated analyses of the same study population were consistent with our findings , with increased mortality rates observed at extremes of a1c levels ( 5 ) . our findings are also consistent with those previously published on data from another large dialysis organization in the u.s . , in which a similar increased mortality rate was seen at both low and high a1c levels ( 4,14 ) . our findings differ somewhat from both those studies in that we found the lowest mortality rate for patients with a1c levels between 7 and 7.9% , whereas kalantar - zadeh et al . ( 4 ) found the lowest rate , after adjustment for malnutrition - inflammation complex syndrome , for patients with a1c levels between 5 and 5.9% . ( 14 ) abstract showed approximately constant rates throughout the 57.9% range after adjusting for malnutrition - inflammation complex syndrome when using a baseline measure and the lowest rate to be in the 77.9% range when using the time - averaged measure . analyses in japanese populations also revealed increased mortality with high a1c levels ( 9,15 ) . thus our findings and those presented by others suggest that intensive glycemic control ( a1c < 6.0% or perhaps < 5% ) may not be optimal in the esrd ) population . the national kidney foundation ( nkf ) released the kidney disease outcomes quality initiative ( kdoqi ) clinical practice guidelines for the management of diabetes in ckd in 2007 ( 8) . these guidelines recommend that the target a1c for persons with diabetes and ckd be set at < 7% , the same as for diabetes patients without ckd . given the limited published literature on this topic among patients with advanced ckd , the nkf workgroup based their recommendation primarily on data obtained from diabetes patients with ckd stages 1 and 2 . new evidence from both clinical trials and observational studies published since release of these guidelines point to the need for a higher a1c target in some patient groups ( 4,6 ) . for instance , the action to control cardiovascular risk in diabetes ( accord ) study , conducted in the non - esrd population , showed that intensive therapy to normalize a1c levels was associated with increased mortality , a result similar to the findings of this study , and did not result in significant reduction in cardiovascular events ( 16 ) . in response to the new evidence , the nkf is updating the kdoqi guidelines . the american diabetes association also updated its 2012 clinical practice recommendations by strengthening the evidence for its recommendation that higher a1c goals may be appropriate in some patients with diabetes ( 7 ) . it should be noted that caveats exist in evaluating a1c measures in the esrd population . first , a reduced life span of erythrocytes , as is common in dialysis patients , may result in lower a1c levels than for non - esrd diabetes patients with the same degree of glycemic control ( 2 ) . furthermore , treatment with exogenous erythropoietin results in an increased proportion of reticulocytes in the circulation , which may be associated with less time for hemoglobin glycosylation to occur ( 3 ) . indeed , recent work suggests the potential for glycated albumin , which , unlike a1c , is not influenced by changes in erythrocyte survival or erythropoiesis - stimulating agent dose , as a measure of glucose control in the esrd population ( 12,17 ) . in addition , there are concerns that hba1c may not reflect glycemic control in the short term because of its prolonged half - life , whereas glycated albumin may potentially reflect short - term changes in plasma glucose ( 17 ) . further study is required to evaluate the use of glycated albumin ( 18,19 ) , however , especially in light of a recent study that suggests alteration in albumin quantitation among hemodialysis patients because of increased oxidative stress ( 20 ) . despite differences in the use of a1c for measuring glycemic control in diabetes patients with and without esrd , the current kdoqi guidelines proposed similar standards for diabetes management as set by the american diabetes association ( 21 ) , with an a1c target below 7% . our findings clearly suggest the continued importance of periodic a1c measurement , because a1c level is strongly associated with mortality . the potential target a1c level appears to differ from that in the general population , however , in that a higher range should be considered for esrd dialysis patients . our findings of a higher target range or a less intensive target may potentially be explained by greater fluctuation in glucose levels in the hemodialysis diabetes population ( 22 ) . it is possible that the net catabolic balance ( 23 ) combined with the frequent occurrence of poor nutritional status of patients on hemodialysis ( 24 ) may require some degree of liberalization of target glucose levels . various competing factors in the chronic dialysis setting may also alter the net balance of glucose control , such as changes in tissue sensitivity to insulin , existence of metabolic acidosis , variations in dextrose concentration in dialysate solution , all of which may all have varying effects on glycemic control ( 22 ) . patients with esrd also have reduced clearance of insulin and certain oral hypoglycemic drugs used to treat diabetes . for example , unmeasured confounders may have biased our estimates of the a1c association with mortality . we also observed a significant percentage of patients with diabetes who had no recorded measurement of a1c levels , and generalization of our findings to these patients may not be appropriate . in addition , there may be a potential for selection bias if the presence or absence of an a1c measurement during the 8-month period is associated with both the level of a1c and mortality . it is encouraging to note , however , that our effect estimates were similar when we restricted the study population to patients who had at least two a1c measurements during the first 8 months of dopps follow - up . our analyses also suggest that 78% of patients with a1c<5% were not receiving glucose - lowering agents . a potential explanation for the low a1c levels among these untreated patients may relate to poor nutritional status , which is highly prevalent among esrd patients ( 23 ) . another possible explanation for the low a1c despite lack of treatment is the concept of burnout of diabetes with onset of esrd , in which some observations have suggested spontaneous decreases of hemoglobin a1c levels among esrd patients ( 25 ) . whereas there is a possibility that patients with a1c < 5% and who were not receiving glucose - lowering agents may actually have been mislabeled as having diabetes , it is unlikely that any such misclassification is selective or biased toward those with low a1c , and any such random misclassification would only have biased our findings toward the null hypothesis . in summary , our findings of a strong association of both high and low a1c levels with elevated mortality suggest the importance of a1c measurement in the management of patients with diabetes undergoing chronic hemodialysis . this analysis supports accumulating evidence that a target range hemoglobin a1c may be indicated in dialysis patients , rather than upper limit cut - point of < 7% , as noted in previous practice guidelines . the dopps is an international population , and the consistency of our findings with those limited to u.s .- specific analyses is reassuring . finally , opportunities for improved use of hypoglycemic agents are suggested by these international data .

objectivelowering hemoglobin a1c to < 7% reduces the risk of microvascular complications of diabetes , but the importance of maintaining this target in diabetes patients with kidney failure is unclear . we evaluated the relationship between a1c levels and mortality in an international prospective cohort study of hemodialysis patients.research design and methodsincluded were 9,201 hemodialysis patients from 12 countries ( dialysis outcomes and practice patterns study 3 and 4 , 20062010 ) with type 1 or type 2 diabetes and at least one a1c measurement during the first 8 months after study entry . associations between a1c and mortality were assessed with cox regression , adjusting for potential confounders.resultsthe association between a1c and mortality was u - shaped . compared with an a1c of 77.9% , the hazard ratios ( 95% ci ) for a1c levels were 1.35 ( 1.091.67 ) for < 5% , 1.18 ( 1.011.37 ) for 55.9% , 1.21 ( 1.051.41 ) for 66.9% , 1.16 ( 0.941.43 ) for 88.9% , and 1.38 ( 1.111.71 ) for 9.0% , after adjustment for age , sex , race , bmi , serum albumin , years of dialysis , serum creatinine , 12 comorbid conditions , insulin use , hemoglobin , ldl cholesterol , country , and study phase . diabetes medications were prescribed for 35% of patients with a1c < 6% and not prescribed for 29% of those with a1c 9%.conclusionsa1c levels strongly predicted mortality in hemodialysis patients with type 1 or type 2 diabetes . mortality increased as a1c moved further from 77.9% ; thus , target a1c in hemodialysis patients may encompass values higher than those recommended by current guidelines . modifying glucose - lowering medicines for dialysis patients to target a1c levels within this range may be a modifiable practice to improve outcomes .

RESEARCH DESIGN AND METHODS Study population Covariates Analyses Sensitivity analyses RESULTS Clinical and demographic characteristics A Sensitivity analyses Impact of nutritional status Diabetes treatment CONCLUSIONS

across the dopps phases , the study design and methodology were structured with the following key elements : 1 ) random selection of dialysis units stratified by type of facility and geographic region in each country ; 2 ) collection of demographic data , diabetes as cause of end - stage renal disease ( esrd ) , and mortality data for all hemodialysis patients in each study unit ; 3 ) collection of detailed patient data from a random selection of 20 to 40 patients within each dialysis unit at study entry and at 4-month intervals ; 4 ) collection of kidney disease quality of life information ; and 5 ) collection of facility practice information , determined from questionnaires completed annually by the dialysis unit s medical director ( medical directors survey ) and by the unit s nurse manager . the methodologies of sample selection and data collection were substantially similar in phases 3 and 4 , with possible differences in operational aspects , such as the proportion of data collected electronically . the dopps 3 ( 20052008 ) and dopps 4 ( 20082011 ) populations included 12,954 patients with type 1 or type 2 diabetes . patients were coded as having diabetes if they had a diagnosis of type 1 or type 2 diabetes , if they had received medications for diabetes ( insulin or oral ) before enrollment , if they had diabetic gastroparesis , or if they were marked as diabetic on the patient census , which is a complete listing of patients dialyzed in the participating facility . an additional 296 patients were excluded because of missing dates for age , study entry , or study exit . the population for this study consisted of 9,201 patients with type 1 or type 2 diabetes after exclusion of patients with no a1c measurements during the first 8 months of dopps follow - up . the primary analyses were performed with the first available a1c value in those with at least one measurement during the first 8 months of data collection . covariate data were collected at study entry on patient age , sex , race ( black vs. other races ) , bmi , albumin , years of dialysis , creatinine , 12 comorbid conditions , insulin use , oral diabetes medication use , ldl cholesterol , hemoglobin , country , and study phase . missing data for covariates ( potential confounders ) , described in the footnote of table 1 , were accounted for by using multiple imputation on the population of patients with at least one valid a1c measurement , as implemented by the iveware program ( 10 ) , and analyzed with the mianalyze procedure in sas stat 9.2 ( 11 ) . in general , the relationships between a1c and mortality were similar among all three methods . for these models , patients were identified as having poor nutritional status if they had any one or more of the following factors ( n = 2,037 ) : bmi < 19 kg / m , weight loss during the first 8 months of dopps follow - up at a rate equivalent to 10% of body weight per year ; albumin < 3.0 mg / dl , or cachexia . standard descriptive statistics were used to characterize the dopps patients with type 1 or type 2 diabetes . all models were adjusted for patient age , sex , race ( black vs. other races ) , bmi , hemoglobin , albumin , years of dialysis , creatinine ( because this is a marker of dietary protein intake and muscle mass and is a predictor of clinical outcome in esrd patients ) , 12 comorbid conditions , insulin use , ldl cholesterol , country , and study phase . sensitivity analyses were performed by comparing the results obtained through the approach described here with models , with mean a1c during the first 8 months of dopps follow - up as the main predictor and restricting to 6,669 patients with at least two a1c measurements during that baseline period . another sensitivity analysis tested the impact of dialysis information on the adjusted relationship between a1c and mortality . across the dopps phases , the study design and methodology were structured with the following key elements : 1 ) random selection of dialysis units stratified by type of facility and geographic region in each country ; 2 ) collection of demographic data , diabetes as cause of end - stage renal disease ( esrd ) , and mortality data for all hemodialysis patients in each study unit ; 3 ) collection of detailed patient data from a random selection of 20 to 40 patients within each dialysis unit at study entry and at 4-month intervals ; 4 ) collection of kidney disease quality of life information ; and 5 ) collection of facility practice information , determined from questionnaires completed annually by the dialysis unit s medical director ( medical directors survey ) and by the unit s nurse manager . the dopps 3 ( 20052008 ) and dopps 4 ( 20082011 ) populations included 12,954 patients with type 1 or type 2 diabetes . patients were coded as having diabetes if they had a diagnosis of type 1 or type 2 diabetes , if they had received medications for diabetes ( insulin or oral ) before enrollment , if they had diabetic gastroparesis , or if they were marked as diabetic on the patient census , which is a complete listing of patients dialyzed in the participating facility . an additional 296 patients were excluded because of missing dates for age , study entry , or study exit . the population for this study consisted of 9,201 patients with type 1 or type 2 diabetes after exclusion of patients with no a1c measurements during the first 8 months of dopps follow - up . the primary analyses were performed with the first available a1c value in those with at least one measurement during the first 8 months of data collection . covariate data were collected at study entry on patient age , sex , race ( black vs. other races ) , bmi , albumin , years of dialysis , creatinine , 12 comorbid conditions , insulin use , oral diabetes medication use , ldl cholesterol , hemoglobin , country , and study phase . missing data for covariates ( potential confounders ) , described in the footnote of table 1 , were accounted for by using multiple imputation on the population of patients with at least one valid a1c measurement , as implemented by the iveware program ( 10 ) , and analyzed with the mianalyze procedure in sas stat 9.2 ( 11 ) . in general , the relationships between a1c and mortality were similar among all three methods . for these models , patients were identified as having poor nutritional status if they had any one or more of the following factors ( n = 2,037 ) : bmi < 19 kg / m , weight loss during the first 8 months of dopps follow - up at a rate equivalent to 10% of body weight per year ; albumin < 3.0 mg / dl , or cachexia . standard descriptive statistics were used to characterize the dopps patients with type 1 or type 2 diabetes . all models were adjusted for patient age , sex , race ( black vs. other races ) , bmi , hemoglobin , albumin , years of dialysis , creatinine ( because this is a marker of dietary protein intake and muscle mass and is a predictor of clinical outcome in esrd patients ) , 12 comorbid conditions , insulin use , ldl cholesterol , country , and study phase . sensitivity analyses were performed by comparing the results obtained through the approach described here with models , with mean a1c during the first 8 months of dopps follow - up as the main predictor and restricting to 6,669 patients with at least two a1c measurements during that baseline period . another sensitivity analysis tested the impact of dialysis information on the adjusted relationship between a1c and mortality . table 1 shows clinical and demographic characteristics of the patients with type 1 or type 2 diabetes according to the number of a1c measurements made during the initial 8-month baseline period , as well as the first available a1c level among those with at least one a1c measurement during that period . patients with more a1c measurements during the first 8 months were treated more frequently with insulin or oral medicine than those with fewer a1c measurements and differed with respect to a number of other demographic and comorbid conditions . among patients with at least one measurement of a1c , those with higher initial a1c values were also treated more frequently with insulin than were those with lower values ; on average they were younger , more likely to be black , higher in bmi , and were likely to have initiated dialysis more recently . among patients with at least one measurement of a1c , average follow - up was 1.4 years ( 1st99th percentile , 8 days3.3 years ) . among these patients ( the main analysis ) , the adjusted mortality rate was lowest at a1c levels of 77.9% ( fig . a : risk of mortality by initial a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . b : risk of mortality by mean a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . our sensitivity analysis used the mean a1c during the first 8 months of dopps follow - up , and this showed a death rate of 0.20 deaths / year ( 1,337 deaths/6,679 patient - years ) . when examined in a cox model , the mortality rates were similar among patients with zero , one , or two measures of a1c during the initial 8 months of follow - up when restricted to patients who survived through all 8 months ( hr for 0 vs. 2 , 1.01 ; 95% ci 0.891.15 ; , 1 vs. 2 , 1.09 ; , 0.951.25 ) , with adjustment for phase and country . another sensitivity analysis , excluding bmi , weight loss , and cholesterol and performing a complete case analysis on the remaining variables , yielded similar effect estimates , with the largest difference in the hr being at a1c < 5% ; namely 1.41 ( 1.131.77 ) in the complete case model instead of 1.30 ( 1.051.61 ) in the imputed data . in the examination of whether nutritional status modified the relationship between a1c and mortality , markers of poor nutritional status were more common in a1c categories below 6% ( table 2 ) . , there was little association with mortality for patients with a1c < 9% , but the rate increased for patients with a1c 9% . in contrast , the association for patients with indicators of poor nutrition demonstrated higher mortality rates for patients with a1c < 7% and 8% . counts of patients with indicators of poor nutrition by mean a1c status risk of mortality by initial a1c among patients with and without indicators of poor nutrition , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . the pattern of dose - response associations between patients who were treated with oral diabetes medicines or insulin and those patients who were not treated with these medicines were similar ; however , the mortality rate among patients with low a1c levels was somewhat higher among patients receiving oral medicine or insulin . as expected , the proportion of patients being prescribed oral diabetes medicines or insulin during the baseline period was positively associated with a1c level . the percentage of patients on any diabetes medicine was 22% for patients with a1c levels below 5% and 35% for patients with a1c levels below 6% . this percentage rose to 71% for patients with a1c levels at 9% or above , leaving 29% of these patients untreated . table 1 shows clinical and demographic characteristics of the patients with type 1 or type 2 diabetes according to the number of a1c measurements made during the initial 8-month baseline period , as well as the first available a1c level among those with at least one a1c measurement during that period . patients with more a1c measurements during the first 8 months were treated more frequently with insulin or oral medicine than those with fewer a1c measurements and differed with respect to a number of other demographic and comorbid conditions . among patients with at least one measurement of a1c , those with higher initial a1c values were also treated more frequently with insulin than were those with lower values ; on average they were younger , more likely to be black , higher in bmi , and were likely to have initiated dialysis more recently . among these patients ( the main analysis ) , the adjusted mortality rate was lowest at a1c levels of 77.9% ( fig . a : risk of mortality by initial a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . b : risk of mortality by mean a1c , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . our sensitivity analysis used the mean a1c during the first 8 months of dopps follow - up , and this showed a death rate of 0.20 deaths / year ( 1,337 deaths/6,679 patient - years ) . when examined in a cox model , the mortality rates were similar among patients with zero , one , or two measures of a1c during the initial 8 months of follow - up when restricted to patients who survived through all 8 months ( hr for 0 vs. 2 , 1.01 ; 95% ci 0.891.15 ; , 1 vs. 2 , 1.09 ; , 0.951.25 ) , with adjustment for phase and country . another sensitivity analysis , excluding bmi , weight loss , and cholesterol and performing a complete case analysis on the remaining variables , yielded similar effect estimates , with the largest difference in the hr being at a1c < 5% ; namely 1.41 ( 1.131.77 ) in the complete case model instead of 1.30 ( 1.051.61 ) in the imputed data . in the examination of whether nutritional status modified the relationship between a1c and mortality , markers of poor nutritional status were more common in a1c categories below 6% ( table 2 ) . there was little association with mortality for patients with a1c < 9% , but the rate increased for patients with a1c 9% . in contrast , the association for patients with indicators of poor nutrition demonstrated higher mortality rates for patients with a1c < 7% and 8% . counts of patients with indicators of poor nutrition by mean a1c status risk of mortality by initial a1c among patients with and without indicators of poor nutrition , adjusted for age , sex , race , bmi , years of dialysis , albumin , creatinine , 10 comorbid conditions , insulin use , hemoglobin , hdl cholesterol , country , and study phase . the pattern of dose - response associations between patients who were treated with oral diabetes medicines or insulin and those patients who were not treated with these medicines were similar ; however , the mortality rate among patients with low a1c levels was somewhat higher among patients receiving oral medicine or insulin . as expected , the proportion of patients being prescribed oral diabetes medicines or insulin during the baseline period was positively associated with a1c level . the percentage of patients on any diabetes medicine was 22% for patients with a1c levels below 5% and 35% for patients with a1c levels below 6% . this percentage rose to 71% for patients with a1c levels at 9% or above , leaving 29% of these patients untreated . a1c levels strongly predict all - cause mortality in hemodialysis patients with type 1 or type 2 diabetes . in the current study , mortality was lowest at a1c levels of 77.9% and increased progressively for either lower or higher a1c levels . the relationship between low a1c and mortality appeared to be even stronger in patients with indicators of poor nutritional status , including low serum albumin , low bmi , or presence of cachexia . careful attention to the use of diabetes medicines , which our data indicate are frequently prescribed to hemodialysis patients with a1c < 6% and frequently not prescribed to those with a1c levels 9% , is a readily modifiable practice that may improve clinical outcomes . our findings differ from previous studies that did not show a relationship between a1c levels and mortality . elevated a1c levels were also found not to be associated with mortality in a retrospective cohort study of maintenance hemodialysis patients in canada ( 13 ) . our findings differ somewhat from both those studies in that we found the lowest mortality rate for patients with a1c levels between 7 and 7.9% , whereas kalantar - zadeh et al . ( 4 ) found the lowest rate , after adjustment for malnutrition - inflammation complex syndrome , for patients with a1c levels between 5 and 5.9% . these guidelines recommend that the target a1c for persons with diabetes and ckd be set at < 7% , the same as for diabetes patients without ckd . for instance , the action to control cardiovascular risk in diabetes ( accord ) study , conducted in the non - esrd population , showed that intensive therapy to normalize a1c levels was associated with increased mortality , a result similar to the findings of this study , and did not result in significant reduction in cardiovascular events ( 16 ) . first , a reduced life span of erythrocytes , as is common in dialysis patients , may result in lower a1c levels than for non - esrd diabetes patients with the same degree of glycemic control ( 2 ) . despite differences in the use of a1c for measuring glycemic control in diabetes patients with and without esrd , the current kdoqi guidelines proposed similar standards for diabetes management as set by the american diabetes association ( 21 ) , with an a1c target below 7% . we also observed a significant percentage of patients with diabetes who had no recorded measurement of a1c levels , and generalization of our findings to these patients may not be appropriate . in addition , there may be a potential for selection bias if the presence or absence of an a1c measurement during the 8-month period is associated with both the level of a1c and mortality . it is encouraging to note , however , that our effect estimates were similar when we restricted the study population to patients who had at least two a1c measurements during the first 8 months of dopps follow - up . our analyses also suggest that 78% of patients with a1c<5% were not receiving glucose - lowering agents . whereas there is a possibility that patients with a1c < 5% and who were not receiving glucose - lowering agents may actually have been mislabeled as having diabetes , it is unlikely that any such misclassification is selective or biased toward those with low a1c , and any such random misclassification would only have biased our findings toward the null hypothesis . in summary , our findings of a strong association of both high and low a1c levels with elevated mortality suggest the importance of a1c measurement in the management of patients with diabetes undergoing chronic hemodialysis . this analysis supports accumulating evidence that a target range hemoglobin a1c may be indicated in dialysis patients , rather than upper limit cut - point of < 7% , as noted in previous practice guidelines .

the extraordinary transmission of light through thin metallic films with periodic nanohole arrays ( nhas ) has attracted substantial attention in the past decade . this phenomenon is attributed to the diffractive excitation of surface plasmon polaritons ( spps ) , and it enables efficient tunneling of light through ( otherwise opaque ) metallic film in narrow wavelength regimes . the transmission characteristics of metallic nhas can be controlled by shape , depth , and periodicity of the nanoholes as well as by the type of metal and refractive index of the surrounding medium . plasmonic nhas have been prepared by a range of modern fabrication techniques such as focused ion beam milling , laser interference lithography , electron beam lithography , and template stripping . the relative simplicity of design and fabrication of plasmonic nhas have paved the way for their applications in detection and observation of biomolecules by using direct refractometric format , fluorescence spectroscopy , surface - enhanced raman spectroscopy , and surface - enhanced infrared absorption spectroscopy as well for spectral filtering and transparent electrode . conventional designs of plasmonic nanostructures are static , and their optical properties are fixed once they are prepared . in order to actuate resonant coupling to surface plasmons , metallic nanostructures have been interfaced with inorganic materials with electro - optically or magneto - optically modulated refractive index . in parallel , organic materials have been explored for actuating of surface plasmon resonance by refractive index variations associated with the photochromic effects and liquid crystal phase transitions as well as by mechanical changes induced by specific molecular binding and by responsive polymers . among the systems that utilize responsive polymers , poly(n - isopropylacrylamide ) ( pnipaam ) based polymers ( in particular copolymers with carboxylic acid groups ) hold a prominent position due to their strong volumetric change upon swelling and collapsing by variation of the temperature around its lower critical solution temperature ( lcst ) of 32 c . this polymer was explored in form of brushes that surrounded or directly capped metallic nanoparticles in order to reversibly tune localized surface plasmon resonance . in addition , pnipaam microgels were coupled with au nanoparticles . moreover , pnipaam - based networks were used as a host for au nanoparticles that displayed tunable localized surface plasmon resonance wavelength originating from near field plasmonic coupling . the swelling and collapsing of pnipaam - based architectures is driven by water diffusion , and the response in the millisecond range was measured for about micrometer thick cross - linked pnipaam - based hydrogel layer by long - range surface plasmons on the top of a indium tin oxide microheater . pnipaam brush with a thickness of 30 nm was characterized to respond faster in about 150 s , when rapid plasmonic heating was employed . this work presents a new plasmonic structure that is tunable by thermoresponsive pnipaam - based hydrogel and that can be prepared by template stripping . it consists of a thin au film with nha that allows for diffractive coupling to propagating spps . this structure is tethered to a solid surface by a cross - linked pnipaam - based hydrogel cushion which responds to temperature variations . it can rapidly swell and collapse , which triggers a flow of water through the plasmonic pores , and it strongly modulates the refractive index by altereing the polymer fraction in the hydrogel film , leading to changes in the transmission spectrum mediated by surface plasmon modes . off - stoichiometry thiol ene polymer ( ostemer 321 ) was purchased from mercene laboratories ( sweden ) . polydimethylsiloxane ( pdms , sylgard 184 ) was purchased from dow corning ( usa ) . pnipaam - based terpolymer ( composed of n - isoproprylacrylamide , methacrylic acid , and 4-methacryloyloxybenzophenone in a ratio of 94:5:1 ) was synthesized as reported before . ( 3-thiopropyl)oxybenzophenone ( benzophenone - thiol ) was synthesized as described in the literature . the electron beam lithography system els-7000 from elionix ( japan ) was used to structure neb22 resist layer with a thickness of 220 nm on a 600 m thick silicon wafer with native oxide . the exposure dose of 178 c cm and a current of 800 pa were used and the resist was developed by o - xylene . then , au layer was deposited by vacuum evaporation with denton explorer e - beam ( usa ) at deposition rate of 2 s. finally , the lift - off process was accomplished by removing the neb22 resist by soaking the wafer in remover 1165 . the wafer was structured by a series of pads carrying a nha , each with an area of 1.2 1.2 mm . the silicon wafer with au nha was incubated in an ethanolic 1 mm benzophenone - thiol solution for 24 h in order to form a self - assembled monolayer . afterward , the surface was rinsed with ethanol and dried in a stream of air . thin pnipaam layer was prepared by spin - coating an ethanolic pnipaam solution with a polymer concentration of 3 wt % and a spin rate of 2000 rpm for 2 min . subsequently , the polymer layer was dried in a vacuum oven ( vt6025 , thermo scientific ) overnight at t = 50 c . pnipaam - based polymer was simultaneously cross - linked and attached to au surface by using a uv light at = 365 nm with an irradiation dose of 25 j cm ( uv - lamp bio - link 365 from vilber , germany ) . the cross - linked polymer network was rinsed thoroughly with water to remove loosely bound pnipaam polymer chains , and it was finally dried with a stream of air . a drop of ostemer 321 epoxy was placed onto the surface of a bk7 glass substrate and contacted with a flat block of pdms in order to spread homogeneously . then the ostemer 321 polymer was precured by uv light at = 365 nm ( irradiation dose of 3 j cm ) through the pdms block . afterward , the pdms block was peeled off , yielding the glass substrate with the rubbery ostemer layer on its top . the ostemer surface was pressed against the pnipaam film covering au nha on the top of a silicon wafer . in order to establish a bond between pnipaam and oestemer , the silicon wafer was then stripped off using a sharp blade leaving the au nha bonded to the glass substrate via the pnipaam - based layer . au nha structures were characterized by scanning electron microscope ( sem ) evo from carl zeiss ( germany ) that was operated at a low voltage of 5 kv . atomic force microscope ( afm ) pico plus from molecular imaging ( usa ) was used in tapping mode to observe the morphology of the dry structure . in addition , substrates with the nha in contact with air and water were analyzed by optical microscope ( bx51 m with camera sc30 from olympus soft imaging solution , germany ) . white light emitted from a halogen lamp lsh102 from lot - oriel ( germany ) was coupled to a multimode optical fiber m26l02 from thorlabs ( uk ) and focused by an achromatic lens ( f = 6 cm , 14 kla 001 from cvi melles griot , germany ) at the investigated structure . the focused light beam passed through the substrate under angles between = 0 and 4. the investigated structure was clamped to a transparent flow cell in order to flow aqueous samples over its surface . temperature - dependent measurements were performed by using a peltier device that was integrated to the flow cell . a translation stage was used to select illuminated area that carried either au nha or a flat au layer as reference . transmitted light was coupled via a lens ( f810sma-635 from thorlabs , uk ) into a multimode optical fiber ( m26l02 from thorlabs , uk ) that was connected to a high - resolution spectrometer ( sr-303i - b from andor , uk ) . the spectra measured on the au nha were normalized with that obtained on a reference flat au surface . the finite difference time domain ( fdtd ) model implemented by lumerical inc . ( canada ) was used for simulations of the near field and far field optical properties of the investigated structure . a unit cell with the width equal to the period was used and periodic bloch boundary condition in x- and y - axes was applied ( cartesian coordinates defined in figure 1a ) . in the z direction , a perfectly matched layer was placed at the distances of 1 m above and below the layer stack . a transmission monitor was placed 0.7 m below the au layer . a 2d monitor in the xz - plane as illustrated in figure 1a , the investigated plasmonic structure consists of an au film with rectangular nha that is attached to a pnipaam - based hydrogel layer . when brought in contact with an aqueous environment , water flows thought the pores in the au membrane into the underneath hydrogel which swells . the pnipaam polymer exhibits thermoresponsive properties that cause the polymer network to collapse when the structure is heated above the lcst expelling water through the porous au membrane . the au nha structure with a thickness of dm = 50 1 nm was prepared on a silicon wafer with native oxide by using electron beam lithography . in order to covalently attach the pnipaam - based hydrogel film to the au nha surface , it was first modified by a self - assembled monolayer of a thiol functionalized with benzophenone groups that served as a linker for attachment of pnipaam - based polymer . the modified au surface was subsequently coated by a film of the pnipaam - based terpolymer that carries benzophenone groups as photo - cross - linking units , as well as carboxylic acid substituents for improved swelling and for subsequent chemical modification ( e.g. , with biological ligands ) . after the pnipaam polymer layer was dried , it was exposed to uv in order to cross - link the polymer chains and simultaneously attach them to the au surface by exploiting the photoreactivity of the benzophenone moieties . afterward , the au nha with the pnipaam network layer was bonded to a uv - precured ostemer layer on a glass substrate ( see figure 1b ) . this rubbery film was pressed against the surface of the pnipaam film and attached via the reactive epoxy groups at elevated temperature . finally , the stack of layers was stripped from the si wafer to yield the surface of the au nha film on the top of a cross - linked pnipaam - based hydrogel cushion . ( a ) schematics of the investigated structure that comprise au film with nha that is attached to a glass substrate by a responsive pnipaam - based hydrogel cushion . ( b ) preparation procedure of the structure by using template stripping from a si wafer . as seen in figure 2a , the developed process allowed successful stripping of the au nha film with an underlying hydrogel layer . sem revealed that the arrays of nanoholes retained their initial dimensions before stripping with a hole diameter of d = 208 8 nm and a period of = 406 13 nm . optical microscopy was used to image a corner of rectangular structured pad , as depicted in figure 2b . the dark area in this image represents the nha structure , and the brighter , more reflective region corresponds to the flat au surface . figure 2c shows an optical microscope image of the structured area that was brought in contact with water at a temperature of t = 22 c . it indicates that the au layer wrinkles due to the swelling of the underneath pnipaam hydrogel film . such wrinkling was observed for other hydrogel films , and it is associated with stress - induced buckling , as the polymer is confined in the plane of the substrate , and thus swelling is predominantly permitted only in the direction perpendicular to the surface . afterward , the surface was dried and water rapidly diffused through the nha pores out of the hydrogel film . interestingly , the about 50 nm thick au film did not exhibit cracks and defects after the swelling and drying cycle as seen in figure 2d , acquired by optical microscopy . in addition , afm was used for the characterization of au nha on the top of the hydrogel cushion after it was swollen and subsequently dried . the acquired image in figure 2e shows that most of the pores in the au film were filled with the pnipaam - based polymer network , which partially protrudes above the au surface ( visible by the brighter color of the pores in height mode ) . in addition , one can see that the au surface between the pores was smooth ( root - mean - square roughness of 0.5 nm ) and without cracks . it is worth mentioning that the observed low roughness at the upper interface is comparable to that of si wafer as observed for other template stripped gold surfaces . at a larger scale the au surface became slightly undulated after swelling and collapsing of the pnipaam - based hydrogel , which is probably associated with the buckling and rearrangement of pnipaam polymer chains below the au membrane after swelling and drying . nevertheless , this large - scale undulation did not affect the regularity and integrity of the nanoscale dimensions of the individual pores . observation of the stripped surface of the au nha by using ( a ) sem and ( b ) optical microscopy with 100-fold magnification . ( c ) optical microscopy images of an au nha surface with a structured pad ( dark area ) when swollen in water and ( d ) subsequently dried . ( e ) afm observation of the stripped au surface with nhas after exposure to water followed by drying . optical waveguide spectroscopy was used to characterize thickness and swelling of the pnipaam polymer film ( analysis of measured data presented in the supporting information ) . these measurements were performed for a pnipaam layer that was prepared by using identical protocol on a flat au surface . this thickness of a dry film in contact with air was measured as 212 nm , and its refractive index was ni = 1.48 . when the film is brought in contact with water at t = 22 c , the volume fraction of the polymer chains in the hydrogel network is reduced by its swelling . therefore , the corresponding refractive index of the pnipaam layer decreased to ni = 1.36 ( which is close to that of water [ n0 = 1.33 ] at the used wavelength of = 633 nm ) , and the thickness increased to about 1.2 m . at a temperature of t = 40 c , hydrogel film collapsed , and its refractive index decreased to ni = 1.46 , which is about 0.1 refractive index units ( riu ) higher than that determined in its highly swollen state at t = 22 c . it should be noted that the thickness of the swollen and collapsed pnipaam hydrogel layer is much higher than the penetration depth of spp field on a gold surface at wavelengths in the visible and near - infrared parts of spectrum . as indicated in figure 1a , the thin au film on the top of pnipaam layer supports spp modes at its both interfaces . the mode that travels at the outer interface ( in contact with air or with aqueous solution with refractive index n0 ) is referred to as sppo . the mode supported by the opposite interface ( in contact with pnipaam polymer with a refractive index ni ) is named sppi . the periodic nha on the au film allows for diffractive coupling of light into the sppo and sppi via ( 1 , 0 ) , ( 0 , 1 ) and higher orders . as presented in figure 3 , this coupling manifests itself as distinct resonant features in the transmission spectra . these spectra were measured with a polychromatic light beam that was focused onto the nha , and the transmitted beam was analyzed by a spectrometer . obtained spectra were normalized by those measured on a reference pad with a flat , nonporous 50 nm thick au film . two fano resonances can be observed for the structure in contact with air in figure 3a . they appear as a spectral band where the transmission decreases at lower wavelengths and increases at higher wavelengths . the resonance associated with the excitation of sppo mode at the outer interface ( between au and air ) exhibits a minimum at o < 550 nm ( this resonance is spectrally broad as the wavelengths are close to the plasma frequency of au ) . the second resonance due to the excitation of the sppi mode at the inner interface ( between au and dry pnipaam ) is manifested as minimum reflectivity at around i = 720 nm . as the same figure shows , the sppi resonance blue - shifts after the structure is brought in contact with water at a temperature of t = 22 c . the sppi resonance wavelength i is decreased by about 15 nm , which can be attributed to the swelling of pnipaam cushion by water diffusing through the nanoholes , accompanied by a decrease in its refractive index from ni . the minimum of the sppo resonance at o red - shifts by about 50 nm as the refractive index of the medium above the au nha structure changes from n0 = 1 ( air ) to 1.33 ( water ) . the controlled permeability of the au membrane with nhas was tested by a series of refractometric experiments where the upper and bottom interfaces were optically probed by resonantly excited sppo and sppi modes , respectively . aqueous samples with water spiked by ethylene glycol at concentrations between 0 and 40 vol % were used in order to change the refractive index of the outer medium between n0 = 1.33 and 1.37 . transmission spectra for gradually increasing n0 due to the ethylene glycol were measured for the swollen pnipaam cushion at a temperature of t = 22 c . as presented in figure 3b , both sppo and sppi resonances shift to longer wavelengths when increasing the concentration of ethylene glycol . the sppo resonance shifts by o = 9.7 nm with the increase of ethylene glycol concentration to 40 vol % . this change corresponds to the refractive index change of n0 = 0.04 riu which yields the refractive index sensitivity of sppo mode of s = o/n0 = 242 nm riu . this value is identical to the gold grating - based spr ( 250 nm riu for the first order coupling at a wavelength of 600 nm ) , and it is comparable to values typically observed on au nha ( 400 nm riu at wavelength of 650 nm ) . the sppi resonance responds to ethylene glycol stronger , and it shifts by i = 26.5 nm for the maximum concentration of 40 vol % . this observation indicates that the ethylene glycol can diffuse through the nha into the underneath swollen hydrogel . the fact that i is almost 3 times higher than o is probably due to a partial collapse of pnipaam triggered by ethylene glycol that provides an additional change in ni . in order to prove the identity of transmission features that were assumed to be associated with the excitation of sppi and sppo , the refractive index was individually changed at the inner and outer side of the au nha structure . first , the refractive index change ni was induced by the collapse of pnipaam cushion that is probed by sppi . in order to do so , the temperature of the water flowing over the nha structure was gradually raised from t = 22 to 40 c . figure 3c reveals that the sppo resonance ( that probes the outer water medium ) shifts weakly with temperature . the measured maximum shift of o = 2.5 nm can be attributed to the effect of collapsing hydrogel that partially protrudes through the nanoholes in au film . an order of magnitude stronger shift of i = 24.5 nm was observed for the sppi resonance due to an outward flux of water through the nha pores and a subsequent collapse of the underlying hydrogel . the dependence of i on temperature presented in the supporting information ( figure s2 ) confirms that the collapse of the pnipaam cushion occurs at the lsct of 31 c similar to the plain pnipaam film in direct contact with an aqueous medium without the metallic structure on top . assuming that the refractive index sensitivity of the sppi resonance is similar to that of sppo , the measured spectral shift can be converted to refractive index change due to the pnipaam cushion collapse of ni = i / s 0.1 . interestingly , this value is identical to that measured for swelling of pnipaam film without au nha on the top ( see figure s1 ) . second , the structure was probed by sppo and sppi modes when refractive index at the upper interface n0 was varied . it was utilized by collapsing pnipaam cushion at t = 40 c and repeating the experiment with the flow of aqueous samples spiked with ethylene glycol over the structure . the measured data in figure 3d indicate that the sppo resonance responds similar as at t = 22 c ( see figure 3b ) . a strong resonant wavelength shift of o = 7.5 nm was measured due to the increase in refractive index by ethylene glycol concentration of 40 vol % . this value is slightly lower than that measured for swollen pnipaam cushion which can be ascribed to the collapsed polymer in the pore which partially occupies space probed in sppo mode . this observation confirms that the hydrogel in its collapsed state prevents diffusion of molecules through the au pores to the underneath interface that is probed by sppi . measured transmission spectra for the au nha on top of a responsive pnipaam hydrogel cushion ( au nha - hydrogel ) ( a ) in contact with air and water at t = 22 c . ( b ) the swollen hydrogel cushion in contact with water spiked with ethylene glycol ( eg , n0 = 1.331.37 ) at t = 22 c . ( c ) au nha - hydrogel in water ( n0 = 1.33 ) with temperature varied in the range of t = 2245 c in order to actuate the pnipaam cushion . ( d ) the collapsed cushion in contact with water spiked with ethylene glycol ( n0 = 1.331.37 ) at t = 40 c . the experimental data were verified by simulating transmission spectrum for the investigated au nha - hydrogel geometry . in these simulations the au film was sandwiched between two semi - infinite dielectrics with refractive index n0 and ni . the transmission spectra presented in figure 4a for the dry ( ni = 1.48 and n0 = 1 ) and swollen ( ni = 1.36 and n0 = 1.33 ) geometry qualitatively agree with those measured at t = 22 c ( compare with figure 3a ) . for the dry structure , simulations predict the sppo resonance occurs at o < 550 nm and the one associated with the sppi mode at around o = 690 nm . for the swollen structure , sppo resonance red - shifts to around o = 570 nm and the one associated with the sppi mode blue - shifts to o = 674 nm . it is worth of noting that the refractive index swollen hydrogel is similar to water which leads to the coupling of the surface plasmons across the au layer . for such refractive index symmetrical configuration sppo mode at lower wavelengths behaves like long - range surface plasmon for which the electric field is weakly confined in the au layer . the sppi mode , on the other hand , behaves like a short - range surface plasmon with the corresponding electric field strongly confined in the au film . when the hydrogel film is collapsed ( n0 = 1.33 and ni = 1.46 ) , the symmetry is broken and individual sppi and sppo modes propagate along individual interfaces . figure 4b illustrates for such a geometry the dispersion relation of sppi and sppo modes represented as absorption dependence on the angle of incidence and wavelength . it reveals complex behavior where resonances associated with the first - order coupling to sppo and sppi split and cross when increasing . in addition , flattening of spp bands for low angle occurs . in order to check for the identity of the used resonances , the near field distribution of the electric field intensity was simulated at the wavelengths 590 and 700 nm , as seen in figures 4c and 4d , respectively . these images confirm that the shorter wavelength resonance sppo is probing the outer interface of the au nha - hydrogel film in contact with water , while the inner interface in contact with the polymer layer is probed by the longer wavelength sppi resonance . simulated transmission spectra for an au nha - hydrogel geometry that is ( a ) in contact with air ( n0 = 1 ) and a collapsed hydrogel - like medium ( ni = 1.48 ) as well as with water ( n0 = 1.33 ) and a swollen hydrogel - like medium ( ni = 1.36 ) , with the optical beam normal to the surface = 0. ( b ) dispersion relation of excited sppi and sppo modes represented as absorption dependence on angle of incidence and wavelength ( n0 = 1.33 , ni = 1.45 ) . simulation of the near field distribution of the electric field intensity |e / e0| being normalized with that of the incident plane wave for a collapsed au nha - hydrogel structure ( n0 = 1.33 , ni = 1.45 ) at ( c ) wavelength of = 590 nm where the sppo is excited and at ( d ) = 700 nm where the sppi occurs . finally , sensitivity of sppo and sppi resonances to the refractive index variations above and below the au nha was theoretically studied . the simulated transmission spectra for ni varying between 1.35 and 1.45 and fixed n0 = 1.33 are presented in figure 5a . these data resembles the temperature - dependent experiment with collapsing the gel ( presented in figure 3c ) they show a strong shift in the sppi resonance of i = 24 nm , which is similar to the experimentally measured value . in accordance with experimental results , the simulations predict less pronounced changes in the sppo resonance of o = 11.5 nm for refractive index change in ni . figure 5b describes the complementary case when the refractive index above the au surface n0 is varied . these simulations are related to the experiment in figure 3d where the refractive index n0 of the liquid medium was changed from 1.33 to 1.37 ( by variation of the water / ethylene glycol mixing ratio ) , while the pnipaam hydrogel cushion with ni = 1.46 was collapsed above the lcst ( t = 40 c ) . the obtained simulation results predict a shift of about o = 13 nm of the sppo resonance wavelength . in accordance with the experimental findings , the response of sppi mode of o we believe that deviation between the experiment and simulations can be mostly attributed to the protrusion of hydrogel present in the pores beyond the upper interface . this leads to additional refractive index changes that are not taken into account in the model . ( a ) transmission spectra of an au nha - hydrogel architecture simulated for increasing refractive index ni from 1.35 to 1.45 at the au hydrogel interface , representing the collapse of pnipaam cushion , and for an aqueous medium at the outer interface ( n0 = 1.33 ) . ( b ) transmission spectra for increasing refractive indices n0 from 1.33 to 1.37 of the liquid medium ( representative of the varying water / ethylene glycol mixtures ) above the au nha hydrogel film with a collapsed pnipaam cushion ( ni = 1.45 ) beneath . a facile template - stripping approach to tether a thin au nha to a thermoresponsive hydrogel cushion is reported , and the optical properties of such structure are characterized in detail . a series of experiments show that water flow through the pores in au nha can be controlled by swelling the underneath hydrogel . when the hydrogel collapses upon temperature increase , water is expelled through the pores , and then they become nonpermeable / closed . in the swollen state the low polymer volume fraction in the hydrogel generates a symmetric refractive index configuration for both interfaces ( au - liquid medium and au - hydrogel ) , which establishes long - range and short - range spps . when the gel is collapsed , the refractive index symmetry is perturbed and distinct spps exist at the individual interfaces . reversible swelling and collapsing of the hydrogel is accompanied by a strong refractive index change of 0.1 , which can be used for tuning the wavelength at which plasmonically enhanced transmission occurs . because of the rapid response to the temperature variations around the lcst , this approach may find its applications in actively tunable optical filters . the reported structure can also be implemented in plasmonic biosensing , where analyzed samples are flowed through the plasmonic nanoholes in order to overcome diffusion - limited mass transfer of target analyte to the surface . in current sensor setups such an architecture is typically realized by using thin nitride membranes which require multiple lithography steps . the presented approach may provide simpler means for device preparation , and it holds potential to harness this intriguing biosensor concept in more practical devices . in conjunction with field intensity enhancement occurring at the nanohole plasmonic hotspot , the more efficient capture of target analyte at the surface offers attractive means to advance sensitivity in direct refractometric detection of biomolecules as well as in sensors relying on plasmonically amplified spectroscopy .

new plasmonic structure with actively tunable optical characteristics based on thermoresponsive hydrogel is reported . it consists of a thin , template - stripped au film with arrays of nanoholes that is tethered to a transparent support by a cross - linked poly(n - isopropylacrylamide ) ( pnipaam)-based polymer network . upon a contact of the porous au surface with an aqueous environment , a rapid flow of water through the pores enables swelling and collapsing of the underlying pnipaam network . the swelling and collapsing could be triggered by small temperature changes around the lower critical solution temperature ( lcst ) of the hydrogel . the process is reversible , and it is associated with strong refractive index changes of n 0.1 , which characteristically alters the spectrum of surface plasmon modes supported by the porous au film . this approach can offer new attractive means for optical biosensors with flow - through architecture and actively tunable plasmonic transmission optical filters .

Introduction Methods Results and Discussion Conclusions

this phenomenon is attributed to the diffractive excitation of surface plasmon polaritons ( spps ) , and it enables efficient tunneling of light through ( otherwise opaque ) metallic film in narrow wavelength regimes . the transmission characteristics of metallic nhas can be controlled by shape , depth , and periodicity of the nanoholes as well as by the type of metal and refractive index of the surrounding medium . plasmonic nhas have been prepared by a range of modern fabrication techniques such as focused ion beam milling , laser interference lithography , electron beam lithography , and template stripping . in parallel , organic materials have been explored for actuating of surface plasmon resonance by refractive index variations associated with the photochromic effects and liquid crystal phase transitions as well as by mechanical changes induced by specific molecular binding and by responsive polymers . among the systems that utilize responsive polymers , poly(n - isopropylacrylamide ) ( pnipaam ) based polymers ( in particular copolymers with carboxylic acid groups ) hold a prominent position due to their strong volumetric change upon swelling and collapsing by variation of the temperature around its lower critical solution temperature ( lcst ) of 32 c . the swelling and collapsing of pnipaam - based architectures is driven by water diffusion , and the response in the millisecond range was measured for about micrometer thick cross - linked pnipaam - based hydrogel layer by long - range surface plasmons on the top of a indium tin oxide microheater . this work presents a new plasmonic structure that is tunable by thermoresponsive pnipaam - based hydrogel and that can be prepared by template stripping . it consists of a thin au film with nha that allows for diffractive coupling to propagating spps . this structure is tethered to a solid surface by a cross - linked pnipaam - based hydrogel cushion which responds to temperature variations . it can rapidly swell and collapse , which triggers a flow of water through the plasmonic pores , and it strongly modulates the refractive index by altereing the polymer fraction in the hydrogel film , leading to changes in the transmission spectrum mediated by surface plasmon modes . pnipaam - based terpolymer ( composed of n - isoproprylacrylamide , methacrylic acid , and 4-methacryloyloxybenzophenone in a ratio of 94:5:1 ) was synthesized as reported before . the wafer was structured by a series of pads carrying a nha , each with an area of 1.2 1.2 mm . pnipaam - based polymer was simultaneously cross - linked and attached to au surface by using a uv light at = 365 nm with an irradiation dose of 25 j cm ( uv - lamp bio - link 365 from vilber , germany ) . the cross - linked polymer network was rinsed thoroughly with water to remove loosely bound pnipaam polymer chains , and it was finally dried with a stream of air . the ostemer surface was pressed against the pnipaam film covering au nha on the top of a silicon wafer . atomic force microscope ( afm ) pico plus from molecular imaging ( usa ) was used in tapping mode to observe the morphology of the dry structure . the focused light beam passed through the substrate under angles between = 0 and 4. the investigated structure was clamped to a transparent flow cell in order to flow aqueous samples over its surface . a 2d monitor in the xz - plane as illustrated in figure 1a , the investigated plasmonic structure consists of an au film with rectangular nha that is attached to a pnipaam - based hydrogel layer . when brought in contact with an aqueous environment , water flows thought the pores in the au membrane into the underneath hydrogel which swells . the pnipaam polymer exhibits thermoresponsive properties that cause the polymer network to collapse when the structure is heated above the lcst expelling water through the porous au membrane . in order to covalently attach the pnipaam - based hydrogel film to the au nha surface , it was first modified by a self - assembled monolayer of a thiol functionalized with benzophenone groups that served as a linker for attachment of pnipaam - based polymer . the modified au surface was subsequently coated by a film of the pnipaam - based terpolymer that carries benzophenone groups as photo - cross - linking units , as well as carboxylic acid substituents for improved swelling and for subsequent chemical modification ( e.g. after the pnipaam polymer layer was dried , it was exposed to uv in order to cross - link the polymer chains and simultaneously attach them to the au surface by exploiting the photoreactivity of the benzophenone moieties . afterward , the au nha with the pnipaam network layer was bonded to a uv - precured ostemer layer on a glass substrate ( see figure 1b ) . finally , the stack of layers was stripped from the si wafer to yield the surface of the au nha film on the top of a cross - linked pnipaam - based hydrogel cushion . ( a ) schematics of the investigated structure that comprise au film with nha that is attached to a glass substrate by a responsive pnipaam - based hydrogel cushion . as seen in figure 2a , the developed process allowed successful stripping of the au nha film with an underlying hydrogel layer . sem revealed that the arrays of nanoholes retained their initial dimensions before stripping with a hole diameter of d = 208 8 nm and a period of = 406 13 nm . the dark area in this image represents the nha structure , and the brighter , more reflective region corresponds to the flat au surface . it indicates that the au layer wrinkles due to the swelling of the underneath pnipaam hydrogel film . such wrinkling was observed for other hydrogel films , and it is associated with stress - induced buckling , as the polymer is confined in the plane of the substrate , and thus swelling is predominantly permitted only in the direction perpendicular to the surface . afterward , the surface was dried and water rapidly diffused through the nha pores out of the hydrogel film . interestingly , the about 50 nm thick au film did not exhibit cracks and defects after the swelling and drying cycle as seen in figure 2d , acquired by optical microscopy . in addition , afm was used for the characterization of au nha on the top of the hydrogel cushion after it was swollen and subsequently dried . the acquired image in figure 2e shows that most of the pores in the au film were filled with the pnipaam - based polymer network , which partially protrudes above the au surface ( visible by the brighter color of the pores in height mode ) . in addition , one can see that the au surface between the pores was smooth ( root - mean - square roughness of 0.5 nm ) and without cracks . at a larger scale the au surface became slightly undulated after swelling and collapsing of the pnipaam - based hydrogel , which is probably associated with the buckling and rearrangement of pnipaam polymer chains below the au membrane after swelling and drying . ( e ) afm observation of the stripped au surface with nhas after exposure to water followed by drying . optical waveguide spectroscopy was used to characterize thickness and swelling of the pnipaam polymer film ( analysis of measured data presented in the supporting information ) . these measurements were performed for a pnipaam layer that was prepared by using identical protocol on a flat au surface . this thickness of a dry film in contact with air was measured as 212 nm , and its refractive index was ni = 1.48 . when the film is brought in contact with water at t = 22 c , the volume fraction of the polymer chains in the hydrogel network is reduced by its swelling . therefore , the corresponding refractive index of the pnipaam layer decreased to ni = 1.36 ( which is close to that of water [ n0 = 1.33 ] at the used wavelength of = 633 nm ) , and the thickness increased to about 1.2 m . at a temperature of t = 40 c , hydrogel film collapsed , and its refractive index decreased to ni = 1.46 , which is about 0.1 refractive index units ( riu ) higher than that determined in its highly swollen state at t = 22 c . the mode supported by the opposite interface ( in contact with pnipaam polymer with a refractive index ni ) is named sppi . the periodic nha on the au film allows for diffractive coupling of light into the sppo and sppi via ( 1 , 0 ) , ( 0 , 1 ) and higher orders . these spectra were measured with a polychromatic light beam that was focused onto the nha , and the transmitted beam was analyzed by a spectrometer . the second resonance due to the excitation of the sppi mode at the inner interface ( between au and dry pnipaam ) is manifested as minimum reflectivity at around i = 720 nm . the sppi resonance wavelength i is decreased by about 15 nm , which can be attributed to the swelling of pnipaam cushion by water diffusing through the nanoholes , accompanied by a decrease in its refractive index from ni . the minimum of the sppo resonance at o red - shifts by about 50 nm as the refractive index of the medium above the au nha structure changes from n0 = 1 ( air ) to 1.33 ( water ) . the controlled permeability of the au membrane with nhas was tested by a series of refractometric experiments where the upper and bottom interfaces were optically probed by resonantly excited sppo and sppi modes , respectively . aqueous samples with water spiked by ethylene glycol at concentrations between 0 and 40 vol % were used in order to change the refractive index of the outer medium between n0 = 1.33 and 1.37 . this value is identical to the gold grating - based spr ( 250 nm riu for the first order coupling at a wavelength of 600 nm ) , and it is comparable to values typically observed on au nha ( 400 nm riu at wavelength of 650 nm ) . the sppi resonance responds to ethylene glycol stronger , and it shifts by i = 26.5 nm for the maximum concentration of 40 vol % . this observation indicates that the ethylene glycol can diffuse through the nha into the underneath swollen hydrogel . the fact that i is almost 3 times higher than o is probably due to a partial collapse of pnipaam triggered by ethylene glycol that provides an additional change in ni . in order to prove the identity of transmission features that were assumed to be associated with the excitation of sppi and sppo , the refractive index was individually changed at the inner and outer side of the au nha structure . first , the refractive index change ni was induced by the collapse of pnipaam cushion that is probed by sppi . the measured maximum shift of o = 2.5 nm can be attributed to the effect of collapsing hydrogel that partially protrudes through the nanoholes in au film . an order of magnitude stronger shift of i = 24.5 nm was observed for the sppi resonance due to an outward flux of water through the nha pores and a subsequent collapse of the underlying hydrogel . the dependence of i on temperature presented in the supporting information ( figure s2 ) confirms that the collapse of the pnipaam cushion occurs at the lsct of 31 c similar to the plain pnipaam film in direct contact with an aqueous medium without the metallic structure on top . assuming that the refractive index sensitivity of the sppi resonance is similar to that of sppo , the measured spectral shift can be converted to refractive index change due to the pnipaam cushion collapse of ni = i / s 0.1 . this observation confirms that the hydrogel in its collapsed state prevents diffusion of molecules through the au pores to the underneath interface that is probed by sppi . measured transmission spectra for the au nha on top of a responsive pnipaam hydrogel cushion ( au nha - hydrogel ) ( a ) in contact with air and water at t = 22 c . in these simulations the au film was sandwiched between two semi - infinite dielectrics with refractive index n0 and ni . for the dry structure , simulations predict the sppo resonance occurs at o < 550 nm and the one associated with the sppi mode at around o = 690 nm . for the swollen structure , sppo resonance red - shifts to around o = 570 nm and the one associated with the sppi mode blue - shifts to o = 674 nm . it is worth of noting that the refractive index swollen hydrogel is similar to water which leads to the coupling of the surface plasmons across the au layer . for such refractive index symmetrical configuration sppo mode at lower wavelengths behaves like long - range surface plasmon for which the electric field is weakly confined in the au layer . the sppi mode , on the other hand , behaves like a short - range surface plasmon with the corresponding electric field strongly confined in the au film . in order to check for the identity of the used resonances , the near field distribution of the electric field intensity was simulated at the wavelengths 590 and 700 nm , as seen in figures 4c and 4d , respectively . these images confirm that the shorter wavelength resonance sppo is probing the outer interface of the au nha - hydrogel film in contact with water , while the inner interface in contact with the polymer layer is probed by the longer wavelength sppi resonance . finally , sensitivity of sppo and sppi resonances to the refractive index variations above and below the au nha was theoretically studied . in accordance with experimental results , the simulations predict less pronounced changes in the sppo resonance of o = 11.5 nm for refractive index change in ni . figure 5b describes the complementary case when the refractive index above the au surface n0 is varied . these simulations are related to the experiment in figure 3d where the refractive index n0 of the liquid medium was changed from 1.33 to 1.37 ( by variation of the water / ethylene glycol mixing ratio ) , while the pnipaam hydrogel cushion with ni = 1.46 was collapsed above the lcst ( t = 40 c ) . in accordance with the experimental findings , the response of sppi mode of o we believe that deviation between the experiment and simulations can be mostly attributed to the protrusion of hydrogel present in the pores beyond the upper interface . this leads to additional refractive index changes that are not taken into account in the model . ( a ) transmission spectra of an au nha - hydrogel architecture simulated for increasing refractive index ni from 1.35 to 1.45 at the au hydrogel interface , representing the collapse of pnipaam cushion , and for an aqueous medium at the outer interface ( n0 = 1.33 ) . ( b ) transmission spectra for increasing refractive indices n0 from 1.33 to 1.37 of the liquid medium ( representative of the varying water / ethylene glycol mixtures ) above the au nha hydrogel film with a collapsed pnipaam cushion ( ni = 1.45 ) beneath . a facile template - stripping approach to tether a thin au nha to a thermoresponsive hydrogel cushion is reported , and the optical properties of such structure are characterized in detail . a series of experiments show that water flow through the pores in au nha can be controlled by swelling the underneath hydrogel . when the hydrogel collapses upon temperature increase , water is expelled through the pores , and then they become nonpermeable / closed . in the swollen state the low polymer volume fraction in the hydrogel generates a symmetric refractive index configuration for both interfaces ( au - liquid medium and au - hydrogel ) , which establishes long - range and short - range spps . reversible swelling and collapsing of the hydrogel is accompanied by a strong refractive index change of 0.1 , which can be used for tuning the wavelength at which plasmonically enhanced transmission occurs . because of the rapid response to the temperature variations around the lcst , this approach may find its applications in actively tunable optical filters . the reported structure can also be implemented in plasmonic biosensing , where analyzed samples are flowed through the plasmonic nanoholes in order to overcome diffusion - limited mass transfer of target analyte to the surface . the presented approach may provide simpler means for device preparation , and it holds potential to harness this intriguing biosensor concept in more practical devices . in conjunction with field intensity enhancement occurring at the nanohole plasmonic hotspot , the more efficient capture of target analyte at the surface offers attractive means to advance sensitivity in direct refractometric detection of biomolecules as well as in sensors relying on plasmonically amplified spectroscopy .

classically , motor responses were seen as the end - result of a one - way route from perception cognition action ; thus , they were thought to reveal little about cognitive processing ( see rosenbaum , 2005 , for discussion ) . however , substantial behavioral evidence now indicates that cognition does not discretely collapse its processing onto movement execution . instead , movement is continually updated by cognitive processing over time ( goodale et al . , 1986 ) . studies in humans , for example , suggest that the process of categorizing a visual stimulus immediately shares its ongoing results with the motor cortex to continuously guide a hand - movement response over time ( e.g. , freeman et al . , 2011 ) . further , human reaching movements suggest that multiple motor plans are prepared in parallel , and that these cascade over time into visually guided action ( song and nakayama , 2006 , 2008 ) . monkey studies show that the hand 's position and velocity are tightly yoked to ongoing changes in the firing of population codes within motor cortex ( paninski et al . , 2004 ) , and when a monkey must generate a hand movement based on a perceptual decision , these motor - cortical population codes are yoked to the evolution of the decision process ( cisek and kalaska , 2005 ) . these findings suggest that manual dynamics are intimately coextensive with mental dynamics , and therefore , hand movements that are sampled fast enough could potentially provide real - time read - outs of internal cognitive processing . the recent tracking of hand movement en route to choices on a screen has opened up new avenues of investigation into a wide range of psychological phenomena . hand trajectories recorded by a computer mouse , or sometimes a wireless remote or position sensor , have proven able to sensitively track participants tentative commitments to various choice alternatives over hundreds of milliseconds . as such , the emergence of a behavioral response can be sampled anywhere from 40 to 120 times per second . this permits insights into fine - grained temporal dynamics , providing unusually high - fidelity , real - time motor traces of the mind . its sensitivity to these dynamics can reveal hidden cognitive states that are otherwise not availed by traditional measures ( song and nakayama , 2006 , 2008 , 2009 ) . more broadly , these real - time dynamics may be used to assess how multiple processes temporally interact and how multiple information sources weigh in on processing across hundreds of milliseconds , thereby resolving critical empirical questions and theoretical impasses across psychology ( e.g. , see spivey , 2007 ) . in language research , debate continues as to whether the language system is a discrete - symbolic architecture or whether it integrates multiple sources of information in parallel to continually constrain its operation . considerable evidence for the parallel , integrative approach came from eye movements , but still some researchers argued that the discontinuity of eye - movement patterns could be accommodated by discrete - symbolic accounts . the link between manual and mental dynamics has recently served as compelling evidence that language processing is indeed , at multiple levels , gradient and parallel . ( 2005 ) asked participants to move the computer mouse from the bottom - center of the screen to the top - left or top - right corners . for instance , a picture of a candle appeared in the top - left and a picture of a candy appeared in the top - right while a spoken phrase was heard ( e.g. , click the candle ) . when the distractor object corresponded to a word whose initial set of phonemes overlapped with the spoken word ( e.g. , a picture of candy , for the spoken word , candle ) , hand movements showed a continuous attraction toward the distractor before settling into the correct alternative . thus , the ongoing accrual of the spoken word partially activated multiple lexical representations ( e.g. , both candle and candy ) , causing movements to partially gravitate toward candy before settling into candle . this showed that the language system continually integrates the acoustic - phonetic input of a spoken word , triggering parallel competing representations that resolve over time . in some cases , however , comprehending spoken language is more difficult and would benefit from help of the visual system . consider the following syntactically ambiguous sentence : typically , listeners resolve such ambiguities by incorporating available visual cues ( e.g. , is there an apple on a towel or an apple by itself ? ) . in a series of studies , participants were given several objects on a screen while they heard a sentence instructing them to move one object to another . for example , hearing the above sentence , participants would use a computer mouse to click on an image displaying an apple on a towel , and to then drag it onto an image of a box . before reaching the box , however , participants hand movements exhibited a continuous attraction toward the object associated with the incorrect syntactic parse ( in this case , an empty towel ) . consistent with analogous eye - tracking studies ( e.g. , tanenhaus et al . , 1995 ) , the attraction effect was not seen when a second relevant object was present ( e.g. , an apple on a napkin ) . this provided strong support for constraint - based accounts of language processing by demonstrating partially active syntactic alternatives competing over time with a weighing - in of visual , contextual , and linguistic factors ( farmer et al . importantly , hand movements were able to reveal competition on a trial - by - trial basis , thereby evading longstanding concerns with analogous eye - movement results that were limited by discrete oculomotor output . for instance , dale and duran ( 2011 ) had participants verify simple statements by clicking on a true or false response . statements involving a negation ( e.g. , cats are not dogs ) led hand movements to exhibit sharp shifts in direction , initially pursuing one response and then rapidly redirecting toward the other , especially for negated true statements . thus , during sentence processing , participants experienced an abrupt shift in their evolving decision . classic work suggests that negation is an operator that rapidly reverses the truth / falsity of an interpretation and predicts shifts in cognitive dynamics , consistent with other work showing that hand movement may index such rapid transitions ( e.g. , resulaj et al . , 2009 ) . for example , they have provided new insights into social categorization . in one series of studies , participants were presented with sex - typical ( e.g. , masculine male ) and sex - atypical ( e.g. , feminine male ) faces and asked to categorize the target 's sex by clicking on a male or hand trajectories were continuously attracted to the opposite sex - category before settling into the correct response . this was evidence that social categorization involves dynamic competition , where partially active category representations ( male and female ) continuously compete over time to stabilize onto one categorical outcome ( freeman et al . , 2008 ; also see dale et al . these findings led to a new model of social categorization , proposing that all available facial , vocal , and bodily cues ( among other constraints ) simultaneously weigh in on multiple partially active category representations , and that these dynamically evolve over time into stable categorical perceptions ( freeman and ambady , 2011b ) . hand movements again showed continuous - attraction effects for atypical targets , but also revealed important dissociations in how the processing of white - specifying and black - specifying cues evolves across the categorization time - course . moreover , as the amount of racial ambiguity increased , hand trajectories showed corresponding increases in attraction toward the unselected race - category and increases in motor complexity . this showed that the competition inherent to social categorization sensitively increases and decreases with the amount of ambiguity challenging the perceptual system ( freeman et al . , 2010 ) . further , hand movements have revealed not only that multiple facial cues are dynamically integrated , but also that cues across sensory modalities ( facial and vocal cues ) are dynamically integrated as well ( freeman and ambady , 2011a ) . hand movements have also been used to temporally dissociate the integration of specific cues , such as facial shape and pigmentation ( freeman and ambady , in press ) . freeman and ambady ( 2011a ) found that when categorizing a face 's sex , the simultaneous processing of a sex - atypical voice led participants mouse trajectories to be continuously attracted to the opposite sex - category response before settling into the response consistent with the face 's correct sex . mean mouse trajectories from one study are depicted ( aggregated across male and female targets ) . in this figure , trajectories for all targets were remapped rightward , with the opposite sex - category on the left and the sex - category consistent with the face 's sex on the right . a sample male face stimulus is displayed ( all male and female face stimuli were somewhat sex - ambiguous ) . a voice stimulus typical for the face 's sex ( masculine ) is shown on the right ( audio waveform depicted in blue ) , next to the mean trajectory for sex - typical trials . its atypical ( feminine ) counterpart is shown on the left , next to the mean trajectory for sex - atypical trials ( audio waveform depicted in purple ) . during an actual trial , a single face was centered at the bottom of the screen while the voice stimulus played . the bar graph shows trajectories maximum deviation toward the opposite sex - category from a direct line between trajectories start and end points ( error bars denote se of the mean ) . the continuous - attraction effect shows that ongoing voice - processing results were dynamically integrated with ongoing face - processing results over time . for instance , freeman and ambady ( 2009 ) found that , before participants clicked on a stereotypically masculine attribute ( e.g. , aggressive ) for male targets or stereotypically feminine attribute ( e.g. , caring ) for female targets , their hands partially swerved toward the attribute stereotypically associated with the opposite sex when facial cues partly overlapped with that sex ( e.g. , masculine women and feminine men ) . thus , not only do facial cues associated with an alternate category trigger a partially active representation of that category , but this partial activation cascades into the triggering of associated stereotypes . hand movements can also reveal other types of partial parallel activations otherwise not seen by traditional measures . for example , although white participants almost always explicitly reported liking black people , an inspection of the hand trajectories leading up to those like judgments revealed a parallel consideration to indicate that they dislike them ( wojnowicz et al . , such a parallel activation of multiple attitudes that continually coalesce into explicit judgments had important theoretical implications for models of social evaluation . ( 2008 ) had participants judge the truth of questions by moving their mouse to yes or thus , hand movements revealed parallel competition across the process of high - level decision - making and showed how this competition is affected by certainty in graded fashion . hand movements also revealed a strong confirmation bias throughout the response , because answers of low probability had overall slower trajectories . duran et al . ( 2010 ) used the same basic design , and revealed that this truth bias after participants were presented with the final word , they were cued to respond either falsely or truthfully . trajectories during false responses had greater sample entropy , reached peak velocity later in the movement , and had a steeper curve toward the competing option while en route to the false response . as such , hand trajectories revealed the ongoing process of overcoming the truth during deceptive responding . 2010 ) had participants answer autobiographical questions either truthfully or falsely by clicking a yes or no response on the screen . trajectories velocity profiles are plotted as a function of time for truthful responses clicking no ( dashed line with circles ) , truthful responses clicking yes ( dashed line with triangles ) , false responses clicking no ( solid line with circles ) , and false responses clicking yes ( solid line with triangles ) . this , among other differences between false and truthful responses in movement complexity and curvature toward the opposite response , suggests the presence of a truth bias that pulls processing off course during the production of a deceptive response . ( 2008 ) studied how growing knowledge , across many trials , covaries with manual dynamics during a learning task . they had participants learn random pairs of shapes in a paired - associate memory design . participants were shown a shape at the bottom of the screen , and instructed to pick one of two in the upper - most corners that was associated with that bottom shape . the researchers found that hand movements ( measured with a wireless nintendo wii remote ) covaried in a gradient way with emerging knowledge of the pairs . moreover , growing knowledge correlated with stronger force responding on the remote , and when new shapes appeared ( unbeknownst to participants ) this bodily confidence was modulated in response . thus , hand movements revealed the longer timescale dynamics of the knowledge itself , as the participants exhibited learning across the task . interestingly , at even longer timescales ( e.g. , hours ) , gesturing patterns of hand movement have long been used to index learning and transitions of knowledge , especially in children ( goldin - meadow and alibali , 2002 ) . in language research , debate continues as to whether the language system is a discrete - symbolic architecture or whether it integrates multiple sources of information in parallel to continually constrain its operation . considerable evidence for the parallel , integrative approach came from eye movements , but still some researchers argued that the discontinuity of eye - movement patterns could be accommodated by discrete - symbolic accounts . the link between manual and mental dynamics has recently served as compelling evidence that language processing is indeed , at multiple levels , gradient and parallel . ( 2005 ) asked participants to move the computer mouse from the bottom - center of the screen to the top - left or top - right corners . for instance , a picture of a candle appeared in the top - left and a picture of a candy appeared in the top - right while a spoken phrase was heard ( e.g. , click the candle ) . when the distractor object corresponded to a word whose initial set of phonemes overlapped with the spoken word ( e.g. , a picture of candy , for the spoken word , candle ) , hand movements showed a continuous attraction toward the distractor before settling into the correct alternative . thus , the ongoing accrual of the spoken word partially activated multiple lexical representations ( e.g. , both candle and candy ) , causing movements to partially gravitate toward candy before settling into candle . this showed that the language system continually integrates the acoustic - phonetic input of a spoken word , triggering parallel competing representations that resolve over time . in some cases , however , comprehending spoken language is more difficult and would benefit from help of the visual system . consider the following syntactically ambiguous sentence : typically , listeners resolve such ambiguities by incorporating available visual cues ( e.g. , is there an apple on a towel or an apple by itself ? ) . in a series of studies , participants were given several objects on a screen while they heard a sentence instructing them to move one object to another . for example , hearing the above sentence , participants would use a computer mouse to click on an image displaying an apple on a towel , and to then drag it onto an image of a box . before reaching the box , however , participants hand movements exhibited a continuous attraction toward the object associated with the incorrect syntactic parse ( in this case , an empty towel ) . consistent with analogous eye - tracking studies ( e.g. , tanenhaus et al . , 1995 ) , the attraction effect was not seen when a second relevant object was present ( e.g. , an apple on a napkin ) . this provided strong support for constraint - based accounts of language processing by demonstrating partially active syntactic alternatives competing over time with a weighing - in of visual , contextual , and linguistic factors ( farmer et al . importantly , hand movements were able to reveal competition on a trial - by - trial basis , thereby evading longstanding concerns with analogous eye - movement results that were limited by discrete oculomotor output . for instance , dale and duran ( 2011 ) had participants verify simple statements by clicking on a true or false response . statements involving a negation ( e.g. , cats are not dogs ) led hand movements to exhibit sharp shifts in direction , initially pursuing one response and then rapidly redirecting toward the other , especially for negated true statements . thus , during sentence processing , participants experienced an abrupt shift in their evolving decision . classic work suggests that negation is an operator that rapidly reverses the truth / falsity of an interpretation and predicts shifts in cognitive dynamics , consistent with other work showing that hand movement may index such rapid transitions ( e.g. , resulaj et al . , 2009 ) . hand movements have also been critical in uncovering the temporal dynamics of social cognition . for example , they have provided new insights into social categorization . in one series of studies , participants were presented with sex - typical ( e.g. , masculine male ) and sex - atypical ( e.g. , feminine male ) faces and asked to categorize the target 's sex by clicking on a male or hand trajectories were continuously attracted to the opposite sex - category before settling into the correct response . this was evidence that social categorization involves dynamic competition , where partially active category representations ( male and female ) continuously compete over time to stabilize onto one categorical outcome ( freeman et al . , 2008 ; also see dale et al . , 2007 ) . these findings led to a new model of social categorization , proposing that all available facial , vocal , and bodily cues ( among other constraints ) simultaneously weigh in on multiple partially active category representations , and that these dynamically evolve over time into stable categorical perceptions ( freeman and ambady , 2011b ) . hand movements again showed continuous - attraction effects for atypical targets , but also revealed important dissociations in how the processing of white - specifying and black - specifying cues evolves across the categorization time - course . moreover , as the amount of racial ambiguity increased , hand trajectories showed corresponding increases in attraction toward the unselected race - category and increases in motor complexity . this showed that the competition inherent to social categorization sensitively increases and decreases with the amount of ambiguity challenging the perceptual system ( freeman et al . , 2010 ) . further , hand movements have revealed not only that multiple facial cues are dynamically integrated , but also that cues across sensory modalities ( facial and vocal cues ) are dynamically integrated as well ( freeman and ambady , 2011a ) . hand movements have also been used to temporally dissociate the integration of specific cues , such as facial shape and pigmentation ( freeman and ambady , in press ) . freeman and ambady ( 2011a ) found that when categorizing a face 's sex , the simultaneous processing of a sex - atypical voice led participants mouse trajectories to be continuously attracted to the opposite sex - category response before settling into the response consistent with the face 's correct sex . mean mouse trajectories from one study are depicted ( aggregated across male and female targets ) . in this figure , trajectories for all targets were remapped rightward , with the opposite sex - category on the left and the sex - category consistent with the face 's sex on the right . a sample male face stimulus is displayed ( all male and female face stimuli were somewhat sex - ambiguous ) . a voice stimulus typical for the face 's sex ( masculine ) is shown on the right ( audio waveform depicted in blue ) , next to the mean trajectory for sex - typical trials . its atypical ( feminine ) counterpart is shown on the left , next to the mean trajectory for sex - atypical trials ( audio waveform depicted in purple ) . during an actual trial , a single face was centered at the bottom of the screen while the voice stimulus played . the bar graph shows trajectories maximum deviation toward the opposite sex - category from a direct line between trajectories start and end points ( error bars denote se of the mean ) . the continuous - attraction effect shows that ongoing voice - processing results were dynamically integrated with ongoing face - processing results over time . for instance , freeman and ambady ( 2009 ) found that , before participants clicked on a stereotypically masculine attribute ( e.g. , aggressive ) for male targets or stereotypically feminine attribute ( e.g. , caring ) for female targets , their hands partially swerved toward the attribute stereotypically associated with the opposite sex when facial cues partly overlapped with that sex ( e.g. , masculine women and feminine men ) . thus , not only do facial cues associated with an alternate category trigger a partially active representation of that category , but this partial activation cascades into the triggering of associated stereotypes . hand movements can also reveal other types of partial parallel activations otherwise not seen by traditional measures . for example , although white participants almost always explicitly reported liking black people , an inspection of the hand trajectories leading up to those like judgments revealed a parallel consideration to indicate that they dislike them ( wojnowicz et al . , 2009 ) . such a parallel activation of multiple attitudes that continually coalesce into explicit judgments had important theoretical implications for models of social evaluation . ( 2008 ) had participants judge the truth of questions by moving their mouse to yes or thus , hand movements revealed parallel competition across the process of high - level decision - making and showed how this competition is affected by certainty in graded fashion . hand movements also revealed a strong confirmation bias throughout the response , because answers of low probability had overall slower trajectories . duran et al . ( 2010 ) used the same basic design , and revealed that this truth bias persists when people answer autobiographical questions falsely . after participants were presented with the final word , they were cued to respond either falsely or truthfully . trajectories during false responses had greater sample entropy , reached peak velocity later in the movement , and had a steeper curve toward the competing option while en route to the false response . as such , hand trajectories revealed the ongoing process of overcoming the truth during deceptive responding . 2010 ) had participants answer autobiographical questions either truthfully or falsely by clicking a yes or no response on the screen . trajectories velocity profiles are plotted as a function of time for truthful responses clicking no ( dashed line with circles ) , truthful responses clicking yes ( dashed line with triangles ) , false responses clicking no ( solid line with circles ) , and false responses clicking yes ( solid line with triangles ) . this , among other differences between false and truthful responses in movement complexity and curvature toward the opposite response , suggests the presence of a truth bias that pulls processing off course during the production of a deceptive response . ( 2008 ) studied how growing knowledge , across many trials , covaries with manual dynamics during a learning task . they had participants learn random pairs of shapes in a paired - associate memory design . participants were shown a shape at the bottom of the screen , and instructed to pick one of two in the upper - most corners that was associated with that bottom shape . the researchers found that hand movements ( measured with a wireless nintendo wii remote ) covaried in a gradient way with emerging knowledge of the pairs . moreover , growing knowledge correlated with stronger force responding on the remote , and when new shapes appeared ( unbeknownst to participants ) this bodily confidence was modulated in response . thus , hand movements revealed the longer timescale dynamics of the knowledge itself , as the participants exhibited learning across the task . interestingly , at even longer timescales ( e.g. , hours ) , gesturing patterns of hand movement have long been used to index learning and transitions of knowledge , especially in children ( goldin - meadow and alibali , 2002 ) . the work discussed above suggests that the intimate link between manual and mental dynamics may be used to provide critical insights into psychological phenomena . it also emphasizes an integrative approach to these phenomena by demonstrating a deep coextension of processes typically investigated independently , such as action , perception , and both basic and social cognition . insights from simple manual actions may therefore serve as an unforeseen bridge between far - reaching areas of psychological science . this action - dynamics approach to the mind holds particular promise given the practicality of measuring hand movements . there now exists freely available , user - friendly software to run and analyze data from computer mouse - tracking experiments ( freeman and ambady , 2010 ) . thus , rather than measure an ultimate response time , researchers can easily open up such responses into continuous streams of rich cognitive output through nothing more than a computer and mouse . mouse - tracking also permits the recording of online mental processing in young children at an age where online data are often difficult to obtain ( anderson et al . , in press ) . with the capturing of hand movements now just as available and affordable as recording the classic response time , we are hopeful that a growing number of studies will employ the methodology . in this action - dynamics approach , researchers are afforded many ways to measure and analyze hand trajectories . the computer mouse is by far the most practical , but other options include a wireless remote ( e.g. , dale et al . , 2008 ) or a position sensor ( e.g. , song and nakayama , 2008 ) . although the techniques are quite similar , one notable difference is that mouse - tracking captures only 2d movement , whereas the others could potentially capture 3d movement . the techniques also provide different motoric experiences ; for example , tasks using a wireless remote can measure large , free - ranging arm movements . another consideration is that , in mouse - tracking , hand movements are stabilized by a surface . moreover , mouse - tracking can be carried out through online data collection systems like amazon mechanical turk ( dale and duran , 2011 ) . once trajectories are recorded , several measures may be computed , including maximum deviation toward an unselected response ( e.g. , distractor ) , curvature area , switches in direction , or movement complexity . the time - course of the hand 's position , velocity , acceleration , or proximity to a selected or unselected response , can also be examined . each analysis carries its own advantages with respect to the theoretical and practical interests at hand . freeman and ambady ( 2010 ) provide a more complete discussion of measures and analytic techniques for trajectory data . they also describe a user - friendly software package that permits the recording , measurement , and analysis of mouse trajectories from highly customizable experimental tasks . despite the headway made by the work reviewed here , however , the moving hand still has much to reveal about the mind . for example , in a recent critique of the method and its interpretation , van der wel et al . ( 2009 ) discussed whether a model of discrete processing could produce , through ( continuous ) linear combinations of motor plans , the evidence for continuous processing often obtained from hand - tracking experiments . the authors argued that the inference of processing continuity from hand - tracking data is not yet fully warranted . in response , spivey et al . ( 2010 ) argued that the measures drawn from hand trajectories are of sufficient richness that the proposed model of van der wel et al . ( 2009 ) does not capture their full range of nuances . nevertheless , both sets of authors agree that such debate is central to further understanding the manner in which cognition flows into action . there is also not yet a clear understanding of the particular cognitive processes directly indexed by hand movements . for instance , several of the studies described here reported effects of motor competition , but one might wonder whether the competition may be mediated by attentional mechanisms ( e.g. , see rizzolatti et al . , 1987 ) . recent mouse - tracking work has found that such competitive effects can also reflect embodied cognitive processes leaking out into the motor system ( miles et al . , 2010 ) . in addition , one might wonder whether average graded attraction effects , like that seen in figure 1 , reflect parallel competing responses ( e.g. , parallel pursuits toward both female and male ) or perhaps strategic response switching ( e.g. , an initial pursuit toward female , which is then corrected by a later pursuit toward male ) . ( 2007b ) demonstrated distinctly different hand - trajectory distributions for scenarios involving parallel competing responses versus scenarios involving strategic midflight corrections ( see figure 3 ) . ( 2008 ) provided a second demonstration of this in the more typical two - choice mouse - tracking design as well . in short , more work is needed to provide a comprehensive mapping of hand movements in these paradigms with specific underlying cognitive processes . nevertheless , at this early stage of the action - dynamics approach , hand movements do show an unusually sensitive ability to tap into real - time cognition . ( 2007a ) conducted an additional study where participants were presented with three colored squares and asked to click on the green square . in the baseline condition , the top and bottom squares were red and the rightward square was green , corresponding to a condition where no attraction to another object on the screen would be predicted . in the switch condition , the green square originally appeared at the top - center , with the other two squares being red . once the cursor exited the leftward start box , however , the green square turned red , and the leftward square that was originally red became green . this simulates a situation where participants initially commit to an incorrect response , and then subsequently switch their commitment to the correct response . in the competition condition , the bottom square was red , the rightward square was green , and the top - center square was a blue - green . the average trajectory showed a direct pursuit toward the correct response . in the switch condition , the average trajectory showed a strong initial movement toward the original location of the green square , followed by a sharp redirection toward the ultimate location of the green square . in the competition condition , the average trajectory was smooth and showed graded attraction toward the location of the distractor . ( b ) shows the distribution of trial - by - trial deviations for trajectories in the competition condition , illustrating unimodality . ( c ) shows the distribution of trial - by - trial deviations for trajectories pooled across the switch and baseline conditions , illustrating bimodality . the average trajectories and distributions elicited by these conditions show how the hand - tracking methodology can identifiably tap into a variety of cognitive processes ( e.g. , parallel competition , strategic response switching ) . another issue is to relate this methodological approach to the long - established precedent of reaction times and reaction time distributions ( e.g. , van zandt , 2000 ) . the reaction time is still the most widely used across psychological studies , and because of its ease of measurement and interpretation , we would not argue that hand - tracking should serve as any full substitute . instead , approximately continuous hand - tracking data from an unfolding decision provides a unique behavioral junction point for certain theoretical debates . for example , theoretical debate anchored methodologically in reaction time eventually sought mitigation in reaction time distribution analyses ( e.g. , ratcliff et al . , 1999 ) . dynamic data from an action trajectory may , in some cases , more directly reveal the cognitive processes under dispute . these same points may be made about eye - tracking , now widely used as an ongoing measure of cognitive processing ( e.g. , tanenhaus et al . , 1995 ) . the same caveats hold : future work should tease apart the relative merits of all these methodological approaches and determine where they are most usefully employed . a final pressing question is how fine - grained mental dynamics provided by hand movements relate to higher - level phenomena , such as behavior , educational and clinical outcomes , or social interaction . more work is needed to tie together data across these multiple time scales . moreover , future research would benefit from combining hand movements with eye - movement and electroencephalography ( eeg ) data for a more complete characterization of the time - course of perception , cognition , and action , and their many interactions . ultimately , this work could be used to bridge diverse domains of psychological science and help form truly integrative accounts of the mind . 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 .

recently , researchers have measured hand movements en route to choices on a screen to understand the dynamics of a broad range of psychological processes . we review this growing body of research and explain how manual action exposes the real - time unfolding of underlying cognitive processing . we describe how simple hand motions may be used to continuously index participants tentative commitments to different choice alternatives during the evolution of a behavioral response . as such , hand - tracking can provide unusually high - fidelity , real - time motor traces of the mind . these motor traces cast novel theoretical and empirical light onto a wide range of phenomena and serve as a potential bridge between far - reaching areas of psychological science from language , to high - level cognition and learning , to social cognitive processes .

The Link between Manual and Mental Dynamics Doing Psychology by Hand Language Social cognition High-level cognition and learning Further Questions and Conclusions Conflict of Interest Statement

classically , motor responses were seen as the end - result of a one - way route from perception cognition action ; thus , they were thought to reveal little about cognitive processing ( see rosenbaum , 2005 , for discussion ) . however , substantial behavioral evidence now indicates that cognition does not discretely collapse its processing onto movement execution . instead , movement is continually updated by cognitive processing over time ( goodale et al . , 1986 ) . studies in humans , for example , suggest that the process of categorizing a visual stimulus immediately shares its ongoing results with the motor cortex to continuously guide a hand - movement response over time ( e.g. monkey studies show that the hand 's position and velocity are tightly yoked to ongoing changes in the firing of population codes within motor cortex ( paninski et al . , 2004 ) , and when a monkey must generate a hand movement based on a perceptual decision , these motor - cortical population codes are yoked to the evolution of the decision process ( cisek and kalaska , 2005 ) . these findings suggest that manual dynamics are intimately coextensive with mental dynamics , and therefore , hand movements that are sampled fast enough could potentially provide real - time read - outs of internal cognitive processing . the recent tracking of hand movement en route to choices on a screen has opened up new avenues of investigation into a wide range of psychological phenomena . hand trajectories recorded by a computer mouse , or sometimes a wireless remote or position sensor , have proven able to sensitively track participants tentative commitments to various choice alternatives over hundreds of milliseconds . as such , the emergence of a behavioral response can be sampled anywhere from 40 to 120 times per second . this permits insights into fine - grained temporal dynamics , providing unusually high - fidelity , real - time motor traces of the mind . its sensitivity to these dynamics can reveal hidden cognitive states that are otherwise not availed by traditional measures ( song and nakayama , 2006 , 2008 , 2009 ) . more broadly , these real - time dynamics may be used to assess how multiple processes temporally interact and how multiple information sources weigh in on processing across hundreds of milliseconds , thereby resolving critical empirical questions and theoretical impasses across psychology ( e.g. ( 2005 ) asked participants to move the computer mouse from the bottom - center of the screen to the top - left or top - right corners . for instance , a picture of a candle appeared in the top - left and a picture of a candy appeared in the top - right while a spoken phrase was heard ( e.g. , a picture of candy , for the spoken word , candle ) , hand movements showed a continuous attraction toward the distractor before settling into the correct alternative . thus , the ongoing accrual of the spoken word partially activated multiple lexical representations ( e.g. this showed that the language system continually integrates the acoustic - phonetic input of a spoken word , triggering parallel competing representations that resolve over time . in some cases , however , comprehending spoken language is more difficult and would benefit from help of the visual system . , is there an apple on a towel or an apple by itself ? ) in a series of studies , participants were given several objects on a screen while they heard a sentence instructing them to move one object to another . for example , hearing the above sentence , participants would use a computer mouse to click on an image displaying an apple on a towel , and to then drag it onto an image of a box . before reaching the box , however , participants hand movements exhibited a continuous attraction toward the object associated with the incorrect syntactic parse ( in this case , an empty towel ) . consistent with analogous eye - tracking studies ( e.g. , tanenhaus et al . , an apple on a napkin ) . importantly , hand movements were able to reveal competition on a trial - by - trial basis , thereby evading longstanding concerns with analogous eye - movement results that were limited by discrete oculomotor output . for instance , dale and duran ( 2011 ) had participants verify simple statements by clicking on a true or false response . , cats are not dogs ) led hand movements to exhibit sharp shifts in direction , initially pursuing one response and then rapidly redirecting toward the other , especially for negated true statements . for example , they have provided new insights into social categorization . , feminine male ) faces and asked to categorize the target 's sex by clicking on a male or hand trajectories were continuously attracted to the opposite sex - category before settling into the correct response . these findings led to a new model of social categorization , proposing that all available facial , vocal , and bodily cues ( among other constraints ) simultaneously weigh in on multiple partially active category representations , and that these dynamically evolve over time into stable categorical perceptions ( freeman and ambady , 2011b ) . hand movements again showed continuous - attraction effects for atypical targets , but also revealed important dissociations in how the processing of white - specifying and black - specifying cues evolves across the categorization time - course . moreover , as the amount of racial ambiguity increased , hand trajectories showed corresponding increases in attraction toward the unselected race - category and increases in motor complexity . this showed that the competition inherent to social categorization sensitively increases and decreases with the amount of ambiguity challenging the perceptual system ( freeman et al . , 2010 ) . further , hand movements have revealed not only that multiple facial cues are dynamically integrated , but also that cues across sensory modalities ( facial and vocal cues ) are dynamically integrated as well ( freeman and ambady , 2011a ) . hand movements have also been used to temporally dissociate the integration of specific cues , such as facial shape and pigmentation ( freeman and ambady , in press ) . freeman and ambady ( 2011a ) found that when categorizing a face 's sex , the simultaneous processing of a sex - atypical voice led participants mouse trajectories to be continuously attracted to the opposite sex - category response before settling into the response consistent with the face 's correct sex . during an actual trial , a single face was centered at the bottom of the screen while the voice stimulus played . the bar graph shows trajectories maximum deviation toward the opposite sex - category from a direct line between trajectories start and end points ( error bars denote se of the mean ) . the continuous - attraction effect shows that ongoing voice - processing results were dynamically integrated with ongoing face - processing results over time . for instance , freeman and ambady ( 2009 ) found that , before participants clicked on a stereotypically masculine attribute ( e.g. hand movements can also reveal other types of partial parallel activations otherwise not seen by traditional measures . for example , although white participants almost always explicitly reported liking black people , an inspection of the hand trajectories leading up to those like judgments revealed a parallel consideration to indicate that they dislike them ( wojnowicz et al . ( 2008 ) had participants judge the truth of questions by moving their mouse to yes or thus , hand movements revealed parallel competition across the process of high - level decision - making and showed how this competition is affected by certainty in graded fashion . hand movements also revealed a strong confirmation bias throughout the response , because answers of low probability had overall slower trajectories . trajectories during false responses had greater sample entropy , reached peak velocity later in the movement , and had a steeper curve toward the competing option while en route to the false response . as such , hand trajectories revealed the ongoing process of overcoming the truth during deceptive responding . trajectories velocity profiles are plotted as a function of time for truthful responses clicking no ( dashed line with circles ) , truthful responses clicking yes ( dashed line with triangles ) , false responses clicking no ( solid line with circles ) , and false responses clicking yes ( solid line with triangles ) . this , among other differences between false and truthful responses in movement complexity and curvature toward the opposite response , suggests the presence of a truth bias that pulls processing off course during the production of a deceptive response . participants were shown a shape at the bottom of the screen , and instructed to pick one of two in the upper - most corners that was associated with that bottom shape . the researchers found that hand movements ( measured with a wireless nintendo wii remote ) covaried in a gradient way with emerging knowledge of the pairs . thus , hand movements revealed the longer timescale dynamics of the knowledge itself , as the participants exhibited learning across the task . , hours ) , gesturing patterns of hand movement have long been used to index learning and transitions of knowledge , especially in children ( goldin - meadow and alibali , 2002 ) . ( 2005 ) asked participants to move the computer mouse from the bottom - center of the screen to the top - left or top - right corners . for instance , a picture of a candle appeared in the top - left and a picture of a candy appeared in the top - right while a spoken phrase was heard ( e.g. , a picture of candy , for the spoken word , candle ) , hand movements showed a continuous attraction toward the distractor before settling into the correct alternative . thus , the ongoing accrual of the spoken word partially activated multiple lexical representations ( e.g. this showed that the language system continually integrates the acoustic - phonetic input of a spoken word , triggering parallel competing representations that resolve over time . in some cases , however , comprehending spoken language is more difficult and would benefit from help of the visual system . , is there an apple on a towel or an apple by itself ? ) in a series of studies , participants were given several objects on a screen while they heard a sentence instructing them to move one object to another . for example , hearing the above sentence , participants would use a computer mouse to click on an image displaying an apple on a towel , and to then drag it onto an image of a box . before reaching the box , however , participants hand movements exhibited a continuous attraction toward the object associated with the incorrect syntactic parse ( in this case , an empty towel ) . consistent with analogous eye - tracking studies ( e.g. , an apple on a napkin ) . importantly , hand movements were able to reveal competition on a trial - by - trial basis , thereby evading longstanding concerns with analogous eye - movement results that were limited by discrete oculomotor output . for instance , dale and duran ( 2011 ) had participants verify simple statements by clicking on a true or false response . , cats are not dogs ) led hand movements to exhibit sharp shifts in direction , initially pursuing one response and then rapidly redirecting toward the other , especially for negated true statements . hand movements have also been critical in uncovering the temporal dynamics of social cognition . , masculine male ) and sex - atypical ( e.g. , feminine male ) faces and asked to categorize the target 's sex by clicking on a male or hand trajectories were continuously attracted to the opposite sex - category before settling into the correct response . , 2007 ) . hand movements again showed continuous - attraction effects for atypical targets , but also revealed important dissociations in how the processing of white - specifying and black - specifying cues evolves across the categorization time - course . moreover , as the amount of racial ambiguity increased , hand trajectories showed corresponding increases in attraction toward the unselected race - category and increases in motor complexity . this showed that the competition inherent to social categorization sensitively increases and decreases with the amount of ambiguity challenging the perceptual system ( freeman et al . further , hand movements have revealed not only that multiple facial cues are dynamically integrated , but also that cues across sensory modalities ( facial and vocal cues ) are dynamically integrated as well ( freeman and ambady , 2011a ) . hand movements have also been used to temporally dissociate the integration of specific cues , such as facial shape and pigmentation ( freeman and ambady , in press ) . freeman and ambady ( 2011a ) found that when categorizing a face 's sex , the simultaneous processing of a sex - atypical voice led participants mouse trajectories to be continuously attracted to the opposite sex - category response before settling into the response consistent with the face 's correct sex . during an actual trial , a single face was centered at the bottom of the screen while the voice stimulus played . the bar graph shows trajectories maximum deviation toward the opposite sex - category from a direct line between trajectories start and end points ( error bars denote se of the mean ) . for instance , freeman and ambady ( 2009 ) found that , before participants clicked on a stereotypically masculine attribute ( e.g. hand movements can also reveal other types of partial parallel activations otherwise not seen by traditional measures . for example , although white participants almost always explicitly reported liking black people , an inspection of the hand trajectories leading up to those like judgments revealed a parallel consideration to indicate that they dislike them ( wojnowicz et al . ( 2008 ) had participants judge the truth of questions by moving their mouse to yes or thus , hand movements revealed parallel competition across the process of high - level decision - making and showed how this competition is affected by certainty in graded fashion . hand movements also revealed a strong confirmation bias throughout the response , because answers of low probability had overall slower trajectories . trajectories during false responses had greater sample entropy , reached peak velocity later in the movement , and had a steeper curve toward the competing option while en route to the false response . as such , hand trajectories revealed the ongoing process of overcoming the truth during deceptive responding . trajectories velocity profiles are plotted as a function of time for truthful responses clicking no ( dashed line with circles ) , truthful responses clicking yes ( dashed line with triangles ) , false responses clicking no ( solid line with circles ) , and false responses clicking yes ( solid line with triangles ) . this , among other differences between false and truthful responses in movement complexity and curvature toward the opposite response , suggests the presence of a truth bias that pulls processing off course during the production of a deceptive response . participants were shown a shape at the bottom of the screen , and instructed to pick one of two in the upper - most corners that was associated with that bottom shape . the researchers found that hand movements ( measured with a wireless nintendo wii remote ) covaried in a gradient way with emerging knowledge of the pairs . thus , hand movements revealed the longer timescale dynamics of the knowledge itself , as the participants exhibited learning across the task . , hours ) , gesturing patterns of hand movement have long been used to index learning and transitions of knowledge , especially in children ( goldin - meadow and alibali , 2002 ) . the work discussed above suggests that the intimate link between manual and mental dynamics may be used to provide critical insights into psychological phenomena . insights from simple manual actions may therefore serve as an unforeseen bridge between far - reaching areas of psychological science . this action - dynamics approach to the mind holds particular promise given the practicality of measuring hand movements . there now exists freely available , user - friendly software to run and analyze data from computer mouse - tracking experiments ( freeman and ambady , 2010 ) . thus , rather than measure an ultimate response time , researchers can easily open up such responses into continuous streams of rich cognitive output through nothing more than a computer and mouse . mouse - tracking also permits the recording of online mental processing in young children at an age where online data are often difficult to obtain ( anderson et al . with the capturing of hand movements now just as available and affordable as recording the classic response time , we are hopeful that a growing number of studies will employ the methodology . in this action - dynamics approach , researchers are afforded many ways to measure and analyze hand trajectories . , song and nakayama , 2008 ) . although the techniques are quite similar , one notable difference is that mouse - tracking captures only 2d movement , whereas the others could potentially capture 3d movement . another consideration is that , in mouse - tracking , hand movements are stabilized by a surface . moreover , mouse - tracking can be carried out through online data collection systems like amazon mechanical turk ( dale and duran , 2011 ) . once trajectories are recorded , several measures may be computed , including maximum deviation toward an unselected response ( e.g. the time - course of the hand 's position , velocity , acceleration , or proximity to a selected or unselected response , can also be examined . each analysis carries its own advantages with respect to the theoretical and practical interests at hand . despite the headway made by the work reviewed here , however , the moving hand still has much to reveal about the mind . for example , in a recent critique of the method and its interpretation , van der wel et al . ( 2009 ) discussed whether a model of discrete processing could produce , through ( continuous ) linear combinations of motor plans , the evidence for continuous processing often obtained from hand - tracking experiments . the authors argued that the inference of processing continuity from hand - tracking data is not yet fully warranted . ( 2009 ) does not capture their full range of nuances . there is also not yet a clear understanding of the particular cognitive processes directly indexed by hand movements . for instance , several of the studies described here reported effects of motor competition , but one might wonder whether the competition may be mediated by attentional mechanisms ( e.g. recent mouse - tracking work has found that such competitive effects can also reflect embodied cognitive processes leaking out into the motor system ( miles et al . , parallel pursuits toward both female and male ) or perhaps strategic response switching ( e.g. , an initial pursuit toward female , which is then corrected by a later pursuit toward male ) . ( 2007b ) demonstrated distinctly different hand - trajectory distributions for scenarios involving parallel competing responses versus scenarios involving strategic midflight corrections ( see figure 3 ) . ( 2008 ) provided a second demonstration of this in the more typical two - choice mouse - tracking design as well . in short , more work is needed to provide a comprehensive mapping of hand movements in these paradigms with specific underlying cognitive processes . nevertheless , at this early stage of the action - dynamics approach , hand movements do show an unusually sensitive ability to tap into real - time cognition . in the switch condition , the average trajectory showed a strong initial movement toward the original location of the green square , followed by a sharp redirection toward the ultimate location of the green square . in the competition condition , the average trajectory was smooth and showed graded attraction toward the location of the distractor . the average trajectories and distributions elicited by these conditions show how the hand - tracking methodology can identifiably tap into a variety of cognitive processes ( e.g. the reaction time is still the most widely used across psychological studies , and because of its ease of measurement and interpretation , we would not argue that hand - tracking should serve as any full substitute . instead , approximately continuous hand - tracking data from an unfolding decision provides a unique behavioral junction point for certain theoretical debates . dynamic data from an action trajectory may , in some cases , more directly reveal the cognitive processes under dispute . these same points may be made about eye - tracking , now widely used as an ongoing measure of cognitive processing ( e.g. a final pressing question is how fine - grained mental dynamics provided by hand movements relate to higher - level phenomena , such as behavior , educational and clinical outcomes , or social interaction . moreover , future research would benefit from combining hand movements with eye - movement and electroencephalography ( eeg ) data for a more complete characterization of the time - course of perception , cognition , and action , and their many interactions . ultimately , this work could be used to bridge diverse domains of psychological science and help form truly integrative accounts of the mind . 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 .

there were approximately 33.4 million people worldwide living with hiv in 2008 ; of these , 2.7 million were newly infected . these statistics are substantially below those seen during the apparent peak in infections in 1996 . at least part of the reason for the trend toward stabilization of the aids epidemic and increased longevity of hiv - infected individuals can be attributed to the beneficial effect of antiretroviral therapy . decreased risk of viral transmission and the prospect of increased longevity of the infected parent secondary to the use of highly effective antiretroviral therapy pose fewer challenges for infected individuals of reproductive age to have their own children . however , hiv infection may impair fertility [ 24 ] , prompting hiv - infected men and women to seek medical assistance ( i.e. , assisted reproductive technologies , or art ) . further , art is the method of choice for fertile serodiscordant couples trying to conceive ; it reduces the risk of hiv transmission associated with unprotected sexual intercourse . providing art to hiv - infected couples , particularly serodiscordant couples where the man is infected , is problematic for health care providers . hiv is sometimes detected in semen from infected men , even when they are receiving antiretroviral therapy [ 6 , 7 ] . spermatozoa contaminated with hiv can transmit virus to the uninfected woman and to the child [ 2 , 8 ] . sperm washing has been advocated as a method to reduce hiv transmission , in which motile spermatozoa are separated from seminal plasma , nonsperm cells , and hiv [ 9 , 10 ] . this method appears to be successful , with no reported infections in multiple procedures carried out in somewhat more than 2000 couples [ 11 , 12 ] . this was , in large part , the basis for california legislation that permits a sperm wash to minimize hiv transmission during art procedures for serodiscordant couples [ 13 , 14 ] . however , not all infected semen processed by sperm washing can be used . due to the nature of the sperm washing method , detectable hiv titers remain in 46% of washed sperm samples [ 4 , 12 , 15 , 16 ] . reasons for this are not entirely clear , but may be due , at least in part , to viral binding to the sperm fraction . for the most part , neither conventional hiv-1 cd4 receptors nor coreceptors ( cxcr4 and ccr5 ) have been found on human spermatozoa [ 10 , 17 , 18 ] . however , several studies [ 17 , 19 , 20 ] have shown that hiv binds to spermatozoa . alternative receptors had been proposed to account for hiv binding [ 19 , 21 ] . ceballos and coworkers found that hiv-1binds to human spermatozoa through sperm surface heparan sulfate receptors and remains infectious . hiv binding , through alternative receptors or nonspecific mechanisms , may explain the failure of a sperm wash to completely remove hiv from infected semen in all instances . further , at least some proponents of sperm washing to reduce risk of hiv transmission during assisted reproduction advise against its use with intracytoplasmic sperm injection ( icsi ) . this recommendation is based on the possibility that icsi may introduce hiv into the oocyte as a result of hiv attached to the sperm membrane [ 4 , 22 ] . addition of a safe antiviral compound may offer some advantages over sperm washing alone in minimizing the risk of hiv transmission during art . ideally , an effective antiviral agent added at or around the time of sperm washing should result in the following : ( 1 ) processed sperm samples would contain no residual infectious hiv ; ( 2 ) icsi could be used as an assisted reproductive technique in hiv - serodiscordant couples with fewer concerns ; ( 3 ) treatment could be offered to a larger number of serodiscordant couples than possible with the conventional sperm wash protocol , which is biased in favor of very low or undetectable initial viral load [ 8 , 12 ] ; ( 4 ) the risk of viral infection of health care providers who are in direct contact with infected samples would be greatly reduced . non - antiretroviral compounds have been developed , intended for vaginal use , to prevent hiv transmission during sexual intercourse . these compounds , known generically as microbicides , have advanced up to and including clinical trials [ 2326 ] . many belong to a class of compounds known as polyanions , one of which is a poly - acidic oligomer , code - named ppcm ( formerly known as sulfuric acid - modified mandelic acid , or samma ) , developed [ 2732 ] by the topcad program at rush university medical center ( chicago , il ) and licensed by yaso biotechnologies ( phoenix , az ) . polyanions tested to date , including ppcm , are noncytotoxic , broad - spectrum antimicrobial agents , with activities against sexually transmitted pathogens , including hiv-1 , hsv-1 , hsv-2 , papillomavirus , n. gonorrhoeae , and c. trachomatis [ 2729 , 31 , 33 ] . they act in part by interfering with microbial binding to target cells via the heparan sulfate ( or similar ) receptor . ppcm may be , therefore , an attractive candidate as a safe antiviral agent that could be used as an hiv-1 preventive agent during art . however , many polyanions , including ppcm , are contraceptive [ 3437 ] . these compounds would be appropriate for art only if their antimicrobial activities can be separated from their contraceptive activities , and they have no adverse effects on sperm function and integrity . contraceptive activities of polyanions may be due , at least in part , to their abilities to induce premature acrosomal loss . we thought that it may be possible to separate contraceptive and antimicrobial activities of ppcm through manipulation of extracellular ca levels . the purpose of the present study was to examine the feasibility of the use of ppcm to reduce infectivity of hiv-1 in infected semen used for art . inhibition of hiv-1 ( bal ) by ppcm is at least partially irreversible and is independent of added extracellular ca . in the absence of added ca , sperm washing in the presence of ppcm substantially reduces ( 3 - 4 logs ) viral titer of ex - vivo hiv-1 ( bal)-treated whole human semen from healthy donors , as compared with washing alone . this occurs without negatively affecting measures of sperm dna integrity or sperm function , including motility , forward progression , acrosomal integrity , and dna decondensation . isolate , a colloidal density gradient medium for separation of spermatozoa from semen for use in art , was purchased from irvine scientific ( santa ana , ca ) . quinn 's sperm wash medium was purchased from sage in - vitro fertilization , inc . ( trumbull , ct ) . rpmi-1640 medium ( rpmi ) was from irvine scientific ; a custom preparation of rpmi to which ca had not been added was also provided by irvine scientific . hiv-1 ( bal ; lot # 07283002 ) , an expanded laboratory strain , was from the virology quality assessment program ( vqa ) , rush university medical center and was provided by the clinical retrovirology research laboratory , department of immunology and microbiology , rush university medical center , under the direction of dr . reagents for immune complex disruption and elisa determination of hiv-1 antigen p24 ( hereafter referred to as p24 ) levels , and real - time reverse transcription - pcr determination of hiv-1 rna were obtained in kit forms from perkinelmer life sciences , inc . ( hiv-1 p24 elisa kit ; boston , ma ) and abbott molecular , inc.(realtime hiv-1 rna assay ; des plaines , il ; m2000 realtime - pcr - system ) , respectively . it is a poly - acidic oligomer with molecular weight of approximately 1500 da and was synthesized by dr . s. jain , university of illinois health sciences center ( chicago , il ) under the direction of the topcad program by reacting d , l - mandelic acid with sulfuric acid under proprietary conditions . the sodium salt was prepared by reacting the free acid with alcoholic naoh , yielding an off - white powder . semen samples used for hiv infectivity , sperm motility , and acrosomal status assessment were collected from pools of eight healthy donors ( rush university medical center ) and five fertile donors ( andromedix laboratory ) from whom informed consent was given , according to the protocols approved by their respective institutional review boards . for semen used in all experiments except dna testing semen was of generally high quality ; mean semen volume was 3.0 0.24 ( standard error of the mean ; sem , n = 13 ) ml . initial mean ( with 90% confidence limits ) values for sperm count , % motility , % forward progression , and % viable spermatozoa were 83 ( 58.9117.8 ; n = 11 ) 10/ml , 69 ( 64.373.6 ; n = 12)% , 78 ( 72.082.9 ; n = 12)% and 90 ( 85.793.0 ; n = 12)% , respectively . semen used for sdfa and sdd testing was derived from five donors of proven fertility , selected for the ability of their spermatozoa to undergo dna decondensation ( 96% 2% sd ) within 15 minutes of adding stimulus . samples were allowed to stand at ambient temperature for one hour to allow for liquefaction . spermatozoa were separated from seminal plasma by centrifugation through a discontinuous gradient consisting of equal volumes of 45% and 90% isolate ( irvine scientific ) at 200 g for 20 min . spermatozoa at the bottom of the gradient were resuspended in quinn 's sperm wash and washed by centrifugation at 200 g for 10 min . unless otherwise noted , the sperm pellet was suspended in medium ( either bww or rpmi ) , to which ca had not been added , prior to further use . motility measurements of 10 l samples were carried out under light microscopy ( 200x ) with a neubauer hemacytometer by standard operating procedures used by the andrology laboratory of the rush centers for advanced reproductive care , rush university medical center . motile spermatozoa were defined as cells displaying any type ( progressive , in - place ) of movement ; spermatozoa passing through the field of vision were considered forwardly progressing . from 200400 spermatozoa motility was recorded as percentage of total spermatozoa that were moving ; forward progression was recorded as the percentage of motile spermatozoa that was forwardly progressive . sperm viability was measured with eosin y. staining was visualized by bright field light microscopy ( 400x ) . spermatozoa that stained green were considered viable ; spermatozoa that stained red were regarded as nonviable . results for sperm motility , forward progression and viability are presented as mean percentage of postwash control values , together with sem . within the context of this study , acrosomal loss refers to the disruption of the sperm acrosome in response to a treatment or chemical entity . no inference is made as to whether this response is identical to a physiological acrosome reaction , during which the acrosome is also lost . spermatozoa recovered from isolate gradient centrifugation were resuspended in modified biggers , whitten and whittingham ( bww ; ) medium ( less albumin and ca ) . after incubations of washed spermatozoa with 10 mg / ml ppcm or with medium alone for 15 min at ambient temperature , samples were placed onto 1 ml of 11% ( wt / vol ) buffered ficoll ( containing 120 mm nacl and 25 mm hepes , ph 7.4 ) , and centrifuged at 15,000 g - min ( 22c to 24c ) . supernatants were aspirated from the sperm pellets , and the pellets were resuspended into modified bww medium . the supernatants were aspirated , and the pellets were resuspended in 1 ml bww medium . the latter washing step was repeated , at which point , the ppcm concentration in the supernatant , as measured by the absorbance at 266 nm , was undetectable . sperm pellets obtained from the above procedure were resuspended in bww medium that contained 2.3 mm cacl2 . after equilibration for 10 min at 37c , acrosomal loss was induced by addition of stimulus ( ppcm or calcium ionophore a23187 ) . fifteen min after acrosomal loss induction , spermatozoa were fixed with buffered glutaraldehyde , smeared onto slides and stained with rose bengal and bismarck brown for acrosome visualization [ 30 , 39 ] . data are expressed as means , with 90% confidence limits , of percentage of acrosomal loss induced . the sdfa and sdd test were carried out at the andromedix laboratory , woburn , ma . the sdd test was formerly known as the human sperm activation assay ( hsaa , ) . controlssemen was mixed with 2 ml nuclear isolation medium ( nim ) , consisting of 200 mm sucrose , 2.4 mm mgcl2 , 10 mm tris - hcl , 5 mm maleic acid , ph 7.4 , and centrifuged at 1200 g for 10 min to remove seminal plasma . sperm pellets were resuspended in 1.0 ml of nim ( solvent for ppcm ) , prior to the sdfa , or analysis in the sdd test as described by brown et al . . semen was mixed with 2 ml nuclear isolation medium ( nim ) , consisting of 200 mm sucrose , 2.4 mm mgcl2 , 10 mm tris - hcl , 5 mm maleic acid , ph 7.4 , and centrifuged at 1200 g for 10 min to remove seminal plasma . sperm pellets were resuspended in 1.0 ml of nim ( solvent for ppcm ) , prior to the sdfa , or analysis in the sdd test as described by brown et al . . treated samplessperm pellets were resuspended in 1.0 ml of nim containing ppcm ( 2 mg / ml or 10 mg / ml , final concentrations ) . after a 10 min incubation , samples were centrifuged at 1200 g for 5 min ; sperm pellets were resuspended in 1.0 ml nim and the suspension was recentrifuged . the final pellets were either resuspended in 0.5 ml nim and snap - frozen in liquid nitrogen prior to the sdfa , or resuspended in 1.0 ml nim and kept overnight at 4c prior to analysis in the sdd test . sperm pellets were resuspended in 1.0 ml of nim containing ppcm ( 2 mg / ml or 10 mg / ml , final concentrations ) . after a 10 min incubation , samples were centrifuged at 1200 g for 5 min ; sperm pellets were resuspended in 1.0 ml nim and the suspension was recentrifuged . the final pellets were either resuspended in 0.5 ml nim and snap - frozen in liquid nitrogen prior to the sdfa , or resuspended in 1.0 ml nim and kept overnight at 4c prior to analysis in the sdd test . the sdfa utilizes acridine orange ( ao ) as a fluorescent dna probe and employs flow cytometry to measure the intensity of fluorescence . ao is a cell - permeant nucleic acid binding dye that emits green fluorescence when bound to double - stranded ( normal ) dna and red fluorescence when bound to single - stranded ( damaged ) dna . for each sample , 5000 cells were measured and the ratio of red to total fluorescence ( red + green ) was calculated . this value , expressed as a percentage , is the dna fragmentation index ( dfi ) . all averaged data for each semen donor were subjected to arcsine transformation prior to further analysis . for this study , dfi scores 15 were considered normal . a mean score where 15 < dfi < 30 , suggests a marginal loss of dna integrity due to treatment . samples ( see above ) were centrifuged at 1200 g for 10 min and resuspended in permeabilization medium for 5 min . they were sequentially washed with nim containing bovine serum albumin , and treated for one hour with xenopus egg extract isolation medium ( xeim ; 10 mm tris - hcl , 1.5 mm mgcl2 , 100 mm kcl , 50 mm dithiothreitol , ph 7.5 ) . sperm suspensions were centrifuged at 1200 g for 10 min , and the pellets were suspended in xeim to a final cell concentration of 25 10 cells / ml . for each sample , 50,000 permeabilized sperm were incubated in 25 l of xenopus laevis egg extract , to simulate the in vivo environment of the sperm nucleus postfertilization . after 15 min , an aliquot of the sperm - extract mixture was placed on a slide , and 50100 sperm were scored with phase - contract microscopy in real time during a 5 min window . ppcm at several concentrations ranging from 0.1 to 20 mg / ml was added to hiv-1 ( bal ) suspensions ( 5.9 10 tcid50s / ml ) . medium was rpmi-1640 , supplemented with glutamine , gentamycin , human growth factor and interleukin-2 , with or without added ca ( 0.46 mm ca(no3)24(h2o ) ) . after 1 h at ambient temperature , suspensions were diluted 1 : 100 with rpmi ( with or without added ca ) , prior to inoculation into peripheral blood mononuclear cell ( pbmc ) suspensions ( 2.0 10 cells in 0.1 ml ) . after 1 h , pbmcs were washed with fresh medium ( containing added ca ) to remove unbound virus and ppcm , and the pbmc suspensions were incubated for 72 hours at 37c to allow for viral replication . viral p24 levels were measured by elisa with reagents provided in kit form by perkinelmer life sciences , inc . , according to instructions provided by the vendor . values were quantified with a standard curve of hiv-1 p24 concentration as a function of absorbance at 490 nm . the concentration of ppcm required for 50% inhibition of p24 production ( ic50 value , obtained from curves fit to the data with tablecurve 2d ; see statistical analysis ) when ppcm was added to complete rpmi was compared with that obtained when ppcm was added to medium without added ca . inhibition of viral replication by ppcm was compared under two conditions , as follows : ( 1 ) ppcm at concentrations up to 13 mg / ml was added to only hiv-1 ( bal ) , and incubated for 1 h , followed by a 1 : 100 dilution in rpmi , prior to the suspension being inoculated onto pbmcs . subsequent washing medium changes and incubation of pbmcs to allow for viral replication were carried out in the absence of ppcm . ( 2 ) ppcm at concentrations up to 1 mg / ml was added to viral suspensions , incubated for 1 h , and inoculated onto pbmcs that contained the same concentrations of ppcm . washing and medium changes also contained ppcm ( i.e. , ppcm was present throughout the entire procedure ) . other experimental conditions and measurement of p24 levels were the same as described above for effect of added extracellular ca . the ic50 value of ppcm when ppcm was added to only virus was compared with that obtained when ppcm was present throughout the viral infection and replication . neat fresh semen pooled from five donors ( 11.3 ml ) was added to an equal volume of hiv-1 ( bal ) inoculum ( 5 10 tcid50s / ml ) . total inoculum and total number of spermatozoa were 5.65 10 tcid50s and 1.07 10 cells , respectively . after 30 min at ambient temperature , small samples were taken , diluted and added directly to pbmc suspensions ( unprocessed control ) . one volume of either 0.9% saline ( processed control ) or ppcm stock solution ( 20 mg / ml in 0.9% saline , sterile - filtered ) was added to three volumes of the semen / viral suspension . spermatozoa were isolated by centrifuging the mixture through sperm isolate preparation gradients ( 200 g , 25 min ) . 0.2 ml ) were resuspended in 1 ml rpmi without added ca and centrifuged at 200 g for 10 min . sperm pellets ( approximately 0.2 ml ) were used as the viral inoculum , and added to pbmc suspensions . after 1 h , pbmcs were washed to remove unbound virus , spermatozoa and ppcm , and incubated for 72 h in ca - replete rpmi to permit viral replication . at this time , a portion of each sample was taken for p24 measurement ( see above ) . the remainder of each sample was stored at 80c until being used for viral rna measurements by real - time reverse transcription pcr ( m2000 real time hiv assay ) , with automated rna preparation ( m2000sp ) and measurement ( m2000rt ) carried out as instructed by the manufacturer . the assay was standardized against a viral standard from the vqa program ( see materials ) . the assay is designed to achieve an interassay standard deviation of 0.25 log copies / ml at concentrations ranging from 500 to 5 10 copies / ml . data ( copies / ml viral rna ) are expressed as means sem . for data requiring either logarithmic ( sperm count ) or arcsine transformation ( frequencies ; initial sperm motility , forward progression , viability ; acrosomal loss ) , results are expressed as means , with 90% confidence limits . effects of ppcm dose and duration of exposure were evaluated with two - way analysis of variance . differences among individual treatment groups were assessed with the newman - keuls multiple range test . data from dose response experiments were best fit to curves by regression analysis with tablecurve 2d curve - fitting software ( version 5 ; spss statistical software , chicago , il ) , from which appropriate constants ( e.g. , ic50 ; that concentration of agent required for 50% inhibition of hiv infectivity ) were calculated . all values are considered significantly different at the 0.05 level of confidence ; p > .10 is considered not different . dose responses of hiv infectivity as a function of ppcm concentration ( from 1 10 to 6.7 mg / ml ) were measured in the presence and absence of added ca . in these experiments , all samples contained ca during the 72 h incubation of pbmcs to allow for viral replication of infected cells . the ic50 ( 90% confidence limits = 33.766.3% inhibition ) for samples incubated in ca - replete rpmi is 24 g / ml . in ca - deficient medium , the ic50 ( 46.853.2% inhibition ) is 39 g / ml ( figure 1 ) . dose response of hiv infectivity as a function of initial ppcm concentration ( from 1 10 to 13.3 mg / ml ) was measured in samples in which ppcm concentrations were maintained throughout the experimental procedure ( viral incubation , inoculation onto pbmcs , wash to remove free virus and incubation of infected pbmcs to permit viral replication ) . this was compared with the dose - response measured in samples in which ppcm - treated virus was first diluted 1 : 100 prior to inoculation onto pbmcs . in the latter samples , medium used for procedures subsequent to initial viral exposure to ppcm was ppcm - free . dilution and subsequent removal of ppcm by washing has essentially no effect on the ability of ppcm to inhibit hiv infectivity ( figure 2 ) . when the initial concentration of ppcm is maintained throughout all procedures ( no dilution ) , the ic50 ( 33.766.3% inhibition ) for ppcm is 23 g / ml . the ic50 ( 90% confidence limits = 48.651.4% inhibition ) for ppcm when added only to viral suspensions , followed by dilution , is 36 g / ml . sperm exposure to ppcm at concentrations up to 10 mg / ml for periods up to 90 min has little effect on sperm motility , forward progression or viability ( figures 3(a)3(c ) ) . two - way anova showed no effect of either ppcm concentration or duration of exposure to ppcm on motility ( f(2,30 ) = 1.36 and f(4,30 ) = 1.43 , resp . ; p > .10 ) or forward progression ( f(2,30 ) = 1.48 and f(4,30 ) = 1.63 , resp . ; similarly , there is no effect of ppcm concentration on sperm viability ( f(2,30 ) = 1.67 , p > .10 ) , although a small effect ( 11% reduction at 90 min exposure to 10 mg / ml ppcm ) of duration may exist ( f(4,30 ) = 5.03 , p < .01 ) . acrosomal status of sperm suspensions treated with 10 mg / ml ppcm for 15 min in bww medium without added ca is similar to control spermatozoa ( no ppcm treatment ) similarly processed , when spermatozoa from both groups are subsequently washed and added to ca - replete medium ( detailed protocol presented in section 2 ) . percentage of ppcm - treated spermatozoa without acrosomes is 19 ( 90% confidence limits = 18.620.0)% . this is essentially the same as the percentage ( 19 ( 17.721.2)% ) of control spermatozoa without acrosomes ( p > .10 , newman - keuls multiple range test ) . acrosomal loss increases to a similar extent in both ppcm - treated and control samples in response to stimuli ( p > .10 , newman - keuls multiple range test ) . percentage of spermatozoa without acrosomes in response to a maximally stimulating concentration of calcium ionophore a23187 is 32 ( 31.032.5)% and 32 ( 30.932.9)% for ppcm - pretreated and control spermatozoa , respectively . percentage of spermatozoa without acrosomes in response to 0.25 g / ml ppcm ( concentration required to induce 50% maximal acrosomal loss ) is 27 ( 26.327.6)% and 27 ( 26.627.7)% for ppcm - pretreated and control spermatozoa , respectively ( figure 4 ) . pretreatment of spermatozoa with either 2 mg / ml or 10 mg / ml ppcm had no effect on sperm dna fragmentation ( figure 5(a ) ) . fragmentation in spermatozoa pretreated with ppcm is essentially the same as that in spermatozoa pretreated with only medium ( f(2,9 ) = 2.80 ; p > .10 ) . mean dfi for negative control spermatozoa , spermatozoa treated with 2 mg / ml ppcm and spermatozoa treated with 10 mg / ml ppcm are 6 ( 90% confidence limits = 4.56.6 ) , 7 ( 2.912.4 ) and 9 ( 6.113.3 ) , respectively . a similar lack of effect of ppcm was noted on the ability of dna from permeabilized spermatozoa to decondense in response to xenopus laevis egg extract ( f(2,8 ) = 0.59 ; p > .10 ) . mean percentage of control spermatozoa , spermatozoa pretreated with 2 mg / ml ppcm , and spermatozoa pretreated with 10 mg / ml ppcm that showed full decondensation in response to egg extract are 96 ( 95.097.0)% , 98 ( 87.299.5)% , and 96 ( 95.596.9)% , respectively ( figure 5(b ) ) . following incubation with hiv-1 ( bal ) for 30 min , whole semen was treated with either 5 mg / ml ppcm or with an equal volume of medium ( without added ca ) , and incubated at ambient temperature for 45 min ( see section 2 for complete description of experimental protocol ) . in samples without ppcm , gradient centrifugation , followed by two washes by resuspension and recentrifugation , reduced the initial viral titer by approximately 92% , as measured by viral p24 levels . a much greater reduction in viral titer is effected by pretreatment with ppcm . ppcm further reduces viral p24 levels by approximately 3 logs , ( 99.94% reduction , to 0.04 0.03 ( sem ) ng / ml ) as compared with samples subjected to only centrifugation . greater reduction of viral titer by ppcm is obtained when titer is expressed as viral rna ( figure 6(b ) ) . similar to p24 concentrations , gradient centrifugation followed by washing ( without ppcm ) reduces viral rna concentration by 92% ( to 4.91 0.055 10 copies / ml ) . viral rna is further reduced by approximately 4.4 logs ( to 20 3 copies / ml ) by pretreatment with ppcm . reductions in viral titer from that observed in the unprocessed , hiv - incubated semen produced by either sperm washing or ppcm pretreatment followed by sperm washing are significant ( p < .01 , newman - keuls multiple range test ) . the present data support the utility of ppcm as an effective compound for use in art to further reduce the risk of hiv transmission . data collected in other investigations for this and similar compounds suggest that ppcm should be relatively safe for use in art , and should be efficacious in reducing infectivity of hiv ( laboratory strains , clinical isolates and different clades ) and several other sexually transmitted pathogens in vitro [ 27 , 29 , 33 , 36 , 4550 ] . contraceptive activity of ppcm is believed to be due to its ability to induce premature acrosomal loss ; this effect requires physiological concentrations of extracellular ca . acrosomal integrity can be maintained in the presence of ppcm only in the absence of added extracellular ca . it was important to demonstrate that ppcm retains its inhibitory activity against hiv in a ca - independent manner . the present study has shown that the dose - response of ppcm - induced inhibition of hiv infectivity is independent of added ca ( figure 1 ) . ideally , inhibition of hiv by ppcm would be irreversible , or at least pseudoirreversible . this allows for removal of all or most of the agent after viral inactivation , which , in turn , minimizes possible untoward side effects on cellular elements of the fertilization process and subsequent embryonic development . ppcm - induced viral inhibition is at least partially irreversible , since the dose - response of hiv inhibition is unaffected by a 1 : 100 dilution of ppcm after initial interaction with the virus ( figure 2 ) . this occurs by preventing the interaction of the positively charged v3 loop of viral gp120 with negatively charged coreceptors ( e.g. , heparan sulfate or similar ) on target ( host ) cells . studies with other polyanionic microbicides ( e.g. , cellulose sulfate , polystyrene sulfonate , cellulose sulfate phthalate ) suggest that relatively high concentrations of these agents may cause gp41 six - helix bundle formation , viral disintegration and rapid loss of infectivity . the work of mesquita and coworkers suggests possible irreversible inhibition of hiv-1 ( bal ) and hiv-1 ( rf ) infectivity after pretreatment of immobilized virus with ppcm followed by washing , further , a structural variant of ppcm inhibits hiv-1 ( iiib ) and hiv-1 ( bal ) in an irreversible or pseudoirreversible manner . high concentrations of ppcm ( 25 mg / ml ) are relatively noncytotoxic toward spermatozoa , as measured by a lack of effect on sperm motility following short - term exposure ( sander - cramer test for spermicidal activity ) . we have confirmed and extended these studies by measuring sperm motility , forward progression and sperm viability after exposure to ppcm at concentrations as high as 10 mg / ml for periods up to 90 min ( figure 3 ) . no effect was observed on sperm motility or forward progression . a small , but significant effect of duration of exposure on sperm viability the physiological significance of the latter finding is unclear , since no concentration - dependent effect was observed . the small effect of duration of exposure could reflect a small time - dependent decrease in viability , independent of the presence of ppcm . these data suggest that concentrations of and duration of exposure to ppcm that should be efficacious against hiv-1 have no substantial effect on sperm viability or selected aspects of sperm function . earlier studies showing ppcm - induced acrosomal loss as a ca - dependent process , dependent upon physiological concentrations of extracellular ca ( ed50 approx . 0.08 mm ) , were carried out with ppcm at concentrations in the sub- g / ml to g / ml range . the present study examined whether these observations could be extended to ppcm when used at concentrations several orders of magnitude higher ( mg / ml range ) . it was important to evaluate whether previous exposure to ppcm affected subsequent ability of spermatozoa to undergo acrosomal loss in response to stimulus . spermatozoa in ca - deficient medium were pretreated with a high ( 10 mg / ml ) concentration of ppcm , washed to reduce ppcm to nondetectable levels , and resuspended in ca - replete medium . under these conditions , further , similar to that of control samples , ppcm - treated spermatozoa respond to acrosomal loss stimuli , namely calcium ionophore a23187 and ppcm ( figure 4 ) . as a stimulus of acrosomal loss ( in the presence of added extracellular ca ) , ppcm was used at a concentration ( 0.25 g / ml ) predicted to induce 50% maximal acrosomal loss . approximately 60% maximal loss was noted , in spermatozoa pretreated with ppcm as well as in control spermatozoa subjected to the same protocol ( washing by centrifugation ) . the slight increase in acrosomal loss over that predicted may reflect an increased fragility of the acrosomes due to the experimental protocol of repeated centrifugation and resuspension . the data suggest that under appropriate conditions , prior treatment with relatively high concentrations of ppcm is without effect on acrosomal status . in order for ppcm - treated sperm to be used in art procedures , it is important to establish that ppcm does not negatively affect sperm dna integrity and/or sperm functions required for postfertilization restructuring of the sperm nucleus , once inside the egg . to test this , ppcm - treated sperm were analyzed with two tests : the sperm dna fragmentation assay ( sdfa)/sperm chromatin structure assay ( scsa ) , a sperm structure assay used to measure dna integrity ; and the sperm dna decondensation ( sdd ) test ; a sperm function test used to measure overall sperm nuclear integrity . both the scsa and sdd test have clinical relevance in predicting live birth outcome [ 41 , 42 , 53 ] . normal scores in ppcm - treated sperm for both tests support the use of ppcm - washed sperm in art . the two tests were also used to test the hypothesis that the polyanionic nature of ppcm might allow the sperm nuclear membrane to be compromised through ionic interactions with either protamines or histones within the sperm chromatin . protamine packaging greatly condenses the dna , resulting in lower accessibility to dna - damaging agents . if this is incomplete , exposed dna may be vulnerable to damage from agents in the surrounding environment . ppcm - induced interference with protamine - dna interaction would be reflected as highly fragmented dna , and/or delayed sperm dna decondensation / nuclear swelling , as detected with the sdfa and sdd test , respectively . however , ppcm ( 10 mg / ml ) has no effect on either sperm dna fragmentation ( figure 5(a ) ) or dna decondensation ( figure 5(b ) ) . these data are consistent with studies in which ppcm displayed no mutagenic activity , as measured by the ames test . further , they support the safety of using ppcm - treated washed spermatozoa in art to reduce risk of hiv infection . the ability of ppcm to inhibit hiv infectivity in vitro is well - documented [ 27 , 28 , 31 , 49 ] . the present study extended these studies by evaluating the ability of ppcm to inhibit hiv associated with whole semen . infected semen was simulated by incubating uninfected semen with a high titer of hiv-1 ( bal ) . this , rather than semen from hiv - infected men , was used to permit sufficiently high viral titers ( particularly after washing by gradient centrifugation ) to accurately quantify efficacy of ppcm combined with sperm washing beyond that effected by washing alone . with this method , sperm washing by gradient and differential centrifugation reduced initial viral titer by somewhat over 90% , whether measured by viral p24 or rna levels ( figures 6(a ) and 6(b ) ) . the method of sperm processing in the present study is similar to that used clinically by pea and coworkers to reduce viral titers in semen from hiv - infected men . the relatively inefficient removal of virus suggests that washing alone may not be an optimal method to completely remove high levels of viral contamination ; this is also suggested by work presented in a review by englert and coworkers . these data are in agreement with other studies showing persistence of hiv in some samples after washing [ 4 , 16 ] . removal of soluble contamination by washing used in the present study is more efficient than removal of hiv . concentration of ppcm , for example , is reduced from 10 mg / ml to less than 0.1 g / ml ( limit of detection ) . although the present study does not specifically address sperm binding of hiv , the data showing only a 90% reduction in viral titer by washing alone ( figure 6 ) are consistent with hiv binding to spermatozoa , described in other studies [ 17 , 19 , 20 ] . levels of p24 in samples treated with ppcm , followed by washing by gradient and differential centrifugations were reduced by 3 logs , as compared with p24 levels measured after washing alone ( figure 6(a ) ) . reduction of viral rna ( > 4 logs , as compared with washing alone ) was greater than that of p24 ; rna levels in the presence of ppcm were near the limit of detection of the assay . since production of structural and regulatory rna species precedes the production of structural proteins , these data suggest that rna levels may be more sensitive than p24 levels to inhibition by at least some antiviral agents . in some instances , the release of p24 antigen can continue to occur even though hiv mrna transcription and the production of infectious virus particles is inhibited . suggest that the use of proteins such as p24 to measure viral replication could obscure the effectiveness of an antiviral agent that primarily affects the transcription of certain types of viral rna or dna . increased efficacy of ppcm when measured by viral rna as compared with p24 measurements is consistent with mechanisms of inhibition of hiv infectivity in addition to inhibition of viral binding to target cells . the present data show that ppcm can interact with and inactivate hiv associated with spermatozoa . although these studies were restricted to a laboratory strain ( bal ) , results can likely be extended to other strains as well as clinical isolates , since ppcm is active against several hiv strains and isolates [ 28 , 29 , 31 , 33 ] . whether interaction of hiv with spermatozoa in vitro is similar to additional work is required to demonstrate efficacy of ppcm against sperm - associated hiv from hiv - infected men . in spite of proven in vitro efficacy of polyanionic compounds against several sexually transmitted pathogenic microbes , including hiv-1 , none has successfully completed phase 3 clinical efficacy trials for hiv prevention [ 5760 ] . part of the failure could be due to the trials themselves [ 58 , 61 ] . regardless of the reason for the failure of clinical trials , the efficacy of these compounds to prevent hiv and other microbial infections in vitro make them attractive candidates for reducing the risk of viral transmission from infected semen when processed in vitro for art . ppcm could also be useful for preventing transmission of other infections that coexist in hiv - infected individuals , including hepatitis c . other polyanions are active in preventing binding of hepatitis c to target cells , possibly by antagonizing interactions with heparan sulfate , or other heparin - like moieties on the cell surface . under appropriate conditions ( i.e. , use of ca - deficient media , washing to remove compound after treatment prior to using ca - replete media ) , ppcm can effectively reduce hiv infectivity by several orders of magnitude without adversely affecting sperm function . although all data collected to date support the utility of ppcm as an antiviral prophylactic , additional work is clearly indicated to validate its use for art . successful validation and confirmation of lack of embryotoxicity of this method could create a new standard of care regarding the use of ppcm or other polyanionic microbicides to reduce infectivity of , or otherwise inactivate , one or more viruses ( including hiv ) that may be present in semen used in art . ppcm and other polyanionic compounds have broad - spectrum activities against a number of sexually transmitted pathogens ( see section 1 ) . our long - term goal will be to determine if ppcm can reduce the risk of transmission of one or more of several sexually transmitted microbes , in addition to hiv , when spermatozoa used for art are routinely treated in vitro with this agent as part of a washing procedure based on data presented in the present study . we examined the feasibility of improving the method of sperm washing used to reduce risk of hiv transmission during art by adding ppcm , a microbicide for reducing sexually transmitted infections . a method is described in which ppcm , with sperm washing , is 3 - 4 orders of magnitude more efficacious than washing alone , in reducing hiv infectivity , without affecting sperm .

hiv - serodiscordant couples wishing to conceive often seek assisted reproduction , during which spermatozoa from infected men are washed to minimize the risk of hiv transmission to partner and fetus . we sought to improve this method by adding a microbicide , ppcm , as an hiv prophylactic . hiv-1 ( bal ) inhibition by ppcm appears irreversible and independent of added ca2 + . without added ca2 + , ppcm ( 10 mg / ml , 90 min ) , a stimulus of ca2 + -dependent acrosomal loss , has no effect on sperm motility , forward progression , or acrosomal status . ppcm - treated ( 10 mg / ml ) sperm retain their ability to acrosome react when ca2 + is added . sperm dna integrity / function is unaffected by ppcm ( 10 mg / ml ) . adding ppcm ( 5 mg / ml , 30 min ) to washing media reduces infectivity ( viral antigen p24 and rna ) of ex - vivo hiv - infected semen by 3 - 4 logs compared with washing alone . sperm washing with appropriate extracellular ca2 + levels and ppcm is significantly more effective than washing alone at reducing hiv infectivity .

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

at least part of the reason for the trend toward stabilization of the aids epidemic and increased longevity of hiv - infected individuals can be attributed to the beneficial effect of antiretroviral therapy . however , hiv infection may impair fertility [ 24 ] , prompting hiv - infected men and women to seek medical assistance ( i.e. further , art is the method of choice for fertile serodiscordant couples trying to conceive ; it reduces the risk of hiv transmission associated with unprotected sexual intercourse . providing art to hiv - infected couples , particularly serodiscordant couples where the man is infected , is problematic for health care providers . sperm washing has been advocated as a method to reduce hiv transmission , in which motile spermatozoa are separated from seminal plasma , nonsperm cells , and hiv [ 9 , 10 ] . this was , in large part , the basis for california legislation that permits a sperm wash to minimize hiv transmission during art procedures for serodiscordant couples [ 13 , 14 ] . hiv binding , through alternative receptors or nonspecific mechanisms , may explain the failure of a sperm wash to completely remove hiv from infected semen in all instances . further , at least some proponents of sperm washing to reduce risk of hiv transmission during assisted reproduction advise against its use with intracytoplasmic sperm injection ( icsi ) . addition of a safe antiviral compound may offer some advantages over sperm washing alone in minimizing the risk of hiv transmission during art . ideally , an effective antiviral agent added at or around the time of sperm washing should result in the following : ( 1 ) processed sperm samples would contain no residual infectious hiv ; ( 2 ) icsi could be used as an assisted reproductive technique in hiv - serodiscordant couples with fewer concerns ; ( 3 ) treatment could be offered to a larger number of serodiscordant couples than possible with the conventional sperm wash protocol , which is biased in favor of very low or undetectable initial viral load [ 8 , 12 ] ; ( 4 ) the risk of viral infection of health care providers who are in direct contact with infected samples would be greatly reduced . many belong to a class of compounds known as polyanions , one of which is a poly - acidic oligomer , code - named ppcm ( formerly known as sulfuric acid - modified mandelic acid , or samma ) , developed [ 2732 ] by the topcad program at rush university medical center ( chicago , il ) and licensed by yaso biotechnologies ( phoenix , az ) . inhibition of hiv-1 ( bal ) by ppcm is at least partially irreversible and is independent of added extracellular ca . in the absence of added ca , sperm washing in the presence of ppcm substantially reduces ( 3 - 4 logs ) viral titer of ex - vivo hiv-1 ( bal)-treated whole human semen from healthy donors , as compared with washing alone . this occurs without negatively affecting measures of sperm dna integrity or sperm function , including motility , forward progression , acrosomal integrity , and dna decondensation . hiv-1 ( bal ; lot # 07283002 ) , an expanded laboratory strain , was from the virology quality assessment program ( vqa ) , rush university medical center and was provided by the clinical retrovirology research laboratory , department of immunology and microbiology , rush university medical center , under the direction of dr . semen samples used for hiv infectivity , sperm motility , and acrosomal status assessment were collected from pools of eight healthy donors ( rush university medical center ) and five fertile donors ( andromedix laboratory ) from whom informed consent was given , according to the protocols approved by their respective institutional review boards . initial mean ( with 90% confidence limits ) values for sperm count , % motility , % forward progression , and % viable spermatozoa were 83 ( 58.9117.8 ; n = 11 ) 10/ml , 69 ( 64.373.6 ; n = 12)% , 78 ( 72.082.9 ; n = 12)% and 90 ( 85.793.0 ; n = 12)% , respectively . results for sperm motility , forward progression and viability are presented as mean percentage of postwash control values , together with sem . no inference is made as to whether this response is identical to a physiological acrosome reaction , during which the acrosome is also lost . after incubations of washed spermatozoa with 10 mg / ml ppcm or with medium alone for 15 min at ambient temperature , samples were placed onto 1 ml of 11% ( wt / vol ) buffered ficoll ( containing 120 mm nacl and 25 mm hepes , ph 7.4 ) , and centrifuged at 15,000 g - min ( 22c to 24c ) . sperm pellets were resuspended in 1.0 ml of nim ( solvent for ppcm ) , prior to the sdfa , or analysis in the sdd test as described by brown et al . treated samplessperm pellets were resuspended in 1.0 ml of nim containing ppcm ( 2 mg / ml or 10 mg / ml , final concentrations ) . sperm pellets were resuspended in 1.0 ml of nim containing ppcm ( 2 mg / ml or 10 mg / ml , final concentrations ) . ppcm at several concentrations ranging from 0.1 to 20 mg / ml was added to hiv-1 ( bal ) suspensions ( 5.9 10 tcid50s / ml ) . after 1 h at ambient temperature , suspensions were diluted 1 : 100 with rpmi ( with or without added ca ) , prior to inoculation into peripheral blood mononuclear cell ( pbmc ) suspensions ( 2.0 10 cells in 0.1 ml ) . after 1 h , pbmcs were washed with fresh medium ( containing added ca ) to remove unbound virus and ppcm , and the pbmc suspensions were incubated for 72 hours at 37c to allow for viral replication . the concentration of ppcm required for 50% inhibition of p24 production ( ic50 value , obtained from curves fit to the data with tablecurve 2d ; see statistical analysis ) when ppcm was added to complete rpmi was compared with that obtained when ppcm was added to medium without added ca . inhibition of viral replication by ppcm was compared under two conditions , as follows : ( 1 ) ppcm at concentrations up to 13 mg / ml was added to only hiv-1 ( bal ) , and incubated for 1 h , followed by a 1 : 100 dilution in rpmi , prior to the suspension being inoculated onto pbmcs . ( 2 ) ppcm at concentrations up to 1 mg / ml was added to viral suspensions , incubated for 1 h , and inoculated onto pbmcs that contained the same concentrations of ppcm . neat fresh semen pooled from five donors ( 11.3 ml ) was added to an equal volume of hiv-1 ( bal ) inoculum ( 5 10 tcid50s / ml ) . after 1 h , pbmcs were washed to remove unbound virus , spermatozoa and ppcm , and incubated for 72 h in ca - replete rpmi to permit viral replication . for data requiring either logarithmic ( sperm count ) or arcsine transformation ( frequencies ; initial sperm motility , forward progression , viability ; acrosomal loss ) , results are expressed as means , with 90% confidence limits . dose responses of hiv infectivity as a function of ppcm concentration ( from 1 10 to 6.7 mg / ml ) were measured in the presence and absence of added ca . dose response of hiv infectivity as a function of initial ppcm concentration ( from 1 10 to 13.3 mg / ml ) was measured in samples in which ppcm concentrations were maintained throughout the experimental procedure ( viral incubation , inoculation onto pbmcs , wash to remove free virus and incubation of infected pbmcs to permit viral replication ) . this was compared with the dose - response measured in samples in which ppcm - treated virus was first diluted 1 : 100 prior to inoculation onto pbmcs . dilution and subsequent removal of ppcm by washing has essentially no effect on the ability of ppcm to inhibit hiv infectivity ( figure 2 ) . when the initial concentration of ppcm is maintained throughout all procedures ( no dilution ) , the ic50 ( 33.766.3% inhibition ) for ppcm is 23 g / ml . sperm exposure to ppcm at concentrations up to 10 mg / ml for periods up to 90 min has little effect on sperm motility , forward progression or viability ( figures 3(a)3(c ) ) . ; similarly , there is no effect of ppcm concentration on sperm viability ( f(2,30 ) = 1.67 , p > .10 ) , although a small effect ( 11% reduction at 90 min exposure to 10 mg / ml ppcm ) of duration may exist ( f(4,30 ) = 5.03 , p < .01 ) . acrosomal status of sperm suspensions treated with 10 mg / ml ppcm for 15 min in bww medium without added ca is similar to control spermatozoa ( no ppcm treatment ) similarly processed , when spermatozoa from both groups are subsequently washed and added to ca - replete medium ( detailed protocol presented in section 2 ) . percentage of ppcm - treated spermatozoa without acrosomes is 19 ( 90% confidence limits = 18.620.0)% . acrosomal loss increases to a similar extent in both ppcm - treated and control samples in response to stimuli ( p > .10 , newman - keuls multiple range test ) . percentage of spermatozoa without acrosomes in response to 0.25 g / ml ppcm ( concentration required to induce 50% maximal acrosomal loss ) is 27 ( 26.327.6)% and 27 ( 26.627.7)% for ppcm - pretreated and control spermatozoa , respectively ( figure 4 ) . pretreatment of spermatozoa with either 2 mg / ml or 10 mg / ml ppcm had no effect on sperm dna fragmentation ( figure 5(a ) ) . mean dfi for negative control spermatozoa , spermatozoa treated with 2 mg / ml ppcm and spermatozoa treated with 10 mg / ml ppcm are 6 ( 90% confidence limits = 4.56.6 ) , 7 ( 2.912.4 ) and 9 ( 6.113.3 ) , respectively . mean percentage of control spermatozoa , spermatozoa pretreated with 2 mg / ml ppcm , and spermatozoa pretreated with 10 mg / ml ppcm that showed full decondensation in response to egg extract are 96 ( 95.097.0)% , 98 ( 87.299.5)% , and 96 ( 95.596.9)% , respectively ( figure 5(b ) ) . following incubation with hiv-1 ( bal ) for 30 min , whole semen was treated with either 5 mg / ml ppcm or with an equal volume of medium ( without added ca ) , and incubated at ambient temperature for 45 min ( see section 2 for complete description of experimental protocol ) . ppcm further reduces viral p24 levels by approximately 3 logs , ( 99.94% reduction , to 0.04 0.03 ( sem ) ng / ml ) as compared with samples subjected to only centrifugation . greater reduction of viral titer by ppcm is obtained when titer is expressed as viral rna ( figure 6(b ) ) . similar to p24 concentrations , gradient centrifugation followed by washing ( without ppcm ) reduces viral rna concentration by 92% ( to 4.91 0.055 10 copies / ml ) . viral rna is further reduced by approximately 4.4 logs ( to 20 3 copies / ml ) by pretreatment with ppcm . reductions in viral titer from that observed in the unprocessed , hiv - incubated semen produced by either sperm washing or ppcm pretreatment followed by sperm washing are significant ( p < .01 , newman - keuls multiple range test ) . the present data support the utility of ppcm as an effective compound for use in art to further reduce the risk of hiv transmission . contraceptive activity of ppcm is believed to be due to its ability to induce premature acrosomal loss ; this effect requires physiological concentrations of extracellular ca . the present study has shown that the dose - response of ppcm - induced inhibition of hiv infectivity is independent of added ca ( figure 1 ) . ideally , inhibition of hiv by ppcm would be irreversible , or at least pseudoirreversible . ppcm - induced viral inhibition is at least partially irreversible , since the dose - response of hiv inhibition is unaffected by a 1 : 100 dilution of ppcm after initial interaction with the virus ( figure 2 ) . the work of mesquita and coworkers suggests possible irreversible inhibition of hiv-1 ( bal ) and hiv-1 ( rf ) infectivity after pretreatment of immobilized virus with ppcm followed by washing , further , a structural variant of ppcm inhibits hiv-1 ( iiib ) and hiv-1 ( bal ) in an irreversible or pseudoirreversible manner . high concentrations of ppcm ( 25 mg / ml ) are relatively noncytotoxic toward spermatozoa , as measured by a lack of effect on sperm motility following short - term exposure ( sander - cramer test for spermicidal activity ) . we have confirmed and extended these studies by measuring sperm motility , forward progression and sperm viability after exposure to ppcm at concentrations as high as 10 mg / ml for periods up to 90 min ( figure 3 ) . no effect was observed on sperm motility or forward progression . earlier studies showing ppcm - induced acrosomal loss as a ca - dependent process , dependent upon physiological concentrations of extracellular ca ( ed50 approx . the present study examined whether these observations could be extended to ppcm when used at concentrations several orders of magnitude higher ( mg / ml range ) . spermatozoa in ca - deficient medium were pretreated with a high ( 10 mg / ml ) concentration of ppcm , washed to reduce ppcm to nondetectable levels , and resuspended in ca - replete medium . under these conditions , further , similar to that of control samples , ppcm - treated spermatozoa respond to acrosomal loss stimuli , namely calcium ionophore a23187 and ppcm ( figure 4 ) . as a stimulus of acrosomal loss ( in the presence of added extracellular ca ) , ppcm was used at a concentration ( 0.25 g / ml ) predicted to induce 50% maximal acrosomal loss . the data suggest that under appropriate conditions , prior treatment with relatively high concentrations of ppcm is without effect on acrosomal status . in order for ppcm - treated sperm to be used in art procedures , it is important to establish that ppcm does not negatively affect sperm dna integrity and/or sperm functions required for postfertilization restructuring of the sperm nucleus , once inside the egg . to test this , ppcm - treated sperm were analyzed with two tests : the sperm dna fragmentation assay ( sdfa)/sperm chromatin structure assay ( scsa ) , a sperm structure assay used to measure dna integrity ; and the sperm dna decondensation ( sdd ) test ; a sperm function test used to measure overall sperm nuclear integrity . normal scores in ppcm - treated sperm for both tests support the use of ppcm - washed sperm in art . ppcm - induced interference with protamine - dna interaction would be reflected as highly fragmented dna , and/or delayed sperm dna decondensation / nuclear swelling , as detected with the sdfa and sdd test , respectively . however , ppcm ( 10 mg / ml ) has no effect on either sperm dna fragmentation ( figure 5(a ) ) or dna decondensation ( figure 5(b ) ) . further , they support the safety of using ppcm - treated washed spermatozoa in art to reduce risk of hiv infection . infected semen was simulated by incubating uninfected semen with a high titer of hiv-1 ( bal ) . this , rather than semen from hiv - infected men , was used to permit sufficiently high viral titers ( particularly after washing by gradient centrifugation ) to accurately quantify efficacy of ppcm combined with sperm washing beyond that effected by washing alone . with this method , sperm washing by gradient and differential centrifugation reduced initial viral titer by somewhat over 90% , whether measured by viral p24 or rna levels ( figures 6(a ) and 6(b ) ) . the method of sperm processing in the present study is similar to that used clinically by pea and coworkers to reduce viral titers in semen from hiv - infected men . concentration of ppcm , for example , is reduced from 10 mg / ml to less than 0.1 g / ml ( limit of detection ) . levels of p24 in samples treated with ppcm , followed by washing by gradient and differential centrifugations were reduced by 3 logs , as compared with p24 levels measured after washing alone ( figure 6(a ) ) . reduction of viral rna ( > 4 logs , as compared with washing alone ) was greater than that of p24 ; rna levels in the presence of ppcm were near the limit of detection of the assay . increased efficacy of ppcm when measured by viral rna as compared with p24 measurements is consistent with mechanisms of inhibition of hiv infectivity in addition to inhibition of viral binding to target cells . although these studies were restricted to a laboratory strain ( bal ) , results can likely be extended to other strains as well as clinical isolates , since ppcm is active against several hiv strains and isolates [ 28 , 29 , 31 , 33 ] . whether interaction of hiv with spermatozoa in vitro is similar to additional work is required to demonstrate efficacy of ppcm against sperm - associated hiv from hiv - infected men . regardless of the reason for the failure of clinical trials , the efficacy of these compounds to prevent hiv and other microbial infections in vitro make them attractive candidates for reducing the risk of viral transmission from infected semen when processed in vitro for art . ppcm could also be useful for preventing transmission of other infections that coexist in hiv - infected individuals , including hepatitis c . , use of ca - deficient media , washing to remove compound after treatment prior to using ca - replete media ) , ppcm can effectively reduce hiv infectivity by several orders of magnitude without adversely affecting sperm function . we examined the feasibility of improving the method of sperm washing used to reduce risk of hiv transmission during art by adding ppcm , a microbicide for reducing sexually transmitted infections . a method is described in which ppcm , with sperm washing , is 3 - 4 orders of magnitude more efficacious than washing alone , in reducing hiv infectivity , without affecting sperm .

sulfur speciation in aerosol particles plays a crucial role in both earth 's energy balance and acid deposition [ 15 ] . numerous efforts have been made to deepen insight into and clarify the oxidation pathways of sulfate in aerosol samples [ 6 , 7 ] . there is a significant precedent in the use of x - ray absorption near edge structures ( xanes ) to assess the presence of sulfur in particulate matter . over the past decade , several reference publications have indicated that the most stable oxidation state of sulfur in aerosol is + 6 ( s(vi ) ) [ 912 ] . these findings were supported by recent experimental data suggesting that ( nh4)2so4 and caso4 - 2(h2o ) are dominant sulfur species in aerosols . despite of these facts , there are several outstanding questions and information gaps which merit additional study for clarification . such is the case for investigating the role of emission source , diurnal variation , and vertical distribution on sulfur oxidation states and on sulfur k - edge xanes spectra in pm10 . since the conversion of gaseous so2 to particulate so4 can dramatically alter the sulfur oxidation state , all ambient sulfur species are required to preserve in anoxic condition due to its relatively high oxidation rate as previously discussed in several publications [ 1416 ] . despite the spatially dynamic movement of aerosols , the majority of studies so far have concentrated only on sulfur speciation as a result of particle size segregation at ground level . up to now , research was focused predominantly on defining sulfur k - edge xanes spectrum of aerosol samples collected from monitoring sites classified either urban or remote / rural . furthermore , the samples have always been collected as close to the source as possible , while measurement of sulfur oxidation states in samples taken from sites adjacent to the emission sources have been largely neglected . neither have there been studies examining sulfur speciation in pm10 as a function of altitude . the answer of how emission source and proximity and vertical distribution affect sulfur oxidation states in aerosols offers crucial insight for understanding aerosol behavior and pollutant transport , with ultimate implications for mitigation and policy . thus , the overall aims of this study areto qualitatively investigate the impact of diurnal variation on sulfur k - edge xanes spectra of pm10 collected at ground level , to analyze sulfur k - edge xanes spectra in pm10 samples collected adjacent to various emission sources representative of biomass burning , crude oil combustion , diesel emission , timber burning and traffic exhaust in songkla province , southern thailand , andto conduct a qualitative investigation of the altitude effect on sulfur k - edge xanes spectra in pm10 samples collected at urban sites throughout bangkok , chiang - mai and hat - yai , thailand . to qualitatively investigate the impact of diurnal variation on sulfur k - edge xanes spectra of pm10 collected at ground level , to analyze sulfur k - edge xanes spectra in pm10 samples collected adjacent to various emission sources representative of biomass burning , crude oil combustion , diesel emission , timber burning and traffic exhaust in songkla province , southern thailand , and to conduct a qualitative investigation of the altitude effect on sulfur k - edge xanes spectra in pm10 samples collected at urban sites throughout bangkok , chiang - mai and hat - yai , thailand . xanes was performed on the samples , and standards were measured at beam line no . 8 in the siam photon laboratory . photon energy from an x - ray beam transported through the beam line was scanned by insb(111 ) double crystals installed in the monochromator ( see figure 1 ) . the beam size illuminating the sample was 10 mm ( w ) 1 mm ( h ) . figure 2 shows the apparatus setup to measure fluorescent yield from the sample . a 10-cm - long ion chamber filled with a gas mixture of n2 ( 30 mbar ) and he ( 983 mbar ) was employed to measure intensity of the x - ray beam before the sample . the ion chamber absorbed only 10% of the beam intensity through ionization of the gas filling and produced a small electrical current signal proportional to the beam intensity . fluorescence x - rays were emitted by the sample and recorded using a 13-channel germanium detector ( ged ) or a lytle detector ( lyd ) . the ged has an advantage over the lyd in its ability to distinguish x - ray fluorescence energy . with the use of a digital window , similar to the ion chamber , the lyd produces small electrical current signal corresponding not only to fluorescence x - rays by the sulfur present , but also for other elements present in the sample and scattered photons from the primary beam . the sample chamber was flooded with helium gas to reduce x - ray absorption and scattering by air . a thin polypropylene window was used to protect the ged from helium gas , which can diffuse through thin detector seal of beryllium spoiling cryostat vacuum of the detector . the distances from the sample to detector were 7 mm and 9 mm for ged and lyd , respectively . xanes spectra were recorded from 2450 ev to 2520 ev with the energy step of 0.2 ev and energy calibrated using the maximum absorption of iron sulfate at 2481.4 ev . absorbance was calculated by the ratio of photon counts from the fluorescent detector to the ion chamber . all qff samples were applied to the adhesive side of the polyimide tape and placed at a 45 angle to the beam path in the sample chamber . without dilution , similar approach was used for the preparation of environmental samples . the data processing and quantitative xanes analyses were conducted using the athena program in the ifeffit computer package . ambient air samples were collected by using a graseby - anderson high volume air sampler with tsp and pm10 te-6001 , operating at a flow rate of 1.4 m min . tsp and pm10 samples were collected on 47 mm whatman quartz microfiber filters ( qmfs ) . the exposed filters were stored in a refrigerator at about 4c until sulfur speciation analysis to prevent the evaporation of volatile compounds . both the field sampling and weighing of filters were performed in compliance with the us epa 's guideline standard operating procedure for sampling and handling of pm2.5 filters . note that all filters were weighed by mettler toledo ab204-s analytical balance before being sent to synchrotron light research institute ( public organization ) , thailand . pm10 samples were collected every three hours over a 27-hour period ( i.e. , n = 9 , 18.0021.00 , 21.0000.00 , 00.0003.00 , 03.0006.00 , 06.0009.00 , 09.0012.00 , 12.0015.00 , 15.0018.00 , and 18.0021.00 ) at the national institute of development administration ( nida ) , thailand 's most prestigious graduate university under the commission for higher education , the ministry of education in bangkok . diurnal variation of urban baseline samples was taken in an open field located at the south - western side of nida 's bangkapi campus , close to sereethai road , one of the most heavily used roads in this area . since the monitoring site is only 1 km away from the mall shopping center bangkapi , tesco - lotus and makro stores , it seems reasonable to assume that pm10 collected from this site represent aerosols derived from traffic vehicle exhausts . as previously mentioned , all samples were gathered on whatman qm - a - type quartz fiber filters ( qffs ) , and carefully preserved inside a pressure controlled glove box ( pcgb ) purged with nitrogen gas to avoid conversion of gaseous so2 to particulate so4 . all filters were installed inside an anoxic preservation stainless cylinder ( apsc ) filled with nitrogen gas ( see figures 3 and 4 ) and carefully delivered to synchrotron light research institute ( slri ) for sulfur k - edge xanes spectra analysis . sampling was conducted over three consecutive days at ( prince of songkla university ( psu ) , hat - yai campus ) , traffic intersection ( ti ) , corps incinerator ( ci ) , charoen phokphand factory ( cpf ) , songkla lake ( sl ) , rubber manufacturing factory ( rmf ) , bus terminal ( bt ) , waste incinerator ( wi ) , barbeque festival ( bf ) , petkrasem road ( pr ) and kor hong hill ( khh ) stations . the air samples collected at bb and ptb stations represent the most important in terms of air quality . therefore , 3-hour samples were collected three times per day in order to avoid any overloading of air particulate matter at these three stations . all monitoring sites are located in songkhla province , which is situated 950 km south of bangkok with a population of over 1.32 million people . thirteen sampling locations were carefully selected to reflect the complex urban environment a mixture of commercial , residential , and industrial estates that are present in this province . prince of songkla university ( psu)the site is situated about 3 m above ground level in the faculty of environmental management , prince of songkla university , and about 550 m away from the main road that leads to the city center of hat - yai . it is important to note that psu represents the sampling period of october ( 24th26th october , 2007 ) . the site is situated about 3 m above ground level in the faculty of environmental management , prince of songkla university , and about 550 m away from the main road that leads to the city center of hat - yai . it is important to note that psu represents the sampling period of october ( 24th26th october , 2007 ) . songkhla lake monitoring station ( sl)it is situated about 13 km from the northern side of prince of songkla university , south of songkhla lake , and about 14 km from the western side of the gulf of thailand . this sampling station is far from any industrial and traffic emission sources , including chemical and metallurgy , and power plants in this district . songkla 1 ( sl1 ) and songkla 2 ( sl2 ) were used to represent the monitoring periods of july ( from 27th to 29th july , 2007 ) and october ( from 20th to 22nd october , 2007 ) respectively . it is situated about 13 km from the northern side of prince of songkla university , south of songkhla lake , and about 14 km from the western side of the gulf of thailand . this sampling station is far from any industrial and traffic emission sources , including chemical and metallurgy , and power plants in this district . songkla 1 ( sl1 ) and songkla 2 ( sl2 ) were used to represent the monitoring periods of july ( from 27th to 29th july , 2007 ) and october ( from 20th to 22nd october , 2007 ) respectively . korhong hill monitoring stations ( khh ) it is located on the top of kor - hong hill with an elevation of 356 meters . this site represents a mixture of all emission sources in the urban area . it is located on the top of kor - hong hill with an elevation of 356 meters . cluster iithis group contains sampling sites which are categorized as a source of diesel and benzene emissions . this group contains sampling sites which are categorized as a source of diesel and benzene emissions . bus terminal ( bt)this site is located at the south - western side of psu and approximately 1.4 km from the campus . as the majority of buses are diesel fuelled in this area , we considered this site a source of diesel emissions . this site is located at the south - western side of psu and approximately 1.4 km from the campus . as the majority of buses are diesel fuelled in this area , we considered this site a source of diesel emissions . petkrasam road ( pr)this site was located in the heart of the city of hat - yai . this site faces the heaviest burden of traffic congestion with a mixture of diesel and gasoline exhaust emissions . thus , the air mass collected from this area is mainly contaminated with diesel and benzene emissions . this site faces the heaviest burden of traffic congestion with a mixture of diesel and gasoline exhaust emissions . thus , the air mass collected from this area is mainly contaminated with diesel and benzene emissions . traffic intersection ( ti)this station is located at the traffic intersection in front of the main gate of psu near a tesco supermarket . it is on the eastern side and approximately 2.5 km from the hat - yai city center close to an urban residential zone . this station is regarded as a heavy traffic area and mainly contaminated with diesel and benzene emissions . this station is located at the traffic intersection in front of the main gate of psu near a tesco supermarket . it is on the eastern side and approximately 2.5 km from the hat - yai city center close to an urban residential zone . this station is regarded as a heavy traffic area and mainly contaminated with diesel and benzene emissions . charoen phokphand factory ( cp)this site is located at the cp fish cannery . this factory is part of the largest business conglomerate in thailand . rubber sheet manufacturing factory ( rmf1 and rmf2)this station is located at tumbol tungwan , hat - yai district . the rubber sheets are treated with high - temperature and high - pressure steam and then purified with sulfuric acid solution . as para rubber trees are used as fuel for this process , we therefore considered it a source of mixed para rubber tree burning emissions , with latex particles and sulfuric acid aerosols also expected . the air samples were collected from 30th july to 1st august 2007 ( rmf1 ) and from 2nd to 4th august 2007 ( rmf2 ) . the rubber sheets are treated with high - temperature and high - pressure steam and then purified with sulfuric acid solution . as para rubber trees are used as fuel for this process , we therefore considered it a source of mixed para rubber tree burning emissions , with latex particles and sulfuric acid aerosols also expected . the air samples were collected from 30th july to 1st august 2007 ( rmf1 ) and from 2nd to 4th august 2007 ( rmf2 ) . para rubber tree burning ( ptb)this station is located at namom district , songkhla province and can be recognized as a source of para rubber tree burning emissions . the air samples were collected on 18th november , 2007 . this station is located at namom district , songkhla province and can be recognized as a source of para rubber tree burning emissions . biomass burning ( bb)this station is located in a rice field in satingpra district , songkhla province . removing rice straw left after the crop is harvested by burning can limit disease organisms but leads to serious air pollution problems in this region . removing rice straw left after the crop is harvested by burning can limit disease organisms but leads to serious air pollution problems in this region . cluster vthis cluster is classified as old tire , clinical waste and charcoal burning sites . corpse incinerator ( ci)this station is a part of kor - hong temple , located on the northern side and about 1.5 km from ti . since timber and tires are generally used as fuel for corpse incineration , this site is considered an emission source of both timber and tire burning . this station is a part of kor - hong temple , located on the northern side and about 1.5 km from ti . since timber and tires are generally used as fuel for corpse incineration , this site is considered an emission source of both timber and tire burning . waste incinerator ( wi)this site is situated in the city center and belongs to the municipality of the city of hat - yai . since the municipal waste incinerated is a heterogeneous mixture of solid waste and burnt fuels , this site can be recognized as a source of a combination of solid waste burning and diesel exhaust emissions . this site is situated in the city center and belongs to the municipality of the city of hat - yai . since the municipal waste incinerated is a heterogeneous mixture of solid waste and burnt fuels , this site can be recognized as a source of a combination of solid waste burning and diesel exhaust emissions . barbeque festival ( bf)this site is located on the psu campus on the top roof of the faculty of natural resources building . the barbeque festival has become an annual tradition that is held on the second week in august . additionally , it is also important to emphasize that there were no obstructions in the vicinity of the sampling equipment at all sites , which were strategically positioned to be accessible to winds from all directions . this site is located on the psu campus on the top roof of the faculty of natural resources building . the barbeque festival has become an annual tradition that is held on the second week in august . the air samples were collected from 15th to18th august , 2007 . additionally , it is also important to emphasize that there were no obstructions in the vicinity of the sampling equipment at all sites , which were strategically positioned to be accessible to winds from all directions . all three sampling sites are located in the city centers of bangkok , chiang - mai and hat - yai , representing the country 's capital and largest city alongside the largest cities in the northern and southern regions of thailand , respectively . air samples were collected at three different heights for three hour periods ( i.e. , 9.00 am12.00 am ) at bai - yoke suite hotel observatory site ( i.e. , level-1 : 38 m ; level-2 : 158 m ; level-3 : 328 m above ground level ) , at centara duangtawan hotel observatory site ( i.e. , level-1 : 12 m ; level-2 : 52 m ; level-3 : 152 m above ground level ) and at novotel centara hat - yai hotel observatory site ( i.e. , level-1 : 30 m ; level-2 : 60 m ; level-3 : 125 m above ground level ) as monitoring sites for bangkok , chiang - mai and hat - yai in that order ( see table 1 ) . while traffic emissions are considered the main source of air pollutants in the bangkok atmosphere , agricultural burning is the major air pollution problem in chiang - mai . a high concentration of traffic emission residue is expected for bangkok samples , while samples from chiang mai will likely contain evidence of industrial emissions and wood burning . hat - yai is located only 30 km from the west coast of the gulf of thailand , so it is likely that aerosol samples from this site will contain traces of sea salt and biomass burning . figure 5 illustrates the diurnal variation of nine sulfur k - edge xanes spectra of pm10 preserved in apsc and pcgb filled with nitrogen gas prior to xanes analysis at slri . there are three features which are common to each sulfur k - edge xanes spectrum : ( a ) the pre - edge region , which represents the energy emitted at 24502465 ev and points to the area of energies lower than the main absorption edge ; ( b ) the near - edge region , which refers to the 5 ev wide region around the white - line ( i.e. , the main peak ) , located at the post - edge region , which denotes the region greater than 2490 ev outside the absorption edge . based on the results from nine sulfur k - edge xanes spectra pm10 samples all seem to show a relatively flat spectrum of preedge region ( i.e. , 24502650 ev ) , followed by a sharp main peak at 2482 ev , and a large postedge fluctuation that arises in the energy band of 24902510 ev ( see figure 5 ) . these results are consistent with previous reports focusing on sulfur k - edge xanes spectra of aerosols with different particle sizes [ 1113 ] . conclusion that particulate sulfur compounds are dominated by s(vi ) , it can be deduced that the oxidation number of elemental sulfur collected at nida bangkapi campus is also + 6 , and plausibly occupied by sulfate species . figure 6 demonstrates the sulfur k - edge xanes spectra of 15 pm10 samples collected from various emission sources in songkla province . similar to the previous sulfur k - edge xanes spectra detected at nida bangkapi campus , the spectra from bt , pr , tesco , rmf , and cpf display a broad resonance at the preedge region , probably due to a superimposition of more than one resonance . for all 15 spectra , a maximum peak appears at the higher energy of 2482 ev , which is consistent with peaks belonging to diurnal variation samples . despite several deviations in the intensities of the maximum peak between the different emission source samples , some other subtle dissimilarities in post - edge regions are apparent , particularly in the subsequent structure , which appears the post - edge region between 2485 and 2510 ev . previous studies interpreted this phenomenon as a consequence of electron transitions with a d - type shape resonance , which can be influenced by various types of cat ions binding with sulfates [ 20 , 21 ] . these discrepancies among different sulfur k - edge xanes spectra can be taken to characterize the finger print of target aerosols associated with various emission baselines . figure 8 underscores the dominance of the sulfate species in urban aerosols by displaying the relatively similar sulfur k - edge xanes spectra of pm10 collected at different heights for comparison to diurnal variation the analogous spectral features were observed in pm10 collected at chiang - mai , bangkok and hat - yai , irrespective of differences in sampling height . based on the positive correlation between t sulfur and photon energy , it appears sensible to consider the highest peak structures at 2482 ev as an outcome of the majority of s(vi ) recorded in urban pm10 . it is also plausible to assume that ammonium sulfate and gypsum are prime candidates based on the systematics of this region as mentioned in previous study [ 8 , 11 , 20 ] . in addition , earlier studies revealed that the molecular structure has little effects 1s 3p transition energy of that specific sulfur functional group , thus highlighting the usefulness of xanes to categorize the oxidation number of elemental sulfur in pm10 [ 2224 ] . to investigate the anoxic preservation effect on sulfur k - edge xanes spectra of pm10 , qff samples were preserved in apsc filled with nitrogen gas immediately after completing collections . it is also important to note that all filter - cutting processes were conducted inside pcgb filled with nitrogen gas prior to xanes analysis . the sulfur k - edge xanes spectra of pm10 samples collected at different periods demonstrated that the position of the main peak was relatively consistent at 2482 ev without any sign of peak shift within 0.5 ev for numerous aerosol samples , indicating that particulate sulfur compositions are not influenced by diurnal variation . the moderately uniform sulfur k - edge xanes spectra suggest that the overall sulfate species distribution is constant at the traffic site . this finding is consistent with similar works performed by takahashi et al . , indicating that anoxic preservation might have minor importance for the oscillation of sulfur k - edge xanes spectra in pm10 [ 1113 ] . firstly , all sulfur compounds from emission sources in urban atmospheric environment are composed of s(vi ) and conceivably predominated by sulfate species . secondly , the oxidation rate of aerosol sulfur is too fast to convert all particulate sulfur compounds to the most stable oxidation state of + 6 before being transported to the receptor site at nida bangkapi campus . thirdly , all sulfur compounds were completely oxidized to s(vi ) after the deposition of particles on qffs within three hours of sampling period . interestingly , this finding is in good agreement with an earlier study , which applied xanes for the determination and quantification of s(iv ) species in aerosol samples collected in qingdao , northeastern china . higashi et al . concluded that sulfite was the only sulfur which possessed the oxidation number of + 4 detected in aerosols , particularly in particles with larger diameters collected in august 2001 . it is also crucial to stress that no s(iv ) species , however , were detected at the surface of the aerosols as demonstrated by surface - sensitive conversion electron / he ion yield xanes . in the case of pm10 collected at nida bangkapi campus , the moderately high reactive trace gas ( e.g. , nox , o3 , and oh radical ) concentration released from diesel engines and other traffic combustions could oxidize sulfur compounds and conclusively convert all of the sulfur oxidation states to s(vi ) . in addition , the relatively low absorption intensity of sulfur k - edge xanes spectrum of pm10 collected from 12.00 am to 15.00 pm can be described as a consequence of the dilution effect triggered by the lifting of warm air mass at midday ( see figure 5 ) . unlike the results of diurnal variation campaign , the comparatively oscillating post - edge regions were discernible and thus highlighted the intricacy of sulfate compositions in pm10 collected from various emission sources ( figure 6 ) . likewise , the white - line intensity fluctuations are more noticeable in emission source spectra . these unique spectra features can be diagnosed as a divergence in both amount and speciation of s detected in pm10 . interestingly , the unsmooth pre - edge regions were predominant in sulfur k - edge xanes spectra of ci , wi , cpf , psu , bb , and ptb ( figure 6 ) . this can be ascribed to both the mixture of source signals ( i.e. , more variety of sulfur oxidation states ) and the relatively low signal - to - noise ratio of s(vi ) particularly in case of ptb and bb ( i.e. , low white line intensities due to high noise baselines ) . it is also worth mentioning that different sulfur compounds possess distinctive energy white line positions . for example , elemental s , organic polysulfide , thiol , sulfoxide , sulfite , sulfone , sulfonate , ester sulfate , and inorganic sulfate display the peak energy of sulfur k - edge white lines at 2472.5 , 2473.0 , 2473.4 , 2475.8 , 2478.7 , 2480.2 , 2481.3 , 2482.5 , and 2482.5 ev , respectively . therefore , the strong oscillation at the pre - edge reveals the crucial information about various sulfur species in target samples . a further investigation of sulfur k - edge xanes spectral features was conducted for baseline samples ( i.e. , cluster i ) . both sl1 and sl2 showed a considerably high level of reproducibility of pre - edges from 2470 ev to 2480 ev regardless of different sampling periods ( see figures 6 and 7 ) . this pattern of behavior supports the idea that both anthropogenic emissions and seasonal effects played a minor role at this monitoring station due to its distance from the urban district of hat - yai city combined with the dilution effect triggered by clean maritime air mass coming in from the gulf of thailand . in contrast , the spectral plateau in the region of 2473 ev2476 ev detected in wi illustrates an exceptional feature , which can not be explained by the differences in climatic conditions due to the relatively similar sampling period and location ( see figure 7 and table 1 ) . the most convincing explanation , therefore , plausibly attributed this difference to the complex nature of solid waste materials combusted in wi . in addition , the comparatively analogous spectral features of psu , ptb , rmf1 , and rmf2 indicated that para rubber tree combustion might play a major role in governing pm10 concentrations in residential zones , and thus raising concerns about the adverse impacts of para rubber industry on air quality in the city of hat - yai . to the best of our knowledge , this is the first study to examine sulfur k - edge xanes spectra of pm10 at different altitudes under same urban atmospheric conditions . all pm10 collected from three different cities displayed moderately similar spectral features in comparison with those of diurnal variation samples monitored at nida bangkapi campus as illustrated in figure 5 . the relatively high daytime air temperature may be responsible for the upwelling of particles , which can be triggered by the convective heat transfer of air mass from surface boundary layer ( sbl ) to urban boundary layer ( ubl ) . this phenomenon allowed air mass , which is relatively homogeneous vertically , to dominate the urban boundary layer in bangkok , chiang - mai and hat - yai during the observations . despite differences in both all spectral features were consistently identical with the principal composition of s(vi ) as illustrated in figure 8 . several concerns were addressed with respect to factors affecting the sulfur k - edge xanes spectra of pm10 collected from various locations with different sampling altitudes . there are three common features that appear across all sulfur k - edge xanes spectra : ( a ) the pre - edge region , involving the energy of 24502465 ev , which signifies the area at energies lower than the main point of the absorption edge , ( b ) the near - edge region , which refers to the 5 ev - wide region immediately encompassing the white - line region ( i.e. , the main peak ) , which is located at 2482 ev , and ( c ) the post - edge region , which denotes the region over 2490 ev , outside the absorption edge . neither anoxic preservation nor emission source appreciably changed this pattern . further investigation was conducted to assess the alteration of particulate sulfur k - edge xanes spectra as a function of sampling altitude . interestingly , comparatively uniform sulfur k - edge xanes spectra were observed in all pm10 collected from different cities and sampling periods . this study also ascertains that the sulfur oxidation state of pm10 is predominantly overwhelmed by s(vi ) .

this paper focuses on providing new results relating to the impacts of diurnal variation , vertical distribution , and emission source on sulfur k - edge xanes spectrum of aerosol samples . all aerosol samples used in the diurnal variation experiment were preserved using anoxic preservation stainless cylinders ( apscs ) and pressure - controlled glove boxes ( pcgbs ) , which were specially designed to prevent oxidation of the sulfur states in pm10 . further investigation of sulfur k - edge xanes spectra revealed that pm10 samples were dominated by s(vi ) , even when preserved in anoxic conditions . the emission source effect on the sulfur oxidation state of pm10 was examined by comparing sulfur k - edge xanes spectra collected from various emission sources in southern thailand , while vertical distribution effects on the sulfur oxidation state of pm10 were made with samples collected from three different altitudes from rooftops of the highest buildings in three major cities in thailand . the analytical results have demonstrated that neither emission source nor vertical distribution appreciably contribute to the characteristic fingerprint of sulfur k - edge xanes spectrum in pm10 .

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

there is a significant precedent in the use of x - ray absorption near edge structures ( xanes ) to assess the presence of sulfur in particulate matter . over the past decade , several reference publications have indicated that the most stable oxidation state of sulfur in aerosol is + 6 ( s(vi ) ) [ 912 ] . such is the case for investigating the role of emission source , diurnal variation , and vertical distribution on sulfur oxidation states and on sulfur k - edge xanes spectra in pm10 . since the conversion of gaseous so2 to particulate so4 can dramatically alter the sulfur oxidation state , all ambient sulfur species are required to preserve in anoxic condition due to its relatively high oxidation rate as previously discussed in several publications [ 1416 ] . up to now , research was focused predominantly on defining sulfur k - edge xanes spectrum of aerosol samples collected from monitoring sites classified either urban or remote / rural . furthermore , the samples have always been collected as close to the source as possible , while measurement of sulfur oxidation states in samples taken from sites adjacent to the emission sources have been largely neglected . the answer of how emission source and proximity and vertical distribution affect sulfur oxidation states in aerosols offers crucial insight for understanding aerosol behavior and pollutant transport , with ultimate implications for mitigation and policy . thus , the overall aims of this study areto qualitatively investigate the impact of diurnal variation on sulfur k - edge xanes spectra of pm10 collected at ground level , to analyze sulfur k - edge xanes spectra in pm10 samples collected adjacent to various emission sources representative of biomass burning , crude oil combustion , diesel emission , timber burning and traffic exhaust in songkla province , southern thailand , andto conduct a qualitative investigation of the altitude effect on sulfur k - edge xanes spectra in pm10 samples collected at urban sites throughout bangkok , chiang - mai and hat - yai , thailand . to qualitatively investigate the impact of diurnal variation on sulfur k - edge xanes spectra of pm10 collected at ground level , to analyze sulfur k - edge xanes spectra in pm10 samples collected adjacent to various emission sources representative of biomass burning , crude oil combustion , diesel emission , timber burning and traffic exhaust in songkla province , southern thailand , and to conduct a qualitative investigation of the altitude effect on sulfur k - edge xanes spectra in pm10 samples collected at urban sites throughout bangkok , chiang - mai and hat - yai , thailand . xanes was performed on the samples , and standards were measured at beam line no . 8 in the siam photon laboratory . a 10-cm - long ion chamber filled with a gas mixture of n2 ( 30 mbar ) and he ( 983 mbar ) was employed to measure intensity of the x - ray beam before the sample . the ion chamber absorbed only 10% of the beam intensity through ionization of the gas filling and produced a small electrical current signal proportional to the beam intensity . with the use of a digital window , similar to the ion chamber , the lyd produces small electrical current signal corresponding not only to fluorescence x - rays by the sulfur present , but also for other elements present in the sample and scattered photons from the primary beam . a thin polypropylene window was used to protect the ged from helium gas , which can diffuse through thin detector seal of beryllium spoiling cryostat vacuum of the detector . xanes spectra were recorded from 2450 ev to 2520 ev with the energy step of 0.2 ev and energy calibrated using the maximum absorption of iron sulfate at 2481.4 ev . all qff samples were applied to the adhesive side of the polyimide tape and placed at a 45 angle to the beam path in the sample chamber . tsp and pm10 samples were collected on 47 mm whatman quartz microfiber filters ( qmfs ) . pm10 samples were collected every three hours over a 27-hour period ( i.e. , n = 9 , 18.0021.00 , 21.0000.00 , 00.0003.00 , 03.0006.00 , 06.0009.00 , 09.0012.00 , 12.0015.00 , 15.0018.00 , and 18.0021.00 ) at the national institute of development administration ( nida ) , thailand 's most prestigious graduate university under the commission for higher education , the ministry of education in bangkok . diurnal variation of urban baseline samples was taken in an open field located at the south - western side of nida 's bangkapi campus , close to sereethai road , one of the most heavily used roads in this area . since the monitoring site is only 1 km away from the mall shopping center bangkapi , tesco - lotus and makro stores , it seems reasonable to assume that pm10 collected from this site represent aerosols derived from traffic vehicle exhausts . as previously mentioned , all samples were gathered on whatman qm - a - type quartz fiber filters ( qffs ) , and carefully preserved inside a pressure controlled glove box ( pcgb ) purged with nitrogen gas to avoid conversion of gaseous so2 to particulate so4 . all filters were installed inside an anoxic preservation stainless cylinder ( apsc ) filled with nitrogen gas ( see figures 3 and 4 ) and carefully delivered to synchrotron light research institute ( slri ) for sulfur k - edge xanes spectra analysis . sampling was conducted over three consecutive days at ( prince of songkla university ( psu ) , hat - yai campus ) , traffic intersection ( ti ) , corps incinerator ( ci ) , charoen phokphand factory ( cpf ) , songkla lake ( sl ) , rubber manufacturing factory ( rmf ) , bus terminal ( bt ) , waste incinerator ( wi ) , barbeque festival ( bf ) , petkrasem road ( pr ) and kor hong hill ( khh ) stations . prince of songkla university ( psu)the site is situated about 3 m above ground level in the faculty of environmental management , prince of songkla university , and about 550 m away from the main road that leads to the city center of hat - yai . the site is situated about 3 m above ground level in the faculty of environmental management , prince of songkla university , and about 550 m away from the main road that leads to the city center of hat - yai . songkhla lake monitoring station ( sl)it is situated about 13 km from the northern side of prince of songkla university , south of songkhla lake , and about 14 km from the western side of the gulf of thailand . this sampling station is far from any industrial and traffic emission sources , including chemical and metallurgy , and power plants in this district . songkla 1 ( sl1 ) and songkla 2 ( sl2 ) were used to represent the monitoring periods of july ( from 27th to 29th july , 2007 ) and october ( from 20th to 22nd october , 2007 ) respectively . it is situated about 13 km from the northern side of prince of songkla university , south of songkhla lake , and about 14 km from the western side of the gulf of thailand . this sampling station is far from any industrial and traffic emission sources , including chemical and metallurgy , and power plants in this district . this site represents a mixture of all emission sources in the urban area . it is located on the top of kor - hong hill with an elevation of 356 meters . petkrasam road ( pr)this site was located in the heart of the city of hat - yai . thus , the air mass collected from this area is mainly contaminated with diesel and benzene emissions . thus , the air mass collected from this area is mainly contaminated with diesel and benzene emissions . it is on the eastern side and approximately 2.5 km from the hat - yai city center close to an urban residential zone . this factory is part of the largest business conglomerate in thailand . the air samples were collected from 30th july to 1st august 2007 ( rmf1 ) and from 2nd to 4th august 2007 ( rmf2 ) . the air samples were collected from 30th july to 1st august 2007 ( rmf1 ) and from 2nd to 4th august 2007 ( rmf2 ) . corpse incinerator ( ci)this station is a part of kor - hong temple , located on the northern side and about 1.5 km from ti . since timber and tires are generally used as fuel for corpse incineration , this site is considered an emission source of both timber and tire burning . waste incinerator ( wi)this site is situated in the city center and belongs to the municipality of the city of hat - yai . this site is situated in the city center and belongs to the municipality of the city of hat - yai . barbeque festival ( bf)this site is located on the psu campus on the top roof of the faculty of natural resources building . additionally , it is also important to emphasize that there were no obstructions in the vicinity of the sampling equipment at all sites , which were strategically positioned to be accessible to winds from all directions . this site is located on the psu campus on the top roof of the faculty of natural resources building . the barbeque festival has become an annual tradition that is held on the second week in august . the air samples were collected from 15th to18th august , 2007 . additionally , it is also important to emphasize that there were no obstructions in the vicinity of the sampling equipment at all sites , which were strategically positioned to be accessible to winds from all directions . all three sampling sites are located in the city centers of bangkok , chiang - mai and hat - yai , representing the country 's capital and largest city alongside the largest cities in the northern and southern regions of thailand , respectively . air samples were collected at three different heights for three hour periods ( i.e. , level-1 : 12 m ; level-2 : 52 m ; level-3 : 152 m above ground level ) and at novotel centara hat - yai hotel observatory site ( i.e. hat - yai is located only 30 km from the west coast of the gulf of thailand , so it is likely that aerosol samples from this site will contain traces of sea salt and biomass burning . figure 5 illustrates the diurnal variation of nine sulfur k - edge xanes spectra of pm10 preserved in apsc and pcgb filled with nitrogen gas prior to xanes analysis at slri . there are three features which are common to each sulfur k - edge xanes spectrum : ( a ) the pre - edge region , which represents the energy emitted at 24502465 ev and points to the area of energies lower than the main absorption edge ; ( b ) the near - edge region , which refers to the 5 ev wide region around the white - line ( i.e. , the main peak ) , located at the post - edge region , which denotes the region greater than 2490 ev outside the absorption edge . based on the results from nine sulfur k - edge xanes spectra pm10 samples all seem to show a relatively flat spectrum of preedge region ( i.e. , 24502650 ev ) , followed by a sharp main peak at 2482 ev , and a large postedge fluctuation that arises in the energy band of 24902510 ev ( see figure 5 ) . these results are consistent with previous reports focusing on sulfur k - edge xanes spectra of aerosols with different particle sizes [ 1113 ] . conclusion that particulate sulfur compounds are dominated by s(vi ) , it can be deduced that the oxidation number of elemental sulfur collected at nida bangkapi campus is also + 6 , and plausibly occupied by sulfate species . figure 6 demonstrates the sulfur k - edge xanes spectra of 15 pm10 samples collected from various emission sources in songkla province . similar to the previous sulfur k - edge xanes spectra detected at nida bangkapi campus , the spectra from bt , pr , tesco , rmf , and cpf display a broad resonance at the preedge region , probably due to a superimposition of more than one resonance . for all 15 spectra , a maximum peak appears at the higher energy of 2482 ev , which is consistent with peaks belonging to diurnal variation samples . despite several deviations in the intensities of the maximum peak between the different emission source samples , some other subtle dissimilarities in post - edge regions are apparent , particularly in the subsequent structure , which appears the post - edge region between 2485 and 2510 ev . these discrepancies among different sulfur k - edge xanes spectra can be taken to characterize the finger print of target aerosols associated with various emission baselines . figure 8 underscores the dominance of the sulfate species in urban aerosols by displaying the relatively similar sulfur k - edge xanes spectra of pm10 collected at different heights for comparison to diurnal variation the analogous spectral features were observed in pm10 collected at chiang - mai , bangkok and hat - yai , irrespective of differences in sampling height . based on the positive correlation between t sulfur and photon energy , it appears sensible to consider the highest peak structures at 2482 ev as an outcome of the majority of s(vi ) recorded in urban pm10 . in addition , earlier studies revealed that the molecular structure has little effects 1s 3p transition energy of that specific sulfur functional group , thus highlighting the usefulness of xanes to categorize the oxidation number of elemental sulfur in pm10 [ 2224 ] . to investigate the anoxic preservation effect on sulfur k - edge xanes spectra of pm10 , qff samples were preserved in apsc filled with nitrogen gas immediately after completing collections . the sulfur k - edge xanes spectra of pm10 samples collected at different periods demonstrated that the position of the main peak was relatively consistent at 2482 ev without any sign of peak shift within 0.5 ev for numerous aerosol samples , indicating that particulate sulfur compositions are not influenced by diurnal variation . the moderately uniform sulfur k - edge xanes spectra suggest that the overall sulfate species distribution is constant at the traffic site . , indicating that anoxic preservation might have minor importance for the oscillation of sulfur k - edge xanes spectra in pm10 [ 1113 ] . firstly , all sulfur compounds from emission sources in urban atmospheric environment are composed of s(vi ) and conceivably predominated by sulfate species . secondly , the oxidation rate of aerosol sulfur is too fast to convert all particulate sulfur compounds to the most stable oxidation state of + 6 before being transported to the receptor site at nida bangkapi campus . thirdly , all sulfur compounds were completely oxidized to s(vi ) after the deposition of particles on qffs within three hours of sampling period . interestingly , this finding is in good agreement with an earlier study , which applied xanes for the determination and quantification of s(iv ) species in aerosol samples collected in qingdao , northeastern china . in the case of pm10 collected at nida bangkapi campus , the moderately high reactive trace gas ( e.g. , nox , o3 , and oh radical ) concentration released from diesel engines and other traffic combustions could oxidize sulfur compounds and conclusively convert all of the sulfur oxidation states to s(vi ) . in addition , the relatively low absorption intensity of sulfur k - edge xanes spectrum of pm10 collected from 12.00 am to 15.00 pm can be described as a consequence of the dilution effect triggered by the lifting of warm air mass at midday ( see figure 5 ) . unlike the results of diurnal variation campaign , the comparatively oscillating post - edge regions were discernible and thus highlighted the intricacy of sulfate compositions in pm10 collected from various emission sources ( figure 6 ) . likewise , the white - line intensity fluctuations are more noticeable in emission source spectra . these unique spectra features can be diagnosed as a divergence in both amount and speciation of s detected in pm10 . interestingly , the unsmooth pre - edge regions were predominant in sulfur k - edge xanes spectra of ci , wi , cpf , psu , bb , and ptb ( figure 6 ) . , more variety of sulfur oxidation states ) and the relatively low signal - to - noise ratio of s(vi ) particularly in case of ptb and bb ( i.e. for example , elemental s , organic polysulfide , thiol , sulfoxide , sulfite , sulfone , sulfonate , ester sulfate , and inorganic sulfate display the peak energy of sulfur k - edge white lines at 2472.5 , 2473.0 , 2473.4 , 2475.8 , 2478.7 , 2480.2 , 2481.3 , 2482.5 , and 2482.5 ev , respectively . a further investigation of sulfur k - edge xanes spectral features was conducted for baseline samples ( i.e. in contrast , the spectral plateau in the region of 2473 ev2476 ev detected in wi illustrates an exceptional feature , which can not be explained by the differences in climatic conditions due to the relatively similar sampling period and location ( see figure 7 and table 1 ) . in addition , the comparatively analogous spectral features of psu , ptb , rmf1 , and rmf2 indicated that para rubber tree combustion might play a major role in governing pm10 concentrations in residential zones , and thus raising concerns about the adverse impacts of para rubber industry on air quality in the city of hat - yai . to the best of our knowledge , this is the first study to examine sulfur k - edge xanes spectra of pm10 at different altitudes under same urban atmospheric conditions . all pm10 collected from three different cities displayed moderately similar spectral features in comparison with those of diurnal variation samples monitored at nida bangkapi campus as illustrated in figure 5 . several concerns were addressed with respect to factors affecting the sulfur k - edge xanes spectra of pm10 collected from various locations with different sampling altitudes . there are three common features that appear across all sulfur k - edge xanes spectra : ( a ) the pre - edge region , involving the energy of 24502465 ev , which signifies the area at energies lower than the main point of the absorption edge , ( b ) the near - edge region , which refers to the 5 ev - wide region immediately encompassing the white - line region ( i.e. , the main peak ) , which is located at 2482 ev , and ( c ) the post - edge region , which denotes the region over 2490 ev , outside the absorption edge . neither anoxic preservation nor emission source appreciably changed this pattern . further investigation was conducted to assess the alteration of particulate sulfur k - edge xanes spectra as a function of sampling altitude . interestingly , comparatively uniform sulfur k - edge xanes spectra were observed in all pm10 collected from different cities and sampling periods . this study also ascertains that the sulfur oxidation state of pm10 is predominantly overwhelmed by s(vi ) .

the online version of this article ( doi:10.1007/s00894 - 012 - 1747-y ) contains supplementary material , which is available to authorized users . photodynamic therapy ( pdt ) is one of the methods to cure cancer , which uses the combination of a drug ( called a photosensitizer ) and a light [ 13 ] . upon irradiation with the light of an appropriate wave - length , the photosensitizer is excited from its ground state ( usually singlet ) to the excited state - at first singlet , which may subsequently undergo conversion to triplet through inter - system crossing . the excess energy might then be transferred to nearby oxygen molecules and excite them from triplet ground state to singlet , thus producing highly reactive species , able to damage living cells . otherwise , the excitation energy might be dissipated in a form of heat , causing cell damage through over - heating . at the end the working principle of the photodynamic therapy implies several requirements , which should be fulfilled by good photosensitizers . they should effectively absorb light in the range appropriate for medical applications , i.e. , 620850 nm , preferably red - shifted . inversely , their absorption in the range 400600 nm should be low in order to avoid prolonged skin sensitivity to the sunlight . next , the efficiency of singlet to triplet intersystem crossing should be enhanced , which is usually achieved through incorporation of a metal ion into its structure . for the good candidates , it is sought that the singlet - triplet energy gap is higher than the energy needed to excite molecular oxygen ( ca 1 ev ) . last but not least , a high stability of these drugs is needed , both thermodynamic and photostability , as well as their low toxicity in the dark . the application of porphyrin - based photosensitizers ( porphyrins , texaphyrins , chlorins and bacteriochlorins ) as agents for photodynamic therapy has draw attention in the last couple of years due to their good light - absorbing properties . synthetic porphyrins , hematoporphyrins and their analogues served as first photosensitizers , but they showed a number of drawbacks , among which were high retention times in living organisms , causing prolonged skin sensitivity to light and relatively low absorption , resulting in the need of the increased doses . nowadays the researchers focus rather on the naturally occurring porphyrins , e.g. , chlorophylls and bacteriochlorophylls [ 5 , 6 ] , as these have shorter retention times and their decomposition pathways are already established . among different derivatives of porphyrins , bacteriochlorophylls offer remarkable properties , which are mainly due to the fact that their absorption spectrum is considerably red - shifted . therefore the current research concentrates on modifications of structure and properties of the native bacteriochlorophylls ( and chlorophylls ) , which might be important for medical applications , and allow to over - come known drawbacks of synthetic photosensitizers . the replacement of the central magnesium ion for a heavy element ameliorates photophysical behavior of these pigments by enhancing singlet - triplet intersystem crossing due to spin - orbit coupling . for instance , it is shown that the lack of phytyl ( a c20 alcohol moiety attached to chlorophylls and bacteriochlorophylls ) ameliorates their solubility in aqueous media . on the other hand , a number of transition metal ions in form of their complexes found application in anti - tumor therapy . platinum(ii ) complexes are the parent drugs , as they are used for chemotherapy against , e.g. , lung and bladder cancers . the second widely known transition metal in this area is ruthenium . in this context , usually ru(iii ) salts are studied , however , it is believed that in organisms ru(iii ) undergoes reduction to ru(ii ) with ascorbic acid or other reductants [ 12 , 13 ] . among others , pd(ii ) and rh(ii ) are also popular and offer new possibilities for treating new types of cancer cells . it seems to be desirable that these two aspects of metal - containing compounds are combined in a single molecule , which may act in two different ways to fight cancer cells . therefore , the idea to use pt , ru , pd , and rh substituted bacteriochlorophylls appears tempting . on the one hand , the absorption properties of bacteriochlorophylls would be used in standard pdt , while on the other hand the anticancer properties of the metal ions will be employed after the treatment with light . consequently , the aim of the present study is to characterize different metallo - substituted bacteriochlorophylls , as possible candidates for photodynamic therapy . special attention is paid to derivatives , which contain transition metal ions , whose anti - tumor activity is known and new systems , in which they may render useful , are sought . additionally , the set of tested metals is enlarged by mg ( a native metal in bacteriochlorophyll ) , co , ni , cu , zn . the selection of the latter follows from the availability of spectroscopic characterization of their bacteriochlorophyll derivatives , made in relation to the possible application of these species in photodynamic therapy . the most extensive studies of different possible photosensitizers derivatives of porphyrin and texaphyrin with and without central magnesium atom for use in pdt are published by russo and his co - workers [ 1416 ] . additionally , the selected adducts of bacteriochlorins with metals of the first transition row are characterized and their spectral properties are discussed in detail . the main stress was put on the characterization of their absorption spectra , which were rationalized with an aid of time - dependent density functional theory . the present research is focused on the adducts with metals , which already found application in anti - cancer therapy . in this respect our study covers a different range of tested metal ions than in russo s studies . moreover , the macrocyclic ligand , which is used throughout the present study , contains the naturally occurring bacteriochlorin ring substituents , thus they may be synthetized on the base of natural materials . one should mention that the theoretical studies of the spectroscopic properties of the native chlorophylls and bacteriochlorophylls are also published ( e.g. , [ 1821 ] ) , but they were undertaken rather in order to elucidate the details of the light absorption in photosynthesis , and not in view of their potential applications in pdt . an interested reader should be referred to the extensive reviews [ 20 , 22 , 23 ] and references therein . in the present study quantum chemical method based on density functional theory ( dft ) with non - local becke - perdew functional was applied [ 2428 ] . the calculation consisted of full geometry optimizations of the studied structures and the resolution - of - identity ( ri ) algorithm was applied [ 29 , 30 ] . all - electron gaussian type orbitals of def2-tzvp quality were used to define atomic orbitals , while effective core potentials were applied for heavy elements ( ru , rh , pd , and pt ) . the solvation was accounted for by cosmo model with default radii for the elements ( h = 1.30 , c = 2.00 , n = 1.83 , o = 1.72 ) and 2.00 for the studied metal ions . dielectric permittivity equal to 80 was used in order to take into account the nature of the possible aqueous environment in which these potential therapeutic agents will appear in tissues . the present results were obtained with turbomole v. 6.3 and further analyzed with aomix 6.6 program package [ 34 , 35 ] . it consists of the bacteriochlorophyll a molecule bearing all its native substituents , except the long phytyl chain , which is here replaced by a hydrogen atom ( so called a bacteriopheophorbide ligand ) . such a simplification of the model is justified in light of the fact that the lack of phytyl ameliorates solubility of bacteriochlorophyll in aqueous media and is beneficial for its prospect medical application.fig . 1geometry structure of the investigated systems : central metals ( co , ni , cu , zn , ru , rh , pd , and pt ) are marked in green . enumeration of pyrroles starts from the left hand upper corner , clock - wise from a to d geometry structure of the investigated systems : central metals ( co , ni , cu , zn , ru , rh , pd , and pt ) are marked in green . enumeration of pyrroles starts from the left hand upper corner , clock - wise from a to d in the investigated bacteriochlorophyll molecule four pyrroles of the macrocycle are not equivalent , so the symmetry of the molecule is lowered to c1 as compared to c4v in porphyrins or d2h as in bacteriochlorins . this is due to the presence of different substituents in the macrocyle periphery and to the fact that two of four pyrroles are saturated , while the remaining two are not . in the present paper eight bacteriochlorophyll derivatives the studied metal ions cover : co(ii ) , ni(ii ) , cu(ii ) , zn(ii ) , ru(ii ) , rh(ii ) , pd(ii ) , and pt(ii ) . for all of the metals + ii oxidation state is common and ensures the neutral charge of the whole system . as one may notice , the range of the studied central metal ions is wider than those of anti - cancer activity , outlined in the introduction . this is a result of the availability of spectroscopic data , which due to structure - activity relationship models may be compared with computed parameters and help to foresee the properties of derivatives not characterized thus far . such an approach already proved useful in forecasting the physico - chemical characteristics of different groups of molecules [ 36 , 37 ] . in the following , the description of the investigated bacteriochlorophyll derivatives is given , with an emphasis on their structural and electronic properties . the discussion is then shifted to the problem of the stability of the chosen metal ions in bacteriochlorophyll cavity in aqueous environment . the basic structural parameters obtained throughout the present study are listed in table 1 . in all but one of the investigated cases , the ground state of the molecules is low spin , which is singlet or dublet , depending on the type of the central metal ion . the only odd system is ru - bchla , whose triplet lies the lowest on the potential energy surface : singlet and pentet states are higher by 0.39 ev and 1.20 ev , respectively . these findings are in agreement with previous theoretical study , in which different transition metal ions from the fourth period have been examined . while most of the studied systems exhibited low spin , iron - bacteriochlorin was characterized by the intermediate spin . the similarity of the ground state multiplicity is a consequence of the location of the two elements in the periodic table : ruthenium lies directly below iron.table 1computed parameters of studied metallobacteriochlorophylls : bond distances are in , homo , lumo and energy gaps are in ev . additionally , literature data are listed , which are referred to in the manuscript : qx max absorption in nm , i pauling electronegativity , ri ionic radius in metalmgconicuznrurhpdptbond distancesm - na 2.0351.9661.9551.9861.9882.0312.0152.0402.041m - nb 2.1291.9711.9642.0812.1362.0462.0372.0392.028m - nc 2.0211.9321.9191.9651.9812.0001.9832.0042.009m - nd 2.1602.0382.0342.1382.1912.0942.0912.0922.074bond ordersm - na 0.5890.7100.7280.7320.7130.6590.7060.5920.687m - nb 0.4390.7400.7700.6050.5270.6310.6430.5940.752m - nc 0.6020.7320.7540.7390.6860.6690.7150.6000.711m - nd 0.4360.6770.6940.5490.4670.6030.6130.5680.691ehomo 4.834.864.884.894.914.924.954.924.94 4.87 4.89 4.89 4.92 elumo 3.683.693.703.733.723.583.633.673.65 4.31 4.31 4.32 4.70 gap1.151.171.171.161.181.341.321.251.29 0.56 0.59 0.58 0.22 qx max 778767779780773763eex 38.818.740.615.60.622.953.367.583.9i 1.311.881.911.901.652.202.282.202.28ri 0.860.790.630.710.740.780.80 computed parameters of studied metallobacteriochlorophylls : bond distances are in , homo , lumo and energy gaps are in ev . additionally , literature data are listed , which are referred to in the manuscript : qx max absorption in nm , i pauling electronegativity , ri ionic radius in the singlet - triplet energy separation is an important parameter regarding proposed application of the investigated systems for photodynamic therapy . the energy gap should be higher than required to excite molecular oxygen to its singlet state . in this respect both new bacteriochlorophyll derivatives , whose ground state is singlet ( with pt and pd ) , turn up suitable . the singlet triplet energy separation is 1.15 ev and 1.11 ev for pt - bchla and pd - bchla , respectively . the analysis of the geometries of m - bchla systems reveal that all cores of the macrocyclic complexes are planar , independent of the type of the central metal ion . this clearly shows the flexibility of the bacteriochlorin macrocycle and its ability to accommodate ions of different radii . out - of - plane displacement of the central metals in porphyrin - related systems , often reported in literature , is related strictly to the presence of additional ligands , which pull them from the center of the macrocycle [ 3840 ] . four pyrroles , which constitute the core of bacteriochlorin , are not equivalent and so four metal studied systems one may observe a similar pattern : the shortest bond is formed with the nitrogen from the pyrrole c , which is fused with the isocyclic ring , while the longest with the nitrogen to which originally phytyl is attached ( pyrrole d ) . the remaining two bonds are neither of the same length : the shorter connects metal with nitrogen of pyrrole a , while the longer with nitrogen from pyrrole b. one may then measure the size of the central cavity , which has a shorter diameter along the line connecting pyrroles a and c , and a larger diameter connecting pyrroles b and d ; the difference between them may reach up to 9 % , as in zn - bchla . the macrocycle core is the widest in zn - bchla ( two diameters are equal to 3.969 and 4.327 ) , and the tightest in ni - bchla ( 3.874 and 3.998 ) . one should notice then that the central metal is never located in the exact center of the cavity , but is always shifted toward the fragment of the macrocycle of a larger electron density ( pyrrole c fused with the isocyclic ring ) . this feature is also reported by sundholm in his theoretical studies of bacteriochlorophyll b species . it should be born in mind , however , that in real conditions the nuclear position will not be static due to thermal vibrations . all nuclei will be able to occupy a certain volume around the determined point in space , and their average location will be determined by experiment . homo levels are similar and vary from -4.95 ev for rh - bchla to -4.83 ev for mg - bchla , while lumo levels vary from -3.73 ev for cu - bchla to -3.58 ev for ru - bchla . consequently , the homo - lumo gaps of all the studied derivatives are similar and fall in the range 1.151.35 ev . such small differences arise from the fact that frontier orbitals are located mostly on the macrocycle , with small contributions from metal orbitals appearing only in lumos see supplementary material . this finding is in line with the results obtained for model metallobacteriochlorins , where similar character of homos and lumos was observed . the bacteriochlorophyll derivatives covered in this study have additional isocyclic ring , therefore their frontier orbitals contain also its admixture . this gives rise to the before - mentioned shifted position of central metal ions too . the uv vis spectra of a majority of the studied compounds can be found in literature . the qx band is red - shifted , ensuring the use of red light for irradiation , which penetrates deeper into tissues than other wavelengths form the visible spectrum . according to our knowledge , an attempt is then made to somehow predict the absorption edge based on the calculated properties of the remaining species . in order to do so the experimental peak positions of the adsorption band ( which in the literature are related to as qx band ) are then correlated with the homo - lumo gap . the obtained results show a linear relationship between the two values with the correlation coefficient equal to 0.89 . the calculated parameters of the regression line serve to approximate the adsorption band of the bacteriochlorophyll derivatives whose spectra are not published . therefore it is predicted that pt - bchla would absorb at 752 nm , ru - bchla at 738 nm , and rh - bchla at 745 nm . these values confirm the applicability of the abovementioned species to be used as light absorbing agents in pdt.fig . 2linear relationship between uv vis absorption energy and homo - lumo gap linear relationship between uv vis absorption energy and homo - lumo gap as was already underlined , one of the main aspects during the test process of new therapeutic agents is their stability and toxicity . in the case of pharmaceuticals containing transition metal ions one should consider the possibility of the central metal leaching into the body fluids . while some of the metal ions are neutral or even beneficial for human health in moderate concentrations ( here , e.g. , magnesium ) , some are toxic and thus may have detrimental effect on the functioning of the body ( here , e.g. , cobalt or platinum ) . to somehow assess a possibility of leaching of the central metal , the exchange of central metals for protons from environment is considered as described by the following reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{mbchla } + 2{{\mathrm{h}}_5}{{\mathrm{o}}_2}^{+}+n{{\mathrm{h}}_2}\mathrm{o}\to { { \mathrm{h}}_2}\mathrm{bchla } + { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{4+\mathrm{n } } } } \right]}^{2 + } } . $ $ \end{document } it is assumed that two protons , which are initially solvated ( each one with two water molecules ) , replace the central metal ion , which forms an aqua complex afterward . the number of aqua ligands is typical for a given metal ion and equals six for mg , co , cu , ru , rh and four for zn , pt , pd , ni . the idea to relate the metallobacteriochlorophyll stability to the resistance toward the displacement of the central metal by acid is generally accepted in metalloporphyrin research [ 4244 ] . this is because 1 ) it is often difficult to determine equilibrium constants directly and 2 ) metalloporphyrins show a wide range of sensitivity to acid . it is then proposed that the measure of the stability of the metal - ligand adduct is the energetic cost of the abovementioned process , calculated in aqueous phase ( accounted for with cosmo model):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { { \mathrm{e}}_{\mathrm{ex}}}=\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{bchla } } \right)+\mathrm{e}\left ( { { { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{{4+\mathrm{n } } } } } \right]}}^{2 + } } } \right)-\mathrm{e}\left ( { \mathrm{m}\mathrm{bchla } } \right)-2\mathrm{e}\left ( { { { \mathrm{h}}_5}{{\mathrm{o}}_2}^{+ } } \right)-n\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right ) . $ $ \end{document } one should emphasize here , that the stability , as defined in this paper , is related only to the thermodynamic property and does not discuss the kinetic stability of the system . the latter would be connected with the height of energy barrier which should be overcome to reach the products . the analysis of the collected data reveals that the native central atom of bacteriochlorophyll , magnesium , is not stable in the macrocycle cavity . this observation is not surprising , as the low stability of magnesium porphyrins , chlorins and bacteriochlorins is a well - known fact [ 8 , 45 , 46 ] , further reflected by the high cost of these synthetic materials . the energy needed for zn to be replaced by two protons is lower that 1 kcal mol . this means that the application of zn - bchla into the tissues might result in the decomposition of the complex into bacteriochlorophyll and the hydrated zinc ion provided the zn - bchla complex is labile . the other metals are stable while inserted into bacteriochlorophyll cavity , with metal exchange energies being larger than 15 kcal mol . the calculated energy exchange allows to rank them in line with the increased predicted stability : cu < co < ru < ni < rh < pd < pt . traditionally , the stability of metal - substituted porphyrins is defined by so - called stability index which is proportional to pauling electronegativity of an element and inversely proportional to its effective ionic radius . mg derivative is characterized by the lowest stability index and zn - bchla is the second least stable , the same as predicted in the present paper . platinum and palladium derivatives show high stability , while copper and cobalt are in the range of medium values . the largest discrepancy is found for nickel adduct ; it is characterized by the highest stability index , while its exchange energy places it below pd and pt complexes . in view of the potential applications of the studied metal - bacteriochlorins for therapeutic purposes , the high stability of pt , pd , these metal ions are cytotoxic and thus their uncontrolled release in tissues might result in more harm than is gained from their therapeutic properties . on the other hand , if there is a way to release them selectively in tumor cells , one may profit from their anti - cancer activity in situ . indeed , one may imagine that after they serve as photosensitizers in pdt , the large macrocyclic ligand might undergo decomposition , e.g. , upon irradiation or induced by endogenous or exogenous factors . as a result , the therapeutic metal ions would be released from bacteriochlorophyll wrap and present in the exact place for their action through mechanisms , which are proposed for their standard complexes . this type of action , which would profit from these two aspects of metallobacteriochlorophylls , might appear advantageous . on the one hand the photophysical properties of the macrocyclic fragment would be in use during light irradiation phase and on the second hand , their central metals might serve as therapeutic tools to fight with anomalous cells afterward . the basic structural parameters obtained throughout the present study are listed in table 1 . in all but one of the investigated cases , the ground state of the molecules is low spin , which is singlet or dublet , depending on the type of the central metal ion . the only odd system is ru - bchla , whose triplet lies the lowest on the potential energy surface : singlet and pentet states are higher by 0.39 ev and 1.20 ev , respectively . these findings are in agreement with previous theoretical study , in which different transition metal ions from the fourth period have been examined . while most of the studied systems exhibited low spin , iron - bacteriochlorin was characterized by the intermediate spin . the similarity of the ground state multiplicity is a consequence of the location of the two elements in the periodic table : ruthenium lies directly below iron.table 1computed parameters of studied metallobacteriochlorophylls : bond distances are in , homo , lumo and energy gaps are in ev . additionally , literature data are listed , which are referred to in the manuscript : qx max absorption in nm , i pauling electronegativity , ri ionic radius in metalmgconicuznrurhpdptbond distancesm - na 2.0351.9661.9551.9861.9882.0312.0152.0402.041m - nb 2.1291.9711.9642.0812.1362.0462.0372.0392.028m - nc 2.0211.9321.9191.9651.9812.0001.9832.0042.009m - nd 2.1602.0382.0342.1382.1912.0942.0912.0922.074bond ordersm - na 0.5890.7100.7280.7320.7130.6590.7060.5920.687m - nb 0.4390.7400.7700.6050.5270.6310.6430.5940.752m - nc 0.6020.7320.7540.7390.6860.6690.7150.6000.711m - nd 0.4360.6770.6940.5490.4670.6030.6130.5680.691ehomo 4.834.864.884.894.914.924.954.924.94 4.87 4.89 4.89 4.92 elumo 3.683.693.703.733.723.583.633.673.65 4.31 4.31 4.32 4.70 gap1.151.171.171.161.181.341.321.251.29 0.56 0.59 0.58 0.22 qx max 778767779780773763eex 38.818.740.615.60.622.953.367.583.9i 1.311.881.911.901.652.202.282.202.28ri 0.860.790.630.710.740.780.80 computed parameters of studied metallobacteriochlorophylls : bond distances are in , homo , lumo and energy gaps are in ev . additionally , literature data are listed , which are referred to in the manuscript : qx max absorption in nm , i pauling electronegativity , ri ionic radius in the singlet - triplet energy separation is an important parameter regarding proposed application of the investigated systems for photodynamic therapy . the energy gap should be higher than required to excite molecular oxygen to its singlet state . in this respect both new bacteriochlorophyll derivatives , whose ground state is singlet ( with pt and pd ) , turn up suitable . the singlet triplet energy separation is 1.15 ev and 1.11 ev for pt - bchla and pd - bchla , respectively . the analysis of the geometries of m - bchla systems reveal that all cores of the macrocyclic complexes are planar , independent of the type of the central metal ion . this clearly shows the flexibility of the bacteriochlorin macrocycle and its ability to accommodate ions of different radii . out - of - plane displacement of the central metals in porphyrin - related systems , often reported in literature , is related strictly to the presence of additional ligands , which pull them from the center of the macrocycle [ 3840 ] . four pyrroles , which constitute the core of bacteriochlorin , are not equivalent and so four metal studied systems one may observe a similar pattern : the shortest bond is formed with the nitrogen from the pyrrole c , which is fused with the isocyclic ring , while the longest with the nitrogen to which originally phytyl is attached ( pyrrole d ) . the remaining two bonds are neither of the same length : the shorter connects metal with nitrogen of pyrrole a , while the longer with nitrogen from pyrrole b. one may then measure the size of the central cavity , which has a shorter diameter along the line connecting pyrroles a and c , and a larger diameter connecting pyrroles b and d ; the difference between them may reach up to 9 % , as in zn - bchla . the macrocycle core is the widest in zn - bchla ( two diameters are equal to 3.969 and 4.327 ) , and the tightest in ni - bchla ( 3.874 and 3.998 ) . one should notice then that the central metal is never located in the exact center of the cavity , but is always shifted toward the fragment of the macrocycle of a larger electron density ( pyrrole c fused with the isocyclic ring ) . this feature is also reported by sundholm in his theoretical studies of bacteriochlorophyll b species . it is thus due to the presence of macrocycle substituents and the isocyclic ring . the non - equivalency of four metal it should be born in mind , however , that in real conditions the nuclear position will not be static due to thermal vibrations . all nuclei will be able to occupy a certain volume around the determined point in space , and their average location will be determined by experiment . homo levels are similar and vary from -4.95 ev for rh - bchla to -4.83 ev for mg - bchla , while lumo levels vary from -3.73 ev for cu - bchla to -3.58 ev for ru - bchla . consequently , the homo - lumo gaps of all the studied derivatives are similar and fall in the range 1.151.35 ev . such small differences arise from the fact that frontier orbitals are located mostly on the macrocycle , with small contributions from metal orbitals appearing only in lumos see supplementary material . this finding is in line with the results obtained for model metallobacteriochlorins , where similar character of homos and lumos was observed . the bacteriochlorophyll derivatives covered in this study have additional isocyclic ring , therefore their frontier orbitals contain also its admixture . this gives rise to the before - mentioned shifted position of central metal ions too . the uv vis spectra of a majority of the studied compounds can be found in literature . the qx band is red - shifted , ensuring the use of red light for irradiation , which penetrates deeper into tissues than other wavelengths form the visible spectrum . according to our knowledge , only some of the metallobacteriochlorophylls described here are characterized in literature . an attempt is then made to somehow predict the absorption edge based on the calculated properties of the remaining species . in order to do so the experimental peak positions of the adsorption band ( which in the literature are related to as qx band ) are then correlated with the homo - lumo gap . the obtained results show a linear relationship between the two values with the correlation coefficient equal to 0.89 . the calculated parameters of the regression line serve to approximate the adsorption band of the bacteriochlorophyll derivatives whose spectra are not published . therefore it is predicted that pt - bchla would absorb at 752 nm , ru - bchla at 738 nm , and rh - bchla at 745 nm . these values confirm the applicability of the abovementioned species to be used as light absorbing agents in pdt.fig . 2linear relationship between uv vis absorption energy and homo - lumo gap linear relationship between uv vis absorption energy and homo - lumo gap as was already underlined , one of the main aspects during the test process of new therapeutic agents is their stability and toxicity . in the case of pharmaceuticals containing transition metal ions while some of the metal ions are neutral or even beneficial for human health in moderate concentrations ( here , e.g. , magnesium ) , some are toxic and thus may have detrimental effect on the functioning of the body ( here , e.g. , cobalt or platinum ) . to somehow assess a possibility of leaching of the central metal , the exchange of central metals for protons from environment is considered as described by the following reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{mbchla } + 2{{\mathrm{h}}_5}{{\mathrm{o}}_2}^{+}+n{{\mathrm{h}}_2}\mathrm{o}\to { { \mathrm{h}}_2}\mathrm{bchla } + { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{4+\mathrm{n } } } } \right]}^{2 + } } . $ $ \end{document } it is assumed that two protons , which are initially solvated ( each one with two water molecules ) , replace the central metal ion , which forms an aqua complex afterward . the number of aqua ligands is typical for a given metal ion and equals six for mg , co , cu , ru , rh and four for zn , pt , pd , ni . the idea to relate the metallobacteriochlorophyll stability to the resistance toward the displacement of the central metal by acid is generally accepted in metalloporphyrin research [ 4244 ] . this is because 1 ) it is often difficult to determine equilibrium constants directly and 2 ) metalloporphyrins show a wide range of sensitivity to acid . it is then proposed that the measure of the stability of the metal - ligand adduct is the energetic cost of the abovementioned process , calculated in aqueous phase ( accounted for with cosmo model):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { { \mathrm{e}}_{\mathrm{ex}}}=\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{bchla } } \right)+\mathrm{e}\left ( { { { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{{4+\mathrm{n } } } } } \right]}}^{2 + } } } \right)-\mathrm{e}\left ( { \mathrm{m}\mathrm{bchla } } \right)-2\mathrm{e}\left ( { { { \mathrm{h}}_5}{{\mathrm{o}}_2}^{+ } } \right)-n\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right ) . $ $ \end{document } one should emphasize here , that the stability , as defined in this paper , is related only to the thermodynamic property and does not discuss the kinetic stability of the system . the latter would be connected with the height of energy barrier which should be overcome to reach the products . the analysis of the collected data reveals that the native central atom of bacteriochlorophyll , magnesium , is not stable in the macrocycle cavity . this observation is not surprising , as the low stability of magnesium porphyrins , chlorins and bacteriochlorins is a well - known fact [ 8 , 45 , 46 ] , further reflected by the high cost of these synthetic materials . the energy needed for zn to be replaced by two protons is lower that 1 kcal mol . this means that the application of zn - bchla into the tissues might result in the decomposition of the complex into bacteriochlorophyll and the hydrated zinc ion provided the zn - bchla complex is labile . the other metals are stable while inserted into bacteriochlorophyll cavity , with metal exchange energies being larger than 15 kcal mol . the calculated energy exchange allows to rank them in line with the increased predicted stability : cu < co < ru < ni < rh < pd < pt . traditionally , the stability of metal - substituted porphyrins is defined by so - called stability index which is proportional to pauling electronegativity of an element and inversely proportional to its effective ionic radius . mg derivative is characterized by the lowest stability index and zn - bchla is the second least stable , the same as predicted in the present paper . platinum and palladium derivatives show high stability , while copper and cobalt are in the range of medium values . the largest discrepancy is found for nickel adduct ; it is characterized by the highest stability index , while its exchange energy places it below pd and pt complexes . in view of the potential applications of the studied metal - bacteriochlorins for therapeutic purposes , the high stability of pt , pd , rh and ru derivatives might be beneficial . these metal ions are cytotoxic and thus their uncontrolled release in tissues might result in more harm than is gained from their therapeutic properties . on the other hand , if there is a way to release them selectively in tumor cells , one may profit from their anti - cancer activity in situ . indeed , one may imagine that after they serve as photosensitizers in pdt , the large macrocyclic ligand might undergo decomposition , e.g. , upon irradiation or induced by endogenous or exogenous factors . as a result , the therapeutic metal ions would be released from bacteriochlorophyll wrap and present in the exact place for their action through mechanisms , which are proposed for their standard complexes . this type of action , which would profit from these two aspects of metallobacteriochlorophylls , might appear advantageous . on the one hand the photophysical properties of the macrocyclic fragment would be in use during light irradiation phase and on the second hand , their central metals might serve as therapeutic tools to fight with anomalous cells afterward . the present study allowed for the structural characterization of bacteriochlorophylls containing non - native divalent metal ions : co , ni , cu , zn , ru , rh , pd , and pt . the main stress was put on the derivatives with metals , which already found applications in anti - tumor therapy in the form of other complexes , with an aim to combine their properties with the known suitability of bacteriochlorophylls to photodynamic therapy . the performed characterization of a wider group of species allowed for the prediction of unknown properties of these promising agents . the geometry structures of the studied species show the surprising flexibility of bacteriochlorophyll cavity , which manifests itself in an ability to accommodate metal ions of different sizes . the test reaction , which was the exchange of the central metal ion for two protons showed that all studied metal macrocycle adducts are stable in aqueous media , except for mg - bchla . the stability of zn - bchla would be minor and in real conditions would depend mostly on the kinetics of its decomposition . the predicted inertness of the synthetic derivatives with metals of anti - tumor activity ( ru , pt , pd , rh ) might turn out beneficial for therapeutic purposes . they are expected not to decompose during the photodynamic therapy , while one may hopefully control their decomposition in the second phase of a therapy .

a theoretical analysis of bacteriochlorophyll a containing its non - native divalent metal ions : co , ni , cu , zn , ru , rh , pd , and pt , has been carried out by means of density functional theory ( dft ) calculations . the main stress was put on the derivatives with metals , which already found applications as coordination compounds in anti - tumor therapy ( ru , pt , pd , and rh ) . the idea was to combine their cytotoxic properties with the known suitability of bacteriochlorophylls macrocycle for photodynamic therapy . the geometries of the studied systems are compared and reveal a number of similarities . the cores of the modified bacteriochlorophylls are flat , and the introduced metal ions lie in plane of the macrocycle , showing its large ability to accommodate metal ions of different sizes . however , four metal nitrogen bonds , linking the central ions with the macrocycle ligand , are not equivalent . metals are the strongest attached to nitrogens , which come from the pyrrole , which is fused with isocyclic ring . based on the known spectroscopic data , the absorption properties of the proposed systems are predicted . finally , it is found that all studied metal macrocycle adducts are stable in aqueous media . the only exceptions are mg - bchla ( the finding is reflected by experimental facts ) and zn - bchla . the predicted high stability of ru- , rh- , pt- and pd - bacteriochlorophylls might turn out beneficial for therapeutic purposes.electronic supplementary materialthe online version of this article ( doi:10.1007/s00894 - 012 - 1747-y ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Methods and model Results and discussion Characteristics of bacteriochlorophylls containing different central metals Central metal leaching Conclusions Electronic supplementary material

the online version of this article ( doi:10.1007/s00894 - 012 - 1747-y ) contains supplementary material , which is available to authorized users . photodynamic therapy ( pdt ) is one of the methods to cure cancer , which uses the combination of a drug ( called a photosensitizer ) and a light [ 13 ] . upon irradiation with the light of an appropriate wave - length , the photosensitizer is excited from its ground state ( usually singlet ) to the excited state - at first singlet , which may subsequently undergo conversion to triplet through inter - system crossing . therefore the current research concentrates on modifications of structure and properties of the native bacteriochlorophylls ( and chlorophylls ) , which might be important for medical applications , and allow to over - come known drawbacks of synthetic photosensitizers . for instance , it is shown that the lack of phytyl ( a c20 alcohol moiety attached to chlorophylls and bacteriochlorophylls ) ameliorates their solubility in aqueous media . on the other hand , a number of transition metal ions in form of their complexes found application in anti - tumor therapy . therefore , the idea to use pt , ru , pd , and rh substituted bacteriochlorophylls appears tempting . on the one hand , the absorption properties of bacteriochlorophylls would be used in standard pdt , while on the other hand the anticancer properties of the metal ions will be employed after the treatment with light . consequently , the aim of the present study is to characterize different metallo - substituted bacteriochlorophylls , as possible candidates for photodynamic therapy . special attention is paid to derivatives , which contain transition metal ions , whose anti - tumor activity is known and new systems , in which they may render useful , are sought . additionally , the set of tested metals is enlarged by mg ( a native metal in bacteriochlorophyll ) , co , ni , cu , zn . the main stress was put on the characterization of their absorption spectra , which were rationalized with an aid of time - dependent density functional theory . the present research is focused on the adducts with metals , which already found application in anti - cancer therapy . moreover , the macrocyclic ligand , which is used throughout the present study , contains the naturally occurring bacteriochlorin ring substituents , thus they may be synthetized on the base of natural materials . in the present study quantum chemical method based on density functional theory ( dft ) with non - local becke - perdew functional was applied [ 2428 ] . all - electron gaussian type orbitals of def2-tzvp quality were used to define atomic orbitals , while effective core potentials were applied for heavy elements ( ru , rh , pd , and pt ) . the solvation was accounted for by cosmo model with default radii for the elements ( h = 1.30 , c = 2.00 , n = 1.83 , o = 1.72 ) and 2.00 for the studied metal ions . it consists of the bacteriochlorophyll a molecule bearing all its native substituents , except the long phytyl chain , which is here replaced by a hydrogen atom ( so called a bacteriopheophorbide ligand ) . such a simplification of the model is justified in light of the fact that the lack of phytyl ameliorates solubility of bacteriochlorophyll in aqueous media and is beneficial for its prospect medical application.fig . 1geometry structure of the investigated systems : central metals ( co , ni , cu , zn , ru , rh , pd , and pt ) are marked in green . enumeration of pyrroles starts from the left hand upper corner , clock - wise from a to d geometry structure of the investigated systems : central metals ( co , ni , cu , zn , ru , rh , pd , and pt ) are marked in green . enumeration of pyrroles starts from the left hand upper corner , clock - wise from a to d in the investigated bacteriochlorophyll molecule four pyrroles of the macrocycle are not equivalent , so the symmetry of the molecule is lowered to c1 as compared to c4v in porphyrins or d2h as in bacteriochlorins . in the present paper eight bacteriochlorophyll derivatives the studied metal ions cover : co(ii ) , ni(ii ) , cu(ii ) , zn(ii ) , ru(ii ) , rh(ii ) , pd(ii ) , and pt(ii ) . as one may notice , the range of the studied central metal ions is wider than those of anti - cancer activity , outlined in the introduction . this is a result of the availability of spectroscopic data , which due to structure - activity relationship models may be compared with computed parameters and help to foresee the properties of derivatives not characterized thus far . the discussion is then shifted to the problem of the stability of the chosen metal ions in bacteriochlorophyll cavity in aqueous environment . in all but one of the investigated cases , the ground state of the molecules is low spin , which is singlet or dublet , depending on the type of the central metal ion . additionally , literature data are listed , which are referred to in the manuscript : qx max absorption in nm , i pauling electronegativity , ri ionic radius in the singlet - triplet energy separation is an important parameter regarding proposed application of the investigated systems for photodynamic therapy . the analysis of the geometries of m - bchla systems reveal that all cores of the macrocyclic complexes are planar , independent of the type of the central metal ion . this clearly shows the flexibility of the bacteriochlorin macrocycle and its ability to accommodate ions of different radii . out - of - plane displacement of the central metals in porphyrin - related systems , often reported in literature , is related strictly to the presence of additional ligands , which pull them from the center of the macrocycle [ 3840 ] . four pyrroles , which constitute the core of bacteriochlorin , are not equivalent and so four metal studied systems one may observe a similar pattern : the shortest bond is formed with the nitrogen from the pyrrole c , which is fused with the isocyclic ring , while the longest with the nitrogen to which originally phytyl is attached ( pyrrole d ) . the remaining two bonds are neither of the same length : the shorter connects metal with nitrogen of pyrrole a , while the longer with nitrogen from pyrrole b. one may then measure the size of the central cavity , which has a shorter diameter along the line connecting pyrroles a and c , and a larger diameter connecting pyrroles b and d ; the difference between them may reach up to 9 % , as in zn - bchla . the macrocycle core is the widest in zn - bchla ( two diameters are equal to 3.969 and 4.327 ) , and the tightest in ni - bchla ( 3.874 and 3.998 ) . one should notice then that the central metal is never located in the exact center of the cavity , but is always shifted toward the fragment of the macrocycle of a larger electron density ( pyrrole c fused with the isocyclic ring ) . such small differences arise from the fact that frontier orbitals are located mostly on the macrocycle , with small contributions from metal orbitals appearing only in lumos see supplementary material . according to our knowledge , an attempt is then made to somehow predict the absorption edge based on the calculated properties of the remaining species . therefore it is predicted that pt - bchla would absorb at 752 nm , ru - bchla at 738 nm , and rh - bchla at 745 nm . to somehow assess a possibility of leaching of the central metal , the exchange of central metals for protons from environment is considered as described by the following reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathrm{mbchla } + 2{{\mathrm{h}}_5}{{\mathrm{o}}_2}^{+}+n{{\mathrm{h}}_2}\mathrm{o}\to { { \mathrm{h}}_2}\mathrm{bchla } + { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{4+\mathrm{n } } } } \right]}^{2 + } } . the number of aqua ligands is typical for a given metal ion and equals six for mg , co , cu , ru , rh and four for zn , pt , pd , ni . it is then proposed that the measure of the stability of the metal - ligand adduct is the energetic cost of the abovementioned process , calculated in aqueous phase ( accounted for with cosmo model):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { { \mathrm{e}}_{\mathrm{ex}}}=\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{bchla } } \right)+\mathrm{e}\left ( { { { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{{4+\mathrm{n } } } } } \right]}}^{2 + } } } \right)-\mathrm{e}\left ( { \mathrm{m}\mathrm{bchla } } \right)-2\mathrm{e}\left ( { { { \mathrm{h}}_5}{{\mathrm{o}}_2}^{+ } } \right)-n\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right ) . the analysis of the collected data reveals that the native central atom of bacteriochlorophyll , magnesium , is not stable in the macrocycle cavity . this means that the application of zn - bchla into the tissues might result in the decomposition of the complex into bacteriochlorophyll and the hydrated zinc ion provided the zn - bchla complex is labile . mg derivative is characterized by the lowest stability index and zn - bchla is the second least stable , the same as predicted in the present paper . in view of the potential applications of the studied metal - bacteriochlorins for therapeutic purposes , the high stability of pt , pd , these metal ions are cytotoxic and thus their uncontrolled release in tissues might result in more harm than is gained from their therapeutic properties . on the one hand the photophysical properties of the macrocyclic fragment would be in use during light irradiation phase and on the second hand , their central metals might serve as therapeutic tools to fight with anomalous cells afterward . in all but one of the investigated cases , the ground state of the molecules is low spin , which is singlet or dublet , depending on the type of the central metal ion . additionally , literature data are listed , which are referred to in the manuscript : qx max absorption in nm , i pauling electronegativity , ri ionic radius in the singlet - triplet energy separation is an important parameter regarding proposed application of the investigated systems for photodynamic therapy . the analysis of the geometries of m - bchla systems reveal that all cores of the macrocyclic complexes are planar , independent of the type of the central metal ion . this clearly shows the flexibility of the bacteriochlorin macrocycle and its ability to accommodate ions of different radii . out - of - plane displacement of the central metals in porphyrin - related systems , often reported in literature , is related strictly to the presence of additional ligands , which pull them from the center of the macrocycle [ 3840 ] . four pyrroles , which constitute the core of bacteriochlorin , are not equivalent and so four metal studied systems one may observe a similar pattern : the shortest bond is formed with the nitrogen from the pyrrole c , which is fused with the isocyclic ring , while the longest with the nitrogen to which originally phytyl is attached ( pyrrole d ) . the remaining two bonds are neither of the same length : the shorter connects metal with nitrogen of pyrrole a , while the longer with nitrogen from pyrrole b. one may then measure the size of the central cavity , which has a shorter diameter along the line connecting pyrroles a and c , and a larger diameter connecting pyrroles b and d ; the difference between them may reach up to 9 % , as in zn - bchla . the macrocycle core is the widest in zn - bchla ( two diameters are equal to 3.969 and 4.327 ) , and the tightest in ni - bchla ( 3.874 and 3.998 ) . one should notice then that the central metal is never located in the exact center of the cavity , but is always shifted toward the fragment of the macrocycle of a larger electron density ( pyrrole c fused with the isocyclic ring ) . such small differences arise from the fact that frontier orbitals are located mostly on the macrocycle , with small contributions from metal orbitals appearing only in lumos see supplementary material . an attempt is then made to somehow predict the absorption edge based on the calculated properties of the remaining species . therefore it is predicted that pt - bchla would absorb at 752 nm , ru - bchla at 738 nm , and rh - bchla at 745 nm . the number of aqua ligands is typical for a given metal ion and equals six for mg , co , cu , ru , rh and four for zn , pt , pd , ni . the idea to relate the metallobacteriochlorophyll stability to the resistance toward the displacement of the central metal by acid is generally accepted in metalloporphyrin research [ 4244 ] . it is then proposed that the measure of the stability of the metal - ligand adduct is the energetic cost of the abovementioned process , calculated in aqueous phase ( accounted for with cosmo model):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { { \mathrm{e}}_{\mathrm{ex}}}=\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{bchla } } \right)+\mathrm{e}\left ( { { { { \left [ { \mathrm{m}{{{\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right)}}_{{4+\mathrm{n } } } } } \right]}}^{2 + } } } \right)-\mathrm{e}\left ( { \mathrm{m}\mathrm{bchla } } \right)-2\mathrm{e}\left ( { { { \mathrm{h}}_5}{{\mathrm{o}}_2}^{+ } } \right)-n\mathrm{e}\left ( { { { \mathrm{h}}_2}\mathrm{o } } \right ) . the analysis of the collected data reveals that the native central atom of bacteriochlorophyll , magnesium , is not stable in the macrocycle cavity . this means that the application of zn - bchla into the tissues might result in the decomposition of the complex into bacteriochlorophyll and the hydrated zinc ion provided the zn - bchla complex is labile . mg derivative is characterized by the lowest stability index and zn - bchla is the second least stable , the same as predicted in the present paper . in view of the potential applications of the studied metal - bacteriochlorins for therapeutic purposes , the high stability of pt , pd , rh and ru derivatives might be beneficial . the present study allowed for the structural characterization of bacteriochlorophylls containing non - native divalent metal ions : co , ni , cu , zn , ru , rh , pd , and pt . the main stress was put on the derivatives with metals , which already found applications in anti - tumor therapy in the form of other complexes , with an aim to combine their properties with the known suitability of bacteriochlorophylls to photodynamic therapy . the geometry structures of the studied species show the surprising flexibility of bacteriochlorophyll cavity , which manifests itself in an ability to accommodate metal ions of different sizes . the test reaction , which was the exchange of the central metal ion for two protons showed that all studied metal macrocycle adducts are stable in aqueous media , except for mg - bchla . the stability of zn - bchla would be minor and in real conditions would depend mostly on the kinetics of its decomposition . the predicted inertness of the synthetic derivatives with metals of anti - tumor activity ( ru , pt , pd , rh ) might turn out beneficial for therapeutic purposes .

the lack of growth control in tumor cells is a result of alterations in a variety of molecules and regulatory pathways involved in cell cycle control . in particular , the transition from g1 to s phase is deregulated in cancer . progression of g1 phase is promoted by active complexes of cyclin - dependent kinases ( cdks ) and cyclins . among them cdk4 associated with its cofactor cyclin d catalyzes phosphorylation of retinoblastoma family proteins ( prb ) . nuclear proteins prb exhibit numerous threonine or serine residues as phosphorylation sites [ 3 , 4 ] . prb interact as pocket proteins by binding and inhibiting critical regulatory proteins , including transcription factors of e2f family . thus , prb act as suppressors of cellular proliferation and regulators of cell cycle in mammalian cells . phosphorylation of prb by cdk4-cyclin d at specific site ser780 inactivates prb and leads to release of e2f proteins from the inhibitory prb - e2f - complex . as a consequence transcription of e2f - regulated genes e2f family proteins regulate expression of genes , whose protein products are necessary for cell cycle progression , apoptosis , and dna repair . thereby , among other cell cycle dependent gene expressions of e2f-1 or proliferating cell nuclear antigen ( pcna ) , a cofactor of dna polymerases that encircles dna , is activated [ 8 , 9 ] . the disruption and hyperactivation of the cdk4-cyclin d / prb / e2f pathway in most cancer types such as malignant sarcomas , gliomas , and breast carcinomas [ 1 , 1012 ] have made the cdk4-cyclin d complex an attractive molecular target for cancer therapy . specific inhibition of cdk4-cyclin d results in prb hypophosphorylation which prevents cell proliferation and further tumor growth . in recent years , a large number of selective cdk4 inhibitors have been described in medicinal chemistry literature [ 1416 ] . in this [ 17 , 18 ] identified various pyrido-[2,3-d]pyrimidin-7-ones derivatives as selective inhibitors of the cdk4-cyclin d complex ( ic50 232 nm ) . one of these derivatives ( pd 0332991 ) has entered clinical trials for cancer therapy , for example , an accomplished clinical phase i study of oral pd 0332991 in patients with advanced solid tumors , excluding small cell lung cancer and retinoblastoma , and an ongoing study of pd 0332991 in clinical phases i and ii in combination with bortezomib and dexamethasone in patients with multiple myeloma . among this series of compounds , an iodine - containing analog ( compound 30 ) has also been reported as a potent cdk4-cyclin d inhibitor ( ic50 5 nm ) causing inhibition of tumor cell proliferation . the iodine substituent represents an attractive site for an isotopic substitution with several radioiodine isotopes yielding radiolabeled compounds for molecular imaging purposes . within the arsenal of molecular imaging methodologies , positron emission tomography ( pet ) represents an important , noninvasive technique for the development and evaluation of anticancer strategies through visualization and assessment of tissue function and quantification of metabolic pathways in vivo . pet uses short - lived positron emitters like carbon-11 ( t1/2 = 20.39 minutes ) , fluorine-18 ( t1/2 = 109.77 minutes ) and iodine-124 ( t1/2 = 4.18 days ) for labeling of biomolecules . the resulting pet radiotracers can be used for in vivo imaging to provide quantitative kinetic information of physiological processes . at present pet represents the most selective and sensitive method ( at the picomolar to nanomolar level ) to depict physiologic , metabolic , and molecular pathways in vivo . while it lacks the spatial resolution of other imaging techniques like magnetic resonance imaging ( mri ) or computed tomography ( ct ) , it is unrivalled in specificity and kinetic sensitivity . apart from its capability to provide pathophysiological information on human disease , for example , by identification of primary tumors or by tumor staging , pet is also important for the precise assessment of therapeutic efficacy . pet also has become an important technique for the development and evaluation of novel anticancer strategies . moreover , pet has also entered the field of modern drug development and evaluation . it provides a unique platform to study pharmacokinetics , pharmacodynamics , and the mode of action of novel drugs in both animal models and humans . most clinical pet studies use 2-[f]fluoro-2-deoxyglucose ( [ f]fdg ) as the radiotracer of choice for tumor imaging . however , although [ f]fdg - pet allows tumor imaging with high sensitivity , its application is sometimes limited by its lack of specificity as documented by the inability to discriminate between malignancies and inflammation . therefore , alternative pet radiotracers are required for specific tumor imaging and further investigation of tumorigenic processes . we hypothesize that appropriately i - labeled selective cdk4 inhibitors represent a novel promising class of pet radiotracers for imaging and functional characterization of tumorigenesis in vivo by pet . currently , an exact and accurate assessment of cdk4 protein levels and/or activity in tumors or other tissues can only be achieved by laborious analyses ex vivo . moreover , instability of cdk4 mrna and protein leads to further difficulties in terms of the intervals between tissue sampling and time of analysis . in this line , noninvasive monitoring by means of nuclear molecular imaging techniques like pet provides unique opportunities to obtain data on functional cdk4 protein expression or activity during tumorigenesis . moreover , the development and in vivo study of appropriately radiolabeled selective cdk4 inhibitors would provide pharmacological data , which may help to understand their exact physiological actions and metabolic pathways . the aim of our study was the biological , biochemical , and radiopharmacological characterization of two cdk4 inhibitors ( 8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl - phenylamino)-8h - pyrido[2,3-d]-pyrimidin-7-one ( ckia ) and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl)-pyridin-2-yl - amino)-8h - pyrido[2,3-d]pyrimidin-7-one ( ckib ) ) in different tumor cell lines . we investigated cell proliferation , cell cycle distribution , prb phosphorylation , and mrna expression of e2f-1 and pcna in tumor cells after treatment with ckia and ckib . our data demonstrate a definite and specific inhibition of tumor cell proliferation after incubation with ckia and ckib . moreover , radiotracers [ i]ckia and [ i]ckib were used to study uptake in human tumor cells and to investigate in vitro stability . the results are important prerequisites for the design of in vivo biodistribution and imaging studies to further support our hypothesis that radiolabeled cdk4 inhibitors are suitable radiotracers for tumor imaging by means of pet . compounds [ i]ckia and [ i]ckib were prepared following standard radioiododestannylation protocols as reported by eisenhut and mier . compounds ckia and ckib and the corresponding stannylated labeling precursors ( ckiap and ckibp ) were prepared with small modifications according to the procedure described by barvian et al . , and vanderwel et al . . radiosynthesis of [ i]ckia and [ i]ckib were performed by i - iodination of the corresponding trimethyl stannylated precursors by regioselective destannylation under mild conditions using [ i]nai and chloramine - t or iodogen as oxidizing agents ( scheme 1 ) . the preparative synthesis starting from 50 l of ckiap ( 5 mg / ml ) and 36.08 mbq [ i]nai using iodogen precoated tubes as oxidizing agent afforded 11.8 mbq ( 33.6% , decay - corrected ) of [ i]ckia within 104 minutes , including hplc purification . the optimized reaction conditions for the preparative [ i]ckia - synthesis were adopted for the synthesis of [ i]ckib . starting from 50 l of a 5 g/l ckibp solution in dmso and 5% glacial acetic acid in methanol ( 1/3 ) and 28.89 mbq of [ i]nai within 107 minutes 7.78 mbq ( 27.8% , decay - corrected ) of [ i]ckib was obtained , including hplc purification . human tumor cell lines ht-29 , a colorectal adenocarcinoma cell line , fadu , a head and neck squamous cell carcinoma cell line and thp-1 , an acute monocytic leukemia cell line , were obtained from dsmz , braunschweig , germany , and cultured in mccoy 's 5a medium ( ht-29 ) or rpmi 1640 medium ( fadu , thp-1 ) supplemented with 10% fetal bovine serum ( fbs ) and 1% penicillin - streptomycin . thp-1 cells were differentiated with 64 nm 12-o - tetradecanoylphorbol-13-acetate ( tpa ; sigma - aldrich , mnchen , germany ) for 72 hours to macrophage - like cells ( thp-1 macrophage ) . after that , medium containing tpa was removed , and cells were replenished with fresh rpmi medium lacking tpa for respective incubation with ckia and ckib ( 72 hours maximum ) . during this time thp-1 macrophage cells remain differentiated . all cells were cultured at 37c , 5% co2 , and 95% humidity in a co2 incubator ( heracell , heraeus , hanau , germany ) . as a control to classify the effect of ckia or ckib , first cells were cultured until confluence and then cell culture medium with only 0.25% fbs was applied for 72 hours . cell growth inhibition was analyzed for up to 72 hours after treatment of ckia and ckib . 2.5 10 cells were seeded into a cell culture flask and incubated for 24 hours . then 0.1 or 1.0 m of the respective compound was added into the cell culture media ( either every 24 hours or only once ) and cells were counted with the casy model tt cell counter ( schaerfe - system , reutlingen , germany ) after 24 , 48 , and 72 hours of incubation with ckia or ckib . after treatment of 1 10 cells for 24 hours without or with ckia or ckib , cells were washed with pbs and detached from the cell culture flask by addition of trypsin ( ht-29 , fadu ) or accutase ( differentiated thp-1 ) . after centrifugation for 5 minutes at 1000 rpm at 4c cells were washed twice with ice - cold pbs and fixed in 70% ethanol at 4c for at least 1 hour . ethanol - fixed cells were washed again , treated with 1 g / ml ribonuclease i ( sigma - aldrich ) for 30 minutes at 37c and stained with 10 g / ml propidium iodide ( sigma - aldrich ) in the dark . flow cytometry analysis was performed on an facscalibur flow cytometer ( becton dickinson , zrich , switzerland ) by use of the cellquest pro software . for cell cycle analysis , 20000 events had been collected in the single - events region with a total event rate not exceeding 300 events / second . data were analyzed with modfit lt software package . at the end of treatment with ckia or ckib , cells were washed with ice - cold pbs and lysed in lysis buffer ( 50 mm tris - hcl ph 7.5 , 150 mm nacl , 0.5% nonidet p40 with 1 mm sodium orthovanadate , 1 mm naf , 10 mm -glycerophosphate , 1 mm dithiothreitol ( dtt ) , 1 mm phenyl - methyl sulfonyl fluoride , 1 mm pefablock , and 1 g / ml leupeptine ) . cells were centrifuged at 15000 g for 15 minutes at 4c . protein concentration in the supernatants were quantified using the bca protein assay kit ( pierce , rockford , usa ) according to the manufacturer 's recommendations and bovine serum albumin as protein standard . 2550 g of protein were mixed with sample buffer , denatured at 95c for 5 minutes and separated on 7.5% sodium - dodecylsulfate ( sds)-polyacrylamide gels for prb detection . after electrophoresis proteins were transferred to nitrocellulose membranes by electroblotting and blocked for 1 hour at room temperature in tbs / tween ( 50 mm tris - hcl ph 8.0 , 150 mm nacl , 0.05% tween-20 ) containing 5% nonfat milk . membranes were incubated with the primary antibodies phospho - rb(ser780 ) ( 1 : 1000 , no . 9309 , cell signaling technology ) at 4c over night followed by incubation with secondary peroxidase - conjugated antibodies antirabbit igg ( 1 : 10000 , a0545 , sigma - aldrich ) or antimouse igg ( 1 : 10000 , a9044 , sigma - aldrich ) for 1 hour at room temperature . after washing with tbs / tween proteins were visualized by chemiluminescence using the supersignal west pico chemiluminescent substrate ( pierce , rockford , usa ) and exposed to kodak biomax light films ( sigma - aldrich ) for 515 minutes . in order to detect a second protein , blots were stripped by incubation with 62.5 mm tris - hcl ph 6.7 , 2% sds and 0.7% -mercaptoethanol at 55c for 15 minutes . after that , again incubation with primary polyclonal antibody anti--actin igg ( 1 : 1000 , a5060 , sigma - aldrich ) and secondary peroxidase - conjugated antibody antirabbit igg ( 1 : 10 , 000 , a0545 , sigma - aldrich ) , and protein detection was performed as described above . after 24-hour - incubation with ckia or ckib rna was extracted using the mirneasy mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . leon - roth , germany ) according to the manufacturer 's guidelines to prevent genomic dna contamination . the reverse transcription and quantitative real - time pcr of specific mrna was carried out in one step from 50 ng of total rna and intron - spanning primers ( metabion , munich , germany ) using the quantitect sybr green rt - pcr kit ( qiagen ) according to the manufacturer 's instructions . one - step quantitative real - time rt - pcr was performed in a mastercycler ep realplex system ( eppendorf , hamburg , germany ) . then , quantitative real - time pcr was performed for 40 cycles with denaturation at 95c for 20 seconds , annealing at 56 to 59c ( depending on the primers , table 1 ) for 20 seconds and extending at 68c for 30 seconds . all pcr products were checked by melting point analysis upon completion of pcr cycles by heating up from 55 to 95c in 20 minutes . for each cell line and treatment two to four different rna samples were analyzed as triplicates . a negative control without rna template was included in every measurement for the different primers . for each experimental sample , mrna levels were normalized to -actin mrna levels ( threshold cycle -actin threshold cycle mrna of interest = ct ) . therefore primers for the amplification of -actin were included in every measurement to correct for sample - to - sample variations . mrna levels were presented as 2 ( ct = ct ( treatment ) ( ct(control ) ) . stability of [ i]ckia and [ i]ckib was examined after 1 hour at 37c in different physiological buffers ( ph 4.2 , 7.4 , 9.0 ) , cell culture medium , and rodent plasma by hplc analysis . for radiotracer uptake experiments , 5 10 cells were seeded in a cavity of a 24-well plate ( greiner , frickenhausen , germany ) and cultured in a humidified atmosphere of 5% co2 at 37c using the above - mentioned cell culture media . after overnight incubation , 0.5 ml of cell culture medium with about 30 kbq [ i]ckia or [ i]ckib was added and incubation was continued for several time points at 37c and 4c . subsequently , cells were washed three times with ice - cold pbs and lysed in 0.5 ml 0.1 m sodium hydroxide with 1% sds . cell lysates were counted with a cobra ii gamma counter ( canberra - packard , meriden , ct , usa ) . protein levels were quantified using the bca protein assay kit ( pierce , rockford , usa ) according to the manufacturer 's recommendations and bovine serum albumin as protein standard . uptake data for all experiments are expressed as percent of injected dose per mg protein ( % id / mg protein ) . descriptive data were expressed as arithmetic means standard deviations . the number of n in the figure legends represents the number of independent experiments . spearman 's rank correlation coefficient was calculated between data related to cdk4 activity and radiotracer uptake . for all analyses a value of p < .05 was considered as statistically significant . the spss statistical software package ( version 12.0 for windows ; for the characterization of cell cycle inhibitors ckia and ckib three different tumor cell lines were studied with respect to their concentration dependent cell proliferation . cells show with 17 hours the shortest doubling time . for fadu cells a doubling time of 22 hours and for thp-1 cells of 38 hours after 72 hours of incubation with tpa , thp-1 monocyte - like suspension cells adhere almost completely at the bottom of culture flask and differentiate to macrophage - like cells . tpa differentiated thp-1 cells ( thp-1 macrophage ) showed no more cell proliferation and served as control cell model . tumor cell growth studies with ckia ( every 24 hours treatment ) indicated a significantly reduced cell proliferation in all tumor cell lines at 48 and 72 hours after treatment with 0.1 ( < 50% ) and 1 m ( < 25% ) of ckia ( figure 1 , p < .05 ) . nonrecurring treatment with 1 m of ckia for 72 hours provided similar findings compared to treatment every 24 hours . also in thp-1 macrophages , cell number was decreased by 33% with 0.1 m of ckia ( figure 1 ) . generally , ckib showed similar effects on cell proliferation , albeit these effects were only achieved at higher ckib concentrations or by longer incubation time compared to ckia . after 48 hours of treatment with ckib , cell proliferation in ht-29 cells was reduced by 35% ( 0.1 m ) and 61% ( 1.0 m ) , respectively , in fadu cells 29% ( 0.1 m ) and 46% ( 1.0 m ) , respectively , and in thp-1 cells 20% ( 0.1 m ) and 46% ( 1.0 m ) , respectively ( figure 1 , p < .05 ) . 72 hours after treatment with 1.0 m of ckib cell proliferation was decreased to 35 , 45 , or 28% ( ht-29 , fadu , thp-1 ) . cell number of thp-1 macrophage cells for this incubation condition was also significantly depleted . already 24 hours after incubation with ckia the percentage of tumor cells in g1 phase showed a concentration dependent increment of up to 89.9% in ht-29 and 84.5% in fadu tumor cells ( figure 2 , table 2 , p < .05 ) . ckia induced a g1 phase arrest after incubation with 0.05 , 0.1 , and 0.5 m in thp-1 cells . at higher concentration of 1.0 m , however , cell cycle distribution switched back to values obtained for untreated cells . over 89% of ht-29 and thp-1 cells in g1 phase were obtained after 24 hours of incubation with 1.0 m of ckib ( table 2 , p < .05 ) . a concentration dependent alteration of cell cycle distribution after incubation with ckib was also observed in fadu cells , but 24 hours after incubation with 1.0 m of ckib only 70.7% were achieved in g1 phase . thp-1 macrophage cells showed no change in percentage of g1 phase neither after incubation with ckia nor with ckib . to study the interaction of ckia or ckib with cdk4 and the influence on the downstream signaling pathway , detection of prb phosphorylation status in whole cell lysates was performed by western blot . cdk4 specific phosphorylation of prb on ser780 and were normalized to total amount of prb , -actin served as internal control . in all cell lines antibodies phospho - rb(ser780 ) and rb(4h1)mab only recognized endogenous prb species with a size of 110 kda . the total levels of prb remained constant in all cell lines , only incubation with 1 m ckia by trend showed a decrease of total prb . after incubation with ckia a concentration dependent hypophosphorylation of prb(ser780 ) was found ( figure 3 ) . prb(ser780 ) phosphorylation was decreased three- to tenfold after 24 hours of treatment with 0.1 and 1 m ckia in all cell lines . results of prb(ser780 ) phosphorylation analyses suggested a downregulation after 24 hours of incubation with 1 m of ckib in ht-29 cells ( figure 3 ) . no significant change in prb(ser780 ) phosphorylation status of fadu and thp-1 cells incubated with ckib was found . mrna expression of prb effected e2f-1 and pcna genes was measured in relation to the effects of ckia and ckib on cdk4 downstream signaling pathway . for grading and comparison of the studied effects also mrna levels of serum - deprived g1 a substantial downregulation of e2f-1 and pcna mrna expression could be demonstrated after incubation with 1 m of ckia in all cell lines studied ( figure 4 ) . in ht-29 and thp-1 tumor cells an up to 15% reduction of mrna expression for both genes ( e2f-1 and pcna ) compared to control without treatment was detectable . in ht-29 and fadu cells mrna expression of e2f-1 and pcna was in the range of corresponding levels in g1 arrested cells . further analysis of released serum - deprived ht-29 cells after 24 hours incubation with ckia exhibited a consistent downregulation of both e2f-1 and pcna mrna ( data not shown ) . also e2f-1 and pcna mrna expression analyses after 24 hours of incubation with 1 m of ckib indicated a downregulation in all cell lines and thp-1 macrophages . expression of e2f-1 and pcna mrna was decreased by ckib up to 60% in fadu or ht-29 cells , but without statistical significance . preliminary experiments with [ i]ckia and [ i]ckib indicated a sufficient stability of this compounds in various buffers ( ph 4.2 , 7.4 , and 9.0 ) , cell culture media , and rodent plasma samples in the range of 9397% at 37c for 1 hour at minimum . in vitro radiotracer uptake studies in adherent tumor cells using [ i]ckia and [ i]ckib showed substantial uptake in ht-29 and fadu cells ( figure 5 ) . the time - dependent cellular uptake was similar in both cell lines and uptake of [ i]ckia is steadily increased with time up to 5 hours . 117 % id / mg protein in ht-29 and 1033 84 % id / mg protein in fadu cells were obtained . at 4c an obvious lower uptake was detectable in both cell lines ( 258 30 % id / mg protein in ht-29 , 169 14 % id / mg protein in fadu , p < .05 ) . in vitro studies with [ i]ckib demonstrated a radiotracer uptake of 904 43 % id / mg protein in ht-29 and 856 45 % id / mg protein in fadu cells after 2 hours at 37c . radiotracer uptake of [ i]ckib was only marginally increased up to 5 hours at 37c and again radiotracer uptake at 4c was substantially lower compared to 37c ( p < .05 ) . in 2005 , ckia was reported as a specific and potent cdk4 inhibitor with an ic50 value of 5 nm by vanderwel et al . , but in spite of promising properties only marginal examinations concerning inhibition of tumor cell proliferation were performed . the purpose of our study was the further evaluation of the known cdk4 inhibitor ckia and of a new derivative compound ckib concerning their biological , biochemical , and radiopharmacological properties , to proof the feasibility and suitability of these cdk4 inhibitors for radiotracer development and imaging of cell proliferation processes in vivo especially in tumor entities . ckib was designed as a less lipophilic analog of cdk4 inhibitor ckia to reduce nonspecific binding . to the best of our knowledge , for the first time cdk4 inhibitors ckia and ckib were demonstrated to act as tumor cell growth inhibitors via specific inhibition of cdk4/prb / e2f signaling pathway and induction of g1 arrest in cells . for our studies , ht-29 , fadu , and thp-1 cells were used as continuously proliferating cell models . the three tumor cell lines represent common models for human solid tumors ( ht-29 , fadu ) and leukemia ( thp-1 ) . furthermore , tpa differentiated thp-1 cells served as a cellular model that shows no cell proliferation potential due to their arrest in cell cycle . the tumor cell lines studied exhibited different doubling rates , which could influence the impact of cell cycle inhibitors . however , cell proliferation after treatment with ckia or ckib showed no conspicuous differences between rapid proliferating ht-29 or slower proliferating fadu and thp-1 cells , respectively . in all cell lines a significant concentration and time dependence of cell division after 72 hours of incubation with 1.0 m of ckia or ckib a decrease in cell number about 5590% compared to control without treatment could be found in tumor cells . ckia and ckib inhibit cell division , but no decrease of cell number under the seeded cell number was observed after incubation with ckia or ckib . control cells thp-1 macrophage only show decreased cell numbers after 72 hours of incubation with ckib . interestingly , also nonrecurring treatment with 1.0 m of compounds for 72 hours showed similar cell number values as for ever 24 hours treatment , which could be an indication for good availability and/or stability of active ckia and ckib in the cells . it is likely , because of action of ckia and ckib in the nucleus , that these compounds are not accessible for transport outside of cells , for example , by multidrug resistance proteins ( mdr ) . measurement of cell cycle distribution clarified and complemented the observed data of cell growth studies . 24 hours after incubation with ckia or ckib a concentration dependent increment of cells in g1 phase was detectable in all tumor cell lines . unlike to cell growth studies , differences between the tumor cell lines after treatment with a certain concentration of ckia or ckib were found . cell cycle distribution studies demonstrated a lower increment of percentage of fadu cells in g1 phase compared to ht-29 or thp-1 cells . for example , 24 hours after incubation with 0.1 m ckia percentage of cells in g1 phase raised up 10.5% in fadu , 22.5% in ht-29 , and 33.4% in thp-1 cells . surprisingly , at a concentration of 1.0 m of ckia cell cycle distribution values in thp-1 cells were observed similar to untreated cells . , in human breast carcinoma cells mda - mb453 after treatment with other pyrido[2,3-d]pyrimidin-7-one derivatives identified as cdk4 inhibitors . this unexpected characteristic of cell cycle distribution observed for a few cdk4 inhibitors at higher concentrations ( > 1.0 m ) is still not understood and requires further investigations to clarify this phenomenon . the effect of ckia on cell cycle distribution was considerably higher compared to ckib at the same concentration range . nevertheless , both ckia and ckib caused a cell cycle arrest in g1 phase ( g1 85% ) in tumor cell lines ht-29 , fadu , and thp-1 , although a higher concentration of ckib is required to obtain the same extent of cell cycle arrest . only in fadu cells ckib failed to reach an increment of g1 phase exceeding 85% ( achieved for 1 m ckia ) under conditions determined . after incubation with 1.0 m ckib only 70.7% of fadu cells were achieved in g1 phase . to further characterize the cell cycle arrest , we analyzed the cdk4 specific prb phosphorylation status and mrna levels downstream of cdk4-cyclin d / prb pathway . rb gene is mutated in some types of cancer [ 24 , 25 ] and nonfunctional prb evades regulation through cdk - induced phosphorylation . as inhibitors for the cdk4-cyclin d / prb pathway , compounds ckia and ckib should inhibit cell proliferation and prb phosphorylation of cells with functional prb . ht-29 and fadu are tumor cell lines with normal prb status [ 27 , 28 ] , and also in thp-1 cells full length prb is found , which indicate the existence of functional prb in these tumor cell line . more than ten phosphorylation sites of prb are known in vivo . during g1/s transition cdk4/6-cyclin d and cdk2-cyclina / e are involved in prb phosphorylation , but different specificity of serine residues of prb were identified for these different cdk - cyclin - complexes [ 4 , 30 ] . while cdk4-cyclin d efficiently and specifically phosphorylates prb on ser780 and ser795 , cdk2-cyclin a / e does not . analyses of prb phosphorylation resulted in a significantly decreased ser780 phosphorylation in all cell lines studied dependent on concentration of ckia . after 24 hours of incubation with ckib a tendency of prb hypophosphorylation on ser780 in ht-29 and thp-1 macrophage cells was observed . additional experiments with specific antibodies against phospho - ser795 of prb also demonstrated a ckia concentration dependent decrease of prb phosphorylation . the cell cycle regulatory activity of prb is especially mediated through its binding of transcriptional factors like e2f , which regulates growth - promoting genes . prb phosphorylated on ser780 can not bind to e2f transcription factors in vivo and as a consequence of prb release from the prb - e2f complex mrna expression of e2f regulated genes , for example , e2f-1 or pcna is activated [ 8 , 31 ] . a substantial downregulation of both e2f-1 and pcna mrna expression was found after 24 hours of incubation with ckia in all cell lines . the mrna levels in ht-29 and fadu with 1.0 m of ckia are comparable to that of serum - deprived cells arrested in g1 phase . also after treatment with 1.0 m of ckib our data report a distinct inhibition of prb phosphorylation by cdk4-cyclin d as the consequence of ckia and ckib effects in cells . reduced prb phosphorylation leads to a disruption of the e2f cascade , exemplified for e2f-1 and pcna mrna expression . it can be assumed that other genes downstream of cdk4-cyclin d / prb / e2f pathway are affected through ckia and ckib induced inhibition of prb phosphorylation , for example , genes essential for dna duplication and cell cycle control like dna polymerase and cyclin e. the influence of ckia and ckib to the cdk4-cyclin d / prb / e2f pathway finally ends in cell cycle arrest and a stop of cell proliferation . ckia , a pyrido[2,3-d]pyrimidin-7-one derivative , was reported by vanderwel et al . , in 2005 for the first time as a specific and potent cdk4 inhibitor with an ic50 value of 5 nm . all derivative compounds synthesized by this group were classified concerning their inhibition of different kinases by means of ic50 values measured by in vitro kinase assays . ic50 values allow a comparison between different compounds , but no inferential evidence about their properties in cells can be made . furthermore , cell effective concentration is much higher than ic50 value determined by an in vitro kinase assay , due to the two barriers cell membrane and nuclear membrane that have to be overcome , and possible metabolism of the compound within the cell . hence , ic50 values determined by in vitro kinase assays should not be the only discriminating argument for choice of a suitable compound for therapeutic applications . in our opinion , flow cytometry is a meaningful , fast , and reproducible method for evaluation of selective cell cycle inhibitors concerning their effect in cells . previous evaluation of ckia in consideration of tumor cell growth inhibition of human colon carcinoma cell line hct116 , measuring the [ c]-thymidine incorporation , resulted in an ic50 value of 0.104 m . although a direct comparison is not possible because of variations in methods and conditions , this finding correlates very well with our data observed for human colon carcinoma cell line ht-29 by flow cytometry . in ht-29 , an ed50 value of 0.095 m ckia could be calculated for increased percentage of cells in g1 phase . in conclusion , among others flow cytometry would be a good alternative for inhibitor classification . however , vanderwel et al . has focused on other derivatives of pyrido[2,3-d]pyrimidin-7-one , regarding to the reduced selectivity of ckia compared to other compounds investigated . pd 0332991 has been extensively characterized among other highly selective inhibitors concerning biochemical and biological properties in vitro and in vivo [ 15 , 32 ] . in consequence pd the question to be answered is , if exceeding high selectivity of pyrido[2,3-d]pyrimidin-7-one derivatives against other cdks than cdk4 will be of outstanding importance for tumor treatment . an expanded effect on different cdks could also be imaginable for effective tumor cell growth inhibition . in summary , ckia and also the new compound ckib were demonstrated to act as tumor cell growth inhibitors via specific inhibition of cdk4-cyclin d / prb / e2f signaling pathway . the mechanism of action confirms the therapeutic benefit of these compounds in field of cancer treatment , although their lowered selectivity . we hypothesized that potent cdk4 inhibitors , which are suitable for cancer therapy , are also of interest as radiotracers for imaging of cell proliferation processes in vivo and especially for tumor visualization by positron emission tomography ( pet ) . for pet imaging application , beside the knowledge about biological and biochemical effectiveness of potent cdk4 inhibitors also radiopharmacological characterization of respective radiotracers is required . based on the structure of the two compounds ckia and ckib appropriate radiolabeling strategies were designed and radiolabeling with the positron - emitting nuclide iodine-124 was performed ( decay - corrected radiochemical yields : 33.6% [ i]ckia , and 27.8% [ i]ckib , purity : > 98% , resp . ) . using [ i]ckia and [ i]ckib stability and radiotracer uptake studies were performed in ht-29 and fadu cells . in both tumor cell lines a substantial cellular uptake of [ i]ckia and [ i]ckib was observed at 37c . in posterior phase ( > 1 hour ) [ i]ckia showed a higher cellular uptake compared to [ i]ckib . this is consistent with the different therapeutic potency of ckia and ckib observed in tumor cell inhibition studies . at lower temperature of 4c obvious lower uptakes pyrido[2,3-d]pyrimidin-7-one derivatives were predicted to bind atp site of cdks , thus implying a structural similarity to atp . nucleoside transporters ( nts ) mediate the uptake of physiologic nucleosides like adenosine and were also reported to transport anticancer nucleoside drugs . especially , energy - dependent concentrative nucleoside transporters ( cnts ) could be presumed for ckia and ckib transport into cells . since no specific pharmacological inhibitor has been found for cnts yet , it is difficult to confirm this presumption . the identification of specific transporters of pyrido[2,3-d]pyrimidin-7-one derivatives will contribute to understanding of effectiveness of these compounds and especially for radiotracer uptake a valuation of tumor accumulation could be made depending on transporter frequency of tumor species . correlation analysis only showed a weak correlation between e2f-1 mrna expression as measure of cdk4 enzyme activity and cki ( ckia and ckib ) uptake ( = 0.390 , p = .042 , n = 15 ) . according to this , the transport of both radiotracers into the cells would be the rate determining step for cellular uptake of ckia and ckib at least in monolayer cell cultures . a therapeutic active compound must reach and affect a certain target , whereas the agent for imaging only has to be retained by the target . certainly , many other properties of a radiotracer like biodistribution or lipophilicity contribute to a specific tumor accumulation and are relevant for high - contrast images . the inhibitory effect of ckia or ckib would only be of subordinate interest regarding the imaging application of these compounds . nevertheless , high specificity and selectivity are a prerequisite for specific accumulation in tumor cells . however , a less selective or specific compound with better biodistribution properties but comparable tumor uptake could have advantages for the application as radiotracer for imaging of tumors by pet . in summary , our data demonstrate a definite and specific inhibition of tumor cell proliferation when incubated with ckia and ckib due to an arrest of tumor cells in g1 phase . the repression of g1 progression is achieved by inhibition of the cdk4-cyclin d / prb / e2f pathway . the tumor cell growth inhibition observed in vitro point out the potential therapeutic benefit of these compounds , which has to be proven in further experiments in vivo . the radiotracer uptake observed in human tumor cells and the stability of [ i]ckia and [ i]ckib as found in physiological buffers are promising prerequisites for in vivo biodistribution and imaging studies by pet . an interesting effect arising from the radiotracer uptake studies using [ i]ckia and [ i]ckib is the observed energy dependent transport of ckia and ckib , due to the difference of uptake at 37c and 4c . regard , and to further support our hypothesis that radiolabeled cdk4 inhibitors are suitable radiotracers for tumor imaging , comprehensive in vivo experiments of [ i]ckia and [ i]ckib involving biodistribution and small animal pet studies should be performed .

the cyclin - dependent kinase ( cdk)-cyclin d / retinoblastoma ( prb)/e2f cascade , which controls the g1/s transition of cell cycle , has been found to be altered in many neoplasias . inhibition of this pathway by using , for example , selective cdk4 inhibitors has been suggested to be a promising approach for cancer therapy . we hypothesized that appropriately radiolabeled cdk4 inhibitors are suitable probes for tumor imaging and may be helpful studying cell proliferation processes in vivo by positron emission tomography . herein , we report the synthesis and biological , biochemical , and radiopharmacological characterizations of two 124i - labeled small molecule cdk4 inhibitors ( 8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl - phenylamino)-8h - pyrido[2,3-d]-pyrimidin-7-one ( ckia ) and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl)-pyridin-2-yl - amino)-8h - pyrido[2,3-d]pyrimidin-7-one ( ckib ) ) . our data demonstrate a defined and specific inhibition of tumor cell proliferation through ckia and ckib by inhibition of the cdk4/prb / e2f pathway emphasizing potential therapeutic benefit of ckia and ckib . furthermore , radiopharmacological properties of [ 124i]ckia and [ 124i]ckib observed in human tumor cells are promising prerequisites for in vivo biodistribution and imaging studies .

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

progression of g1 phase is promoted by active complexes of cyclin - dependent kinases ( cdks ) and cyclins . as a consequence transcription of e2f - regulated genes e2f family proteins regulate expression of genes , whose protein products are necessary for cell cycle progression , apoptosis , and dna repair . the disruption and hyperactivation of the cdk4-cyclin d / prb / e2f pathway in most cancer types such as malignant sarcomas , gliomas , and breast carcinomas [ 1 , 1012 ] have made the cdk4-cyclin d complex an attractive molecular target for cancer therapy . specific inhibition of cdk4-cyclin d results in prb hypophosphorylation which prevents cell proliferation and further tumor growth . in recent years , a large number of selective cdk4 inhibitors have been described in medicinal chemistry literature [ 1416 ] . one of these derivatives ( pd 0332991 ) has entered clinical trials for cancer therapy , for example , an accomplished clinical phase i study of oral pd 0332991 in patients with advanced solid tumors , excluding small cell lung cancer and retinoblastoma , and an ongoing study of pd 0332991 in clinical phases i and ii in combination with bortezomib and dexamethasone in patients with multiple myeloma . among this series of compounds , an iodine - containing analog ( compound 30 ) has also been reported as a potent cdk4-cyclin d inhibitor ( ic50 5 nm ) causing inhibition of tumor cell proliferation . within the arsenal of molecular imaging methodologies , positron emission tomography ( pet ) represents an important , noninvasive technique for the development and evaluation of anticancer strategies through visualization and assessment of tissue function and quantification of metabolic pathways in vivo . the resulting pet radiotracers can be used for in vivo imaging to provide quantitative kinetic information of physiological processes . at present pet represents the most selective and sensitive method ( at the picomolar to nanomolar level ) to depict physiologic , metabolic , and molecular pathways in vivo . apart from its capability to provide pathophysiological information on human disease , for example , by identification of primary tumors or by tumor staging , pet is also important for the precise assessment of therapeutic efficacy . most clinical pet studies use 2-[f]fluoro-2-deoxyglucose ( [ f]fdg ) as the radiotracer of choice for tumor imaging . therefore , alternative pet radiotracers are required for specific tumor imaging and further investigation of tumorigenic processes . we hypothesize that appropriately i - labeled selective cdk4 inhibitors represent a novel promising class of pet radiotracers for imaging and functional characterization of tumorigenesis in vivo by pet . moreover , the development and in vivo study of appropriately radiolabeled selective cdk4 inhibitors would provide pharmacological data , which may help to understand their exact physiological actions and metabolic pathways . the aim of our study was the biological , biochemical , and radiopharmacological characterization of two cdk4 inhibitors ( 8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl - phenylamino)-8h - pyrido[2,3-d]-pyrimidin-7-one ( ckia ) and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl)-pyridin-2-yl - amino)-8h - pyrido[2,3-d]pyrimidin-7-one ( ckib ) ) in different tumor cell lines . we investigated cell proliferation , cell cycle distribution , prb phosphorylation , and mrna expression of e2f-1 and pcna in tumor cells after treatment with ckia and ckib . our data demonstrate a definite and specific inhibition of tumor cell proliferation after incubation with ckia and ckib . moreover , radiotracers [ i]ckia and [ i]ckib were used to study uptake in human tumor cells and to investigate in vitro stability . the results are important prerequisites for the design of in vivo biodistribution and imaging studies to further support our hypothesis that radiolabeled cdk4 inhibitors are suitable radiotracers for tumor imaging by means of pet . compounds ckia and ckib and the corresponding stannylated labeling precursors ( ckiap and ckibp ) were prepared with small modifications according to the procedure described by barvian et al . radiosynthesis of [ i]ckia and [ i]ckib were performed by i - iodination of the corresponding trimethyl stannylated precursors by regioselective destannylation under mild conditions using [ i]nai and chloramine - t or iodogen as oxidizing agents ( scheme 1 ) . the preparative synthesis starting from 50 l of ckiap ( 5 mg / ml ) and 36.08 mbq [ i]nai using iodogen precoated tubes as oxidizing agent afforded 11.8 mbq ( 33.6% , decay - corrected ) of [ i]ckia within 104 minutes , including hplc purification . the optimized reaction conditions for the preparative [ i]ckia - synthesis were adopted for the synthesis of [ i]ckib . starting from 50 l of a 5 g/l ckibp solution in dmso and 5% glacial acetic acid in methanol ( 1/3 ) and 28.89 mbq of [ i]nai within 107 minutes 7.78 mbq ( 27.8% , decay - corrected ) of [ i]ckib was obtained , including hplc purification . human tumor cell lines ht-29 , a colorectal adenocarcinoma cell line , fadu , a head and neck squamous cell carcinoma cell line and thp-1 , an acute monocytic leukemia cell line , were obtained from dsmz , braunschweig , germany , and cultured in mccoy 's 5a medium ( ht-29 ) or rpmi 1640 medium ( fadu , thp-1 ) supplemented with 10% fetal bovine serum ( fbs ) and 1% penicillin - streptomycin . after that , medium containing tpa was removed , and cells were replenished with fresh rpmi medium lacking tpa for respective incubation with ckia and ckib ( 72 hours maximum ) . cell growth inhibition was analyzed for up to 72 hours after treatment of ckia and ckib . then 0.1 or 1.0 m of the respective compound was added into the cell culture media ( either every 24 hours or only once ) and cells were counted with the casy model tt cell counter ( schaerfe - system , reutlingen , germany ) after 24 , 48 , and 72 hours of incubation with ckia or ckib . after that , again incubation with primary polyclonal antibody anti--actin igg ( 1 : 1000 , a5060 , sigma - aldrich ) and secondary peroxidase - conjugated antibody antirabbit igg ( 1 : 10 , 000 , a0545 , sigma - aldrich ) , and protein detection was performed as described above . stability of [ i]ckia and [ i]ckib was examined after 1 hour at 37c in different physiological buffers ( ph 4.2 , 7.4 , 9.0 ) , cell culture medium , and rodent plasma by hplc analysis . the spss statistical software package ( version 12.0 for windows ; for the characterization of cell cycle inhibitors ckia and ckib three different tumor cell lines were studied with respect to their concentration dependent cell proliferation . tumor cell growth studies with ckia ( every 24 hours treatment ) indicated a significantly reduced cell proliferation in all tumor cell lines at 48 and 72 hours after treatment with 0.1 ( < 50% ) and 1 m ( < 25% ) of ckia ( figure 1 , p < .05 ) . after 48 hours of treatment with ckib , cell proliferation in ht-29 cells was reduced by 35% ( 0.1 m ) and 61% ( 1.0 m ) , respectively , in fadu cells 29% ( 0.1 m ) and 46% ( 1.0 m ) , respectively , and in thp-1 cells 20% ( 0.1 m ) and 46% ( 1.0 m ) , respectively ( figure 1 , p < .05 ) . already 24 hours after incubation with ckia the percentage of tumor cells in g1 phase showed a concentration dependent increment of up to 89.9% in ht-29 and 84.5% in fadu tumor cells ( figure 2 , table 2 , p < .05 ) . a concentration dependent alteration of cell cycle distribution after incubation with ckib was also observed in fadu cells , but 24 hours after incubation with 1.0 m of ckib only 70.7% were achieved in g1 phase . mrna expression of prb effected e2f-1 and pcna genes was measured in relation to the effects of ckia and ckib on cdk4 downstream signaling pathway . for grading and comparison of the studied effects also mrna levels of serum - deprived g1 a substantial downregulation of e2f-1 and pcna mrna expression could be demonstrated after incubation with 1 m of ckia in all cell lines studied ( figure 4 ) . preliminary experiments with [ i]ckia and [ i]ckib indicated a sufficient stability of this compounds in various buffers ( ph 4.2 , 7.4 , and 9.0 ) , cell culture media , and rodent plasma samples in the range of 9397% at 37c for 1 hour at minimum . in vitro radiotracer uptake studies in adherent tumor cells using [ i]ckia and [ i]ckib showed substantial uptake in ht-29 and fadu cells ( figure 5 ) . , but in spite of promising properties only marginal examinations concerning inhibition of tumor cell proliferation were performed . the purpose of our study was the further evaluation of the known cdk4 inhibitor ckia and of a new derivative compound ckib concerning their biological , biochemical , and radiopharmacological properties , to proof the feasibility and suitability of these cdk4 inhibitors for radiotracer development and imaging of cell proliferation processes in vivo especially in tumor entities . to the best of our knowledge , for the first time cdk4 inhibitors ckia and ckib were demonstrated to act as tumor cell growth inhibitors via specific inhibition of cdk4/prb / e2f signaling pathway and induction of g1 arrest in cells . the three tumor cell lines represent common models for human solid tumors ( ht-29 , fadu ) and leukemia ( thp-1 ) . furthermore , tpa differentiated thp-1 cells served as a cellular model that shows no cell proliferation potential due to their arrest in cell cycle . the tumor cell lines studied exhibited different doubling rates , which could influence the impact of cell cycle inhibitors . in all cell lines a significant concentration and time dependence of cell division after 72 hours of incubation with 1.0 m of ckia or ckib a decrease in cell number about 5590% compared to control without treatment could be found in tumor cells . ckia and ckib inhibit cell division , but no decrease of cell number under the seeded cell number was observed after incubation with ckia or ckib . interestingly , also nonrecurring treatment with 1.0 m of compounds for 72 hours showed similar cell number values as for ever 24 hours treatment , which could be an indication for good availability and/or stability of active ckia and ckib in the cells . it is likely , because of action of ckia and ckib in the nucleus , that these compounds are not accessible for transport outside of cells , for example , by multidrug resistance proteins ( mdr ) . measurement of cell cycle distribution clarified and complemented the observed data of cell growth studies . unlike to cell growth studies , differences between the tumor cell lines after treatment with a certain concentration of ckia or ckib were found . for example , 24 hours after incubation with 0.1 m ckia percentage of cells in g1 phase raised up 10.5% in fadu , 22.5% in ht-29 , and 33.4% in thp-1 cells . , in human breast carcinoma cells mda - mb453 after treatment with other pyrido[2,3-d]pyrimidin-7-one derivatives identified as cdk4 inhibitors . this unexpected characteristic of cell cycle distribution observed for a few cdk4 inhibitors at higher concentrations ( > 1.0 m ) is still not understood and requires further investigations to clarify this phenomenon . the effect of ckia on cell cycle distribution was considerably higher compared to ckib at the same concentration range . nevertheless , both ckia and ckib caused a cell cycle arrest in g1 phase ( g1 85% ) in tumor cell lines ht-29 , fadu , and thp-1 , although a higher concentration of ckib is required to obtain the same extent of cell cycle arrest . to further characterize the cell cycle arrest , we analyzed the cdk4 specific prb phosphorylation status and mrna levels downstream of cdk4-cyclin d / prb pathway . as inhibitors for the cdk4-cyclin d / prb pathway , compounds ckia and ckib should inhibit cell proliferation and prb phosphorylation of cells with functional prb . ht-29 and fadu are tumor cell lines with normal prb status [ 27 , 28 ] , and also in thp-1 cells full length prb is found , which indicate the existence of functional prb in these tumor cell line . during g1/s transition cdk4/6-cyclin d and cdk2-cyclina / e are involved in prb phosphorylation , but different specificity of serine residues of prb were identified for these different cdk - cyclin - complexes [ 4 , 30 ] . the cell cycle regulatory activity of prb is especially mediated through its binding of transcriptional factors like e2f , which regulates growth - promoting genes . prb phosphorylated on ser780 can not bind to e2f transcription factors in vivo and as a consequence of prb release from the prb - e2f complex mrna expression of e2f regulated genes , for example , e2f-1 or pcna is activated [ 8 , 31 ] . also after treatment with 1.0 m of ckib our data report a distinct inhibition of prb phosphorylation by cdk4-cyclin d as the consequence of ckia and ckib effects in cells . it can be assumed that other genes downstream of cdk4-cyclin d / prb / e2f pathway are affected through ckia and ckib induced inhibition of prb phosphorylation , for example , genes essential for dna duplication and cell cycle control like dna polymerase and cyclin e. the influence of ckia and ckib to the cdk4-cyclin d / prb / e2f pathway finally ends in cell cycle arrest and a stop of cell proliferation . furthermore , cell effective concentration is much higher than ic50 value determined by an in vitro kinase assay , due to the two barriers cell membrane and nuclear membrane that have to be overcome , and possible metabolism of the compound within the cell . previous evaluation of ckia in consideration of tumor cell growth inhibition of human colon carcinoma cell line hct116 , measuring the [ c]-thymidine incorporation , resulted in an ic50 value of 0.104 m . pd 0332991 has been extensively characterized among other highly selective inhibitors concerning biochemical and biological properties in vitro and in vivo [ 15 , 32 ] . in consequence pd the question to be answered is , if exceeding high selectivity of pyrido[2,3-d]pyrimidin-7-one derivatives against other cdks than cdk4 will be of outstanding importance for tumor treatment . in summary , ckia and also the new compound ckib were demonstrated to act as tumor cell growth inhibitors via specific inhibition of cdk4-cyclin d / prb / e2f signaling pathway . the mechanism of action confirms the therapeutic benefit of these compounds in field of cancer treatment , although their lowered selectivity . we hypothesized that potent cdk4 inhibitors , which are suitable for cancer therapy , are also of interest as radiotracers for imaging of cell proliferation processes in vivo and especially for tumor visualization by positron emission tomography ( pet ) . based on the structure of the two compounds ckia and ckib appropriate radiolabeling strategies were designed and radiolabeling with the positron - emitting nuclide iodine-124 was performed ( decay - corrected radiochemical yields : 33.6% [ i]ckia , and 27.8% [ i]ckib , purity : > 98% , resp . ) in both tumor cell lines a substantial cellular uptake of [ i]ckia and [ i]ckib was observed at 37c . this is consistent with the different therapeutic potency of ckia and ckib observed in tumor cell inhibition studies . especially , energy - dependent concentrative nucleoside transporters ( cnts ) could be presumed for ckia and ckib transport into cells . correlation analysis only showed a weak correlation between e2f-1 mrna expression as measure of cdk4 enzyme activity and cki ( ckia and ckib ) uptake ( = 0.390 , p = .042 , n = 15 ) . according to this , the transport of both radiotracers into the cells would be the rate determining step for cellular uptake of ckia and ckib at least in monolayer cell cultures . in summary , our data demonstrate a definite and specific inhibition of tumor cell proliferation when incubated with ckia and ckib due to an arrest of tumor cells in g1 phase . the repression of g1 progression is achieved by inhibition of the cdk4-cyclin d / prb / e2f pathway . the tumor cell growth inhibition observed in vitro point out the potential therapeutic benefit of these compounds , which has to be proven in further experiments in vivo . the radiotracer uptake observed in human tumor cells and the stability of [ i]ckia and [ i]ckib as found in physiological buffers are promising prerequisites for in vivo biodistribution and imaging studies by pet . an interesting effect arising from the radiotracer uptake studies using [ i]ckia and [ i]ckib is the observed energy dependent transport of ckia and ckib , due to the difference of uptake at 37c and 4c . regard , and to further support our hypothesis that radiolabeled cdk4 inhibitors are suitable radiotracers for tumor imaging , comprehensive in vivo experiments of [ i]ckia and [ i]ckib involving biodistribution and small animal pet studies should be performed .

the bogalusa heart study is being conducted in a biracial ( 65% white and 35% black ) community of bogalusa , la . a panel design , based on repeated ( at 34 years ) cross - sectional surveys of school - aged children and adults who participated in earlier surveys as children , resulted in the formation of a prospective longitudinal cohort with serial observations from child- to adulthood . this study includes subjects ( n = 2,603 ; young to middle - aged adults ; 34% black and 57% females ) who participated in their baseline and follow - up examinations during 19792011 and had fasting blood samples on both examinations . at the baseline examination , individuals with a history of diabetes or who had a fasting glucose level 126 mg / dl ( 7 these subjects were 444 years of age ( mean sd , 17.1 7.0 ) at baseline and 1850 at follow - up ( 33.2 9.3 ) . the study subjects were followed on average for 16 years ( mean sd , 16.2 7.5 ) . with respect to age , race , sex , overall adiposity ( bmi ) , and lipid , glucose , and insulin profile , the baseline characteristics of the study cohort , which represented 26% of the overall original ascertained baseline population , were similar to the characteristics of the subjects who did not participate in the follow - up survey as young adults ( data not shown ) . the follow - up , cross - sectional examination date at which diabetes was identified was used as the date of diagnosis ; otherwise , follow - up was censored at the last follow - up . according to the american diabetes association criteria ( 13 ) , follow - up adult subjects were classified as nondiabetic ( n = 2,459 ) if they had a fasting glucose level < 126 mg / dl ( 7.0 mmol / l ) and diabetic ( n = 144 ) if the fasting glucose level was 126 mg / dl ( 7 mmol / l ) or if they were on medication for diabetes . informed consent was obtained from all participants , and the study was approved by the institutional review board of the tulane university health sciences center . participants were instructed to fast for 12 h before the venipuncture , and compliance was ascertained by an interview on the day of examination . information on personal health history ( e.g. , hypertension , dyslipidemia , or diabetes and medical treatment for these conditions ) was obtained by questionnaires . bmi ( kg / m , weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity . right upper arm length and circumference were used to select the cuff size for blood pressure measurements with mercury sphygmomanometers . two randomly assigned trained nurses or observers measured blood pressure ( three replicates each ) on the right arm while subjects were in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fourth ( children ) or fifth ( adults ) korotkoff , respectively . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . participants were characterized as having a parental history of type 2 diabetes if one or both natural parents reported having the disease at baseline . cholesterol and triglyceride ( tg ) levels were initially measured using chemical procedures on a technicon autoanalyzer ii ( technicon instrument ) according to the laboratory manual of the lipid research clinics program . later , these variables were determined by enzymatic procedures on an abbott vp instrument ( abbott laboratories ) between 1987 and 1996 and on a hitachi 902 automatic analyzer ( roche diagnostics ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention ( cdc ) , which routinely monitors the precision and accuracy of total cholesterol , tgs , and hdl cholesterol ( hdl - c ) measurements since the beginning of this study . serum lipoprotein cholesterols were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 15).the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl - c , 0.86 to 0.98 for ldl cholesterol ( ldl - c ) , and 0.88 to 0.99 for tgs ( 12,14 ) . from 1976 to 1991 , plasma glucose was measured initially by a glucose oxidase method using a beckman glucose analyzer ( beckman instruments ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . plasma - immunoreactive insulin levels were measured by a commercial radioimmunoassay kit ( phadebas ; pharmacia diagnostics ) . the intraclass correlation coefficients between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment formula homa - ir = ( insulin [ u / ml ] glucose [ mmol / l]/22.5 ) . all of the statistical analyses were performed with sas version 9.2 ( sas institute ) . values of tgs , ratio of tgs and hdl - c ( tg / hdl - c ratio ) , glucose , insulin , and homa - ir variables used in the analyses were log transformed . general linear models were used to examine the baseline cardiometabolic risk factor variables , including parental history of diabetes , by status of follow - up young adult diabetes ( nondiabetes / diabetes ) and age - onset diabetes group , adjusted for age , race , and sex . the trends of incidence of diabetes in age onset , stratified by race and sex , were examined using the cochran - armitage trend test . logistic regression models , adjusted for age , race , and sex , were used to examine whether the maternal diabetes measured at the initial survey ( baseline ) predicted diabetes at follow - up by age - onset type 2 diabetes group . models assessing the independent relations between baseline cardiometabolic risk factor variables and young - onset type 2 diabetes by age - onset group were constructed using a backward elimination multivariate cox proportional hazards model with the years of follow - up as the time scale that was used to estimate the hazard ratios ( hrs ) and 95% cis . the baseline independent variables initially included in these models were age , race , sex , race by sex interaction , parental ( or maternal ) history of diabetes ( yes / no ) , bmi , map , tg / hdl - c ratio , and fasting plasma glucose and insulin . nonsignificant terms ( p > 0.05 ) were removed from the model by backward stepwise procedure . before fitting the model , the assumptions of the cox proportional hazards regression model were checked . the schoenfeld residual goodness - of - fit test for each independent variable included in the cox model was performed . because there was no interaction effect between baseline race ( or sex ) and glucose ( or insulin and homa - ir ) levels , the race - sex groups were combined to increase statistical power and to simplify the presentation . to evaluate the discriminatory capability of the models using the area under the roc curve ( c statistic ) , the multivariate c statistic logistic regressions were performed on the association of the selected baseline variables ( parental or maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) with incident diabetes status at the follow - up in young adulthood adjusted for age , race , sex , and map . rocs ( c value and its 95% ci ) were tested for equality by pairwise comparison of each model with the rest . in addition , to assess model discrimination , increment in c statistic in a model with traditional risk factor ( age , race , sex , and map ) and a selected baseline variable compared with traditional risk factor alone was calculated . the bogalusa heart study is being conducted in a biracial ( 65% white and 35% black ) community of bogalusa , la . a panel design , based on repeated ( at 34 years ) cross - sectional surveys of school - aged children and adults who participated in earlier surveys as children , resulted in the formation of a prospective longitudinal cohort with serial observations from child- to adulthood . this study includes subjects ( n = 2,603 ; young to middle - aged adults ; 34% black and 57% females ) who participated in their baseline and follow - up examinations during 19792011 and had fasting blood samples on both examinations . at the baseline examination , individuals with a history of diabetes or who had a fasting glucose level 126 mg / dl ( 7 these subjects were 444 years of age ( mean sd , 17.1 7.0 ) at baseline and 1850 at follow - up ( 33.2 9.3 ) . the study subjects were followed on average for 16 years ( mean sd , 16.2 7.5 ) . with respect to age , race , sex , overall adiposity ( bmi ) , and lipid , glucose , and insulin profile , the baseline characteristics of the study cohort , which represented 26% of the overall original ascertained baseline population , were similar to the characteristics of the subjects who did not participate in the follow - up survey as young adults ( data not shown ) . the follow - up , cross - sectional examination date at which diabetes was identified was used as the date of diagnosis ; otherwise , follow - up was censored at the last follow - up . according to the american diabetes association criteria ( 13 ) , follow - up adult subjects were classified as nondiabetic ( n = 2,459 ) if they had a fasting glucose level < 126 mg / dl ( 7.0 mmol / l ) and diabetic ( n = 144 ) if the fasting glucose level was 126 mg / dl ( 7 mmol / l ) or if they were on medication for diabetes . informed consent was obtained from all participants , and the study was approved by the institutional review board of the tulane university health sciences center . participants were instructed to fast for 12 h before the venipuncture , and compliance was ascertained by an interview on the day of examination . information on personal health history ( e.g. , hypertension , dyslipidemia , or diabetes and medical treatment for these conditions ) was obtained by questionnaires . bmi ( kg / m , weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity . right upper arm length and circumference were used to select the cuff size for blood pressure measurements with mercury sphygmomanometers . two randomly assigned trained nurses or observers measured blood pressure ( three replicates each ) on the right arm while subjects were in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fourth ( children ) or fifth ( adults ) korotkoff , respectively . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . participants were characterized as having a parental history of type 2 diabetes if one or both natural parents reported having the disease at baseline . cholesterol and triglyceride ( tg ) levels were initially measured using chemical procedures on a technicon autoanalyzer ii ( technicon instrument ) according to the laboratory manual of the lipid research clinics program . later , these variables were determined by enzymatic procedures on an abbott vp instrument ( abbott laboratories ) between 1987 and 1996 and on a hitachi 902 automatic analyzer ( roche diagnostics ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention ( cdc ) , which routinely monitors the precision and accuracy of total cholesterol , tgs , and hdl cholesterol ( hdl - c ) measurements since the beginning of this study . serum lipoprotein cholesterols were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 15).the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl - c , 0.86 to 0.98 for ldl cholesterol ( ldl - c ) , and 0.88 to 0.99 for tgs ( 12,14 ) . from 1976 to 1991 , plasma glucose was measured initially by a glucose oxidase method using a beckman glucose analyzer ( beckman instruments ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . plasma - immunoreactive insulin levels were measured by a commercial radioimmunoassay kit ( phadebas ; pharmacia diagnostics ) . the intraclass correlation coefficients between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment formula homa - ir = ( insulin [ u / ml ] glucose [ mmol / l]/22.5 ) . all of the statistical analyses were performed with sas version 9.2 ( sas institute ) . values of tgs , ratio of tgs and hdl - c ( tg / hdl - c ratio ) , glucose , insulin , and homa - ir variables used in the analyses were log transformed . general linear models were used to examine the baseline cardiometabolic risk factor variables , including parental history of diabetes , by status of follow - up young adult diabetes ( nondiabetes / diabetes ) and age - onset diabetes group , adjusted for age , race , and sex . the trends of incidence of diabetes in age onset , stratified by race and sex , were examined using the cochran - armitage trend test . logistic regression models , adjusted for age , race , and sex , were used to examine whether the maternal diabetes measured at the initial survey ( baseline ) predicted diabetes at follow - up by age - onset type 2 diabetes group . models assessing the independent relations between baseline cardiometabolic risk factor variables and young - onset type 2 diabetes by age - onset group were constructed using a backward elimination multivariate cox proportional hazards model with the years of follow - up as the time scale that was used to estimate the hazard ratios ( hrs ) and 95% cis . the baseline independent variables initially included in these models were age , race , sex , race by sex interaction , parental ( or maternal ) history of diabetes ( yes / no ) , bmi , map , tg / hdl - c ratio , and fasting plasma glucose and insulin . nonsignificant terms ( p > 0.05 ) were removed from the model by backward stepwise procedure . before fitting the model , the assumptions of the cox proportional hazards regression model were checked . the schoenfeld residual goodness - of - fit test for each independent variable included in the cox model was performed . because there was no interaction effect between baseline race ( or sex ) and glucose ( or insulin and homa - ir ) levels , the race - sex groups were combined to increase statistical power and to simplify the presentation . to evaluate the discriminatory capability of the models using the area under the roc curve ( c statistic ) , the multivariate c statistic logistic regressions were performed on the association of the selected baseline variables ( parental or maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) with incident diabetes status at the follow - up in young adulthood adjusted for age , race , sex , and map . rocs ( c value and its 95% ci ) were tested for equality by pairwise comparison of each model with the rest . in addition , to assess model discrimination , increment in c statistic in a model with traditional risk factor ( age , race , sex , and map ) and a selected baseline variable compared with traditional risk factor alone was calculated . the characteristics of the study cohort at baseline by onset - diabetes status ( yes / no ) and age - onset group are shown in table 1 . comparisons were made after adjustment for race , sex , and age at baseline . the overall increase with age showed an increased occurrence of diabetes beginning with 17 cases at age 1829 years . at baseline , the diabetes group versus nondiabetes group showed higher levels of bmi , tgs , insulin , and homa index and greater parental history of diabetes across all four groups ; higher levels of glucose ( except for 1829 age - group ) ; lower levels of hdl - c ( except for 4050 age - group ) and higher levels of map ( except for 1829 age - group ) and tg / hdl - c ratio ( except for 4050 age - group ) ; higher levels of ldl - c ( 3039 age - group and total sample only ) ; and higher age ( 4050 age - group and total sample only ) . cardiometabolic risk factor variables at baseline according to age onset of type 2 diabetes in young adults : the bogalusa heart study supplementary fig . 1 illustrates the incidence ( percent ) of onset of type 2 diabetes by race , sex , and age - onset group over an average of 16 years . the incidence of onset of diabetes increased with age , regardless of race and sex ( p for trend 0.01 ) . overall , the incidences of type 2 diabetes were 1.6% ( 17 of 1,044 ) at age 1829 years , 7.7% ( 58 of 755 ) at age 3039 years , and 8.6% ( 69 of 806 ) at age 4050 years the incidence was higher in black women ( 7.2% ) than in white women ( 4.7% ) ( p < 0.05 ) . no such difference was detected at age 1829 years ( p = 0.411 ) . in the total sample , blacks ( 7.1% ) were significantly more incident than whites ( 4.8% ) , as expected ( p < 0.05 ) . no significant sex difference in the progression of type 2 diabetes was observed ( data not shown ) . among 2,603 participants in the study cohort , there were 144 new diabetes cases ( events ) over an average of 16 years . the sum of person - time ( year ) of the population at risk was 10,949 , 13,531 , 17,653 , and 42,133 person - years for age - groups 1829 , 3039 , 4050 , and total sample , respectively . the person - year incidence rate of the onset of diabetes was 1.6 at age 1829 years , 4.3 at age 3039 years , and 3.9 at age 4050 years per 1,000 person - years . the overall incidence rate was 3.4 per 1,000 person - years in the total sample ( data not shown ) . figure 1 displays the prevalence ( percent ) of baseline maternal diabetes by follow - up diabetes status and age - onset group after 16 years . the prevalence of baseline maternal diabetes at ages 1829 , 3039 , and 4050 years and in total sample was 6.1 , 8.6 , 7.5 , and 7.2% , respectively . the prevalence of maternal diabetes was greater among patients with diabetes than those without diabetes , regardless of age - group ( p < 0.01 ) . further , the prevalence of maternal diabetes occurred 1.5-fold more frequently than paternal diabetes among the different age - groups ( p < 0.01 ) . no significant difference in the prevalence of baseline paternal diabetes between the two groups was detected in all age - groups . at follow - up , maternal diabetes consistently showed much greater prevalence among all age - groups and in the total sample , whereas paternal diabetes showed only more prevalence at age - group 3039 and in the total sample ( p < 0.01 ) ( data not shown ) . prevalence of maternal diabetes at baseline by follow - up age - group and diabetes status . p values were compared between the diabetes groups , adjusted for race , sex , and age at baseline . table 2 shows the results of a multivariable adjusted cox proportional hazards model that included race , sex , race by sex interaction , parental history of diabetes , bmi , map , tg / hdl - c ratio , glucose , and insulin at baseline . after adjusting for age at baseline , race ( black versus white ) showed significant hrs of 2.78 and 1.65 of being young - onset type 2 diabetes after 16 years for age - group 3039 and total sample , respectively ; parental history of diabetes ( yes / no ) displayed hrs of 5.24 , 2.44 , and 2.44 for age - groups 1829 and 4050 and total sample , respectively ; bmi showed hrs of 1.11 , 1.07 , and 1.08 for age - groups 3039 and 4050 and total sample , respectively ; tg / hdl - c ratio showed hrs of 1.07 and 1.04 for age - group 4050 and total sample , respectively ; glucose showed hrs of 1.05 for age - groups 3039 and 4050 and total sample ; and insulin showed a hr of 1.05 for age - group 1829 . further , alternate multivariate analyses using maternal diabetes , instead of parental diabetes , gave essentially identical results ( data not shown ) . baseline predictors of follow - up type 2 diabetes by age - onset status : the bogalusa heart study in terms of discriminative values of different selected baseline cardiometabolic variables ( parental or maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) ( table 3 ) , the predictive models produced c values ranging from 0.706 to 0.742 for onset of type 2 diabetes for these measures , which were relatively similar to each other in magnitude . compared with the traditional model including age , race , sex , and map , the diabetic model that added the selected baseline variable above had a significant increment in c statistic for bmi , insulin , glucose , parental and maternal history of diabetes , and tg / hdl - c ratio , in that order . discriminatory value of selected cardiometabolic risk factor variables in predicting onset of type 2 diabetes : the bogalusa heart study the current study explores the natural history of type 2 diabetes in a biracial community based population of relatively young adults , free from a selection bias , and monitored longitudinally over a period of 16 years . the results show that , after adjusting for race and sex , adiposity ( as depicted by bmi ) , tgs , tg / hdl - c ratio , fasting plasma glucose ( except for age - group 1829 at baseline ) , insulin , homa index , and parental history of diabetes measured at baseline were consistently and significantly different between the nondiabetes and diabetes groups across the age - onset groups and in the total sample size . the incidences of onset of diabetes increased with age when stratifying by race and sex groups ( p for trend 0.01 ) , greater in black versus white females and blacks versus whites in the total cohort ( p < 0.05 ) . in a multivariable cox proportional hazards model adjusted for age , baseline parental history of diabetes and fasting plasma insulin were significantly associated with type 2 diabetes in the 16-year follow - up of young adults in the age - onset group 1829 ; whereas race ( black versus white ) , bmi , and glucose were associated with diabetes in the older age - group with onset , 3039 years ; sex ( female versus male ) , parental history of diabetes , bmi , tg / hdl - c ratio , and glucose were associated with diabetes in age - group 4050 ; and race ( black versus white ) , parental history of diabetes , bmi , tg / hdl - c ratio , and glucose were associated with diabetes in the total sample . in terms of discriminative values of different selected baseline variables ( parental and maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) , the predictive models produced c values ranging from 0.706 to 0.742 for onset of type 2 diabetes for these measures . in the current study , the overall incidence rate of type 2 diabetes was lower than that found previously ( 1,911 ) . this difference might be explained by the lower average age in this overall cohort ( 1,911 ) and ethnic difference of our cohort , especially when compared with that in the study of pima indians ( 8) . nonetheless , the observed incidence rate of type 2 diabetes at ages 1829 ( 1,16,17 ) , 3039 ( 17 ) , and 4050 years ( 5 ) did not differ from other previous findings . in the bogalusa sample ( 4 ) , as parents of a children s study , they were younger and blacks were about twice more than whites . the observed incidence rate of young - onset type 2 diabetes increased with age by race - sex group , as might be expected and is consistent with previous observations ( 1,10 ) . the difference of race ( black > white ) in the current study cohort is also in agreement with the earlier reports ( 1,10,11,17 ) . further , compared with other race - sex groups , diabetes was less incident among white females ( 17 ) . aside from the traditional risk factors such as anthropometry , parental history of diabetes , and certain physiologic covariates , the differential distribution of a genetic polymorphism in black and white populations , the greater exposure to an adverse environment in early life , and the reduced availability of vitamin d in dark - skinned populations may contribute to the higher incidence of type 2 diabetes in blacks ( 11 ) . as a whole and as in other studies , the incidence of diabetes increases among older people and in ethnic minority populations ( 1,10 ) . having a parent with diabetes contributed strongly to diabetes risk in our present multivariate model , regardless of overall age and other risk factors . the current study , consistent with earlier reports ( 9,18 ) , demonstrated an excess maternal transmission of type 2 diabetes , and importantly , we noted this association was strongest at the young age of onset , 1829 years . at the young age of children , parents may be too young to manifest type 2 diabetes . indeed , in the current cohort , although parents were also at a younger age , the hr of being type 2 diabetes at age 1829 years was twofold higher than that in other age - groups and in total sample . adding paternal ( or maternal ) diabetes to the traditional model significantly improved discrimination of type 2 diabetes by at least 5% ( area under the curve = 0.721 ) . the importance of parental history at an earlier age suggests the important and more obvious role of genetics in the transmission of diabetes to offspring ( 19 ) . this was also suggested in our earlier glucose tolerance study by parental history ( 4,6,20 ) . as in other studies ( 16,17 ) , our observations have indicated that parental ( or maternal ) diabetes , but not glucose or obesity , and fasting plasma insulin measured at the average childhood age at baseline were the independent predictors of young - onset type 2 diabetes at follow - up age of 1829 years . further , fasting plasma glucose , but not insulin or parental ( or maternal ) diabetes , along with black race and obesity at the baseline adolescence age were predictors of younger - onset diabetes at follow - up age of 3039 years ( 17 ) . in addition , environmental exposure ( 21 ) is increasingly involved in the risk for the development of diabetes currently being observed in the epidemic of obesity ( 1,2 ) , and all conventional cardiometabolic risk factors beginning at an early age were predictors of the later ( 4050 years ) age - onset of diabetes . as a result , the simple and feasible composition of elementary office measurements , knowledge of parental diabetes , and childhood glucose and insulin may predict the young onset of type 2 diabetes ( 3,17 ) . of interest , adiposity , as depicted by bmi , was a strong predictor of diabetes in this study and in many earlier observations ( 1,3,5,7,8,10 ) . indeed , in the current study , the addition of bmi to the traditional model maximally improved the c statistic by 8% as compared with other risk factors . as is well known , obesity is pathologically linked to insulin resistance / hyperinsulinemia and related to development of dysglycemia . this is consistent with our earlier studies in that obesity precedes childhood - detectable hyperinsulinemia / insulin resistance or metabolic syndrome ( 22 ) . moreover , excess central adiposity augments the expression of proinflammatory adipocytokines , including tumor necrosis factor- , and reduces the expression of insulin - sensitizing and anti - inflammatory adiponectin , which causes an increase in insulin resistance ( 23 ) . excess fat and related insulin resistance / hyperinsulinemia increase tg ( very low - density lipoprotein ) levels as a result of abnormal fatty acid metabolism and excess hepatic tg synthesis and/or low clearance of tgs from the circulation ( 24 ) . in turn , increases in ldl - c and decreases in hdl - c levels ensue ( 24 ) . the current study has certain limitations in that it lacks direct assessments of postchallenge glucose , in vivo insulin action and secretion , glycosylated hemoglobin , and body fat mass and distribution . instead , we used well - established simple surrogate measures of glucose homeostasis that are applicable to population studies . previous studies , including our own , have found 90% of the self - reported diabetic information to be valid ( 25 ) . although diabetes cases were excluded at participant entry , type 1 and type 2 diabetes could not be distinguished in insulin users at follow - up because of the lack of measurements of glutamic acid decarboxylase antibodies and fasting c - peptide levels , which are used for clinical diagnosis of diabetes types . even with cdc quality controls , variability in the accuracy of measured variables over time is of concern . however , any potential measurement errors ( drifts ) due to different methodologies , if any , over time would have resulted in the underestimation of the observed relationships . finally , there was uncertainty about the precise time of the onset of diabetes . in summary , these findings indicate that adverse levels of traditional cardiometabolic risk factors , parental ( especially maternal ) diabetes , black race , adiposity , and measures of glucose homeostatis characterize the early natural history of the development of type 2 diabetes . in relatively young to middle age adults , maternal history of type 2 diabetes is an important predictor of diabetes onset , especially at the younger age . the feasible composition of elementary office and insulin measurements with the knowledge of parental history of diabetes should add in the clinical assessment to begin early prevention in children and adolescents to preclude the onset of type 2 diabetes .

objectivethe risk factors for middle - age onset of type 2 diabetes are well known . however , information is scant regarding the age onset of type 2 diabetes and its correlates in community - based black and white relatively young adults.research design and methodsthis prospective cohort study consisted of normoglycemic ( n = 2,459 ) and type 2 diabetic ( n = 144 ) adults aged 1850 years who were followed for an average of 16 years.resultsthe incidence rate of the onset of type 2 diabetes was 1.6 , 4.3 , 3.9 , and 3.4 per 1,000 person - years for age - groups 1829 , 3039 , and 4050 and total sample , respectively . incidences of diabetes increased with age by race and sex groups ( p for trend 0.01 ) ; higher in black females versus white females and blacks versus whites in total sample ( p < 0.05 ) . in a multivariable cox model , baseline parental diabetes ( hazard ratio [ hr ] 5.24 ) and plasma insulin were significantly associated with diabetes incidence at the youngest age ( 1829 years ) ; black race , bmi , and glucose at age 3039 years ; female sex , parental diabetes ( hr 2.44 ) , bmi , ratio of triglycerides and hdl cholesterol ( tg / hdl - c ratio ) , and glucose at age 4050 years ; and black race , parental diabetes ( hr 2.44 ) , bmi , tg / hdl - c ratio , and glucose in whole cohort . further , patients with diabetes , regardless of age onset , displayed a significantly higher prevalence of maternal history of diabetes at baseline ( p < 0.01).conclusionsin relatively young adults , predictability of baseline cardiometabolic risk factors along with race , sex , and parental history of diabetes for the onset of type 2 diabetes varied by age - group . these findings have implications for early prevention and intervention in relatively young adults .

RESEARCH DESIGN AND METHODS Study population General examination Laboratory analyses Statistical analysis RESULTS CONCLUSIONS

this study includes subjects ( n = 2,603 ; young to middle - aged adults ; 34% black and 57% females ) who participated in their baseline and follow - up examinations during 19792011 and had fasting blood samples on both examinations . at the baseline examination , individuals with a history of diabetes or who had a fasting glucose level 126 mg / dl ( 7 these subjects were 444 years of age ( mean sd , 17.1 7.0 ) at baseline and 1850 at follow - up ( 33.2 9.3 ) . with respect to age , race , sex , overall adiposity ( bmi ) , and lipid , glucose , and insulin profile , the baseline characteristics of the study cohort , which represented 26% of the overall original ascertained baseline population , were similar to the characteristics of the subjects who did not participate in the follow - up survey as young adults ( data not shown ) . according to the american diabetes association criteria ( 13 ) , follow - up adult subjects were classified as nondiabetic ( n = 2,459 ) if they had a fasting glucose level < 126 mg / dl ( 7.0 mmol / l ) and diabetic ( n = 144 ) if the fasting glucose level was 126 mg / dl ( 7 mmol / l ) or if they were on medication for diabetes . participants were characterized as having a parental history of type 2 diabetes if one or both natural parents reported having the disease at baseline . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention ( cdc ) , which routinely monitors the precision and accuracy of total cholesterol , tgs , and hdl cholesterol ( hdl - c ) measurements since the beginning of this study . serum lipoprotein cholesterols were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 15).the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl - c , 0.86 to 0.98 for ldl cholesterol ( ldl - c ) , and 0.88 to 0.99 for tgs ( 12,14 ) . values of tgs , ratio of tgs and hdl - c ( tg / hdl - c ratio ) , glucose , insulin , and homa - ir variables used in the analyses were log transformed . general linear models were used to examine the baseline cardiometabolic risk factor variables , including parental history of diabetes , by status of follow - up young adult diabetes ( nondiabetes / diabetes ) and age - onset diabetes group , adjusted for age , race , and sex . the trends of incidence of diabetes in age onset , stratified by race and sex , were examined using the cochran - armitage trend test . logistic regression models , adjusted for age , race , and sex , were used to examine whether the maternal diabetes measured at the initial survey ( baseline ) predicted diabetes at follow - up by age - onset type 2 diabetes group . models assessing the independent relations between baseline cardiometabolic risk factor variables and young - onset type 2 diabetes by age - onset group were constructed using a backward elimination multivariate cox proportional hazards model with the years of follow - up as the time scale that was used to estimate the hazard ratios ( hrs ) and 95% cis . the baseline independent variables initially included in these models were age , race , sex , race by sex interaction , parental ( or maternal ) history of diabetes ( yes / no ) , bmi , map , tg / hdl - c ratio , and fasting plasma glucose and insulin . to evaluate the discriminatory capability of the models using the area under the roc curve ( c statistic ) , the multivariate c statistic logistic regressions were performed on the association of the selected baseline variables ( parental or maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) with incident diabetes status at the follow - up in young adulthood adjusted for age , race , sex , and map . in addition , to assess model discrimination , increment in c statistic in a model with traditional risk factor ( age , race , sex , and map ) and a selected baseline variable compared with traditional risk factor alone was calculated . this study includes subjects ( n = 2,603 ; young to middle - aged adults ; 34% black and 57% females ) who participated in their baseline and follow - up examinations during 19792011 and had fasting blood samples on both examinations . at the baseline examination , individuals with a history of diabetes or who had a fasting glucose level 126 mg / dl ( 7 these subjects were 444 years of age ( mean sd , 17.1 7.0 ) at baseline and 1850 at follow - up ( 33.2 9.3 ) . with respect to age , race , sex , overall adiposity ( bmi ) , and lipid , glucose , and insulin profile , the baseline characteristics of the study cohort , which represented 26% of the overall original ascertained baseline population , were similar to the characteristics of the subjects who did not participate in the follow - up survey as young adults ( data not shown ) . according to the american diabetes association criteria ( 13 ) , follow - up adult subjects were classified as nondiabetic ( n = 2,459 ) if they had a fasting glucose level < 126 mg / dl ( 7.0 mmol / l ) and diabetic ( n = 144 ) if the fasting glucose level was 126 mg / dl ( 7 mmol / l ) or if they were on medication for diabetes . participants were characterized as having a parental history of type 2 diabetes if one or both natural parents reported having the disease at baseline . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention ( cdc ) , which routinely monitors the precision and accuracy of total cholesterol , tgs , and hdl cholesterol ( hdl - c ) measurements since the beginning of this study . serum lipoprotein cholesterols were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 15).the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl - c , 0.86 to 0.98 for ldl cholesterol ( ldl - c ) , and 0.88 to 0.99 for tgs ( 12,14 ) . values of tgs , ratio of tgs and hdl - c ( tg / hdl - c ratio ) , glucose , insulin , and homa - ir variables used in the analyses were log transformed . general linear models were used to examine the baseline cardiometabolic risk factor variables , including parental history of diabetes , by status of follow - up young adult diabetes ( nondiabetes / diabetes ) and age - onset diabetes group , adjusted for age , race , and sex . the trends of incidence of diabetes in age onset , stratified by race and sex , were examined using the cochran - armitage trend test . logistic regression models , adjusted for age , race , and sex , were used to examine whether the maternal diabetes measured at the initial survey ( baseline ) predicted diabetes at follow - up by age - onset type 2 diabetes group . models assessing the independent relations between baseline cardiometabolic risk factor variables and young - onset type 2 diabetes by age - onset group were constructed using a backward elimination multivariate cox proportional hazards model with the years of follow - up as the time scale that was used to estimate the hazard ratios ( hrs ) and 95% cis . the baseline independent variables initially included in these models were age , race , sex , race by sex interaction , parental ( or maternal ) history of diabetes ( yes / no ) , bmi , map , tg / hdl - c ratio , and fasting plasma glucose and insulin . to evaluate the discriminatory capability of the models using the area under the roc curve ( c statistic ) , the multivariate c statistic logistic regressions were performed on the association of the selected baseline variables ( parental or maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) with incident diabetes status at the follow - up in young adulthood adjusted for age , race , sex , and map . in addition , to assess model discrimination , increment in c statistic in a model with traditional risk factor ( age , race , sex , and map ) and a selected baseline variable compared with traditional risk factor alone was calculated . comparisons were made after adjustment for race , sex , and age at baseline . the overall increase with age showed an increased occurrence of diabetes beginning with 17 cases at age 1829 years . at baseline , the diabetes group versus nondiabetes group showed higher levels of bmi , tgs , insulin , and homa index and greater parental history of diabetes across all four groups ; higher levels of glucose ( except for 1829 age - group ) ; lower levels of hdl - c ( except for 4050 age - group ) and higher levels of map ( except for 1829 age - group ) and tg / hdl - c ratio ( except for 4050 age - group ) ; higher levels of ldl - c ( 3039 age - group and total sample only ) ; and higher age ( 4050 age - group and total sample only ) . cardiometabolic risk factor variables at baseline according to age onset of type 2 diabetes in young adults : the bogalusa heart study supplementary fig . 1 illustrates the incidence ( percent ) of onset of type 2 diabetes by race , sex , and age - onset group over an average of 16 years . the incidence of onset of diabetes increased with age , regardless of race and sex ( p for trend 0.01 ) . overall , the incidences of type 2 diabetes were 1.6% ( 17 of 1,044 ) at age 1829 years , 7.7% ( 58 of 755 ) at age 3039 years , and 8.6% ( 69 of 806 ) at age 4050 years the incidence was higher in black women ( 7.2% ) than in white women ( 4.7% ) ( p < 0.05 ) . in the total sample , blacks ( 7.1% ) were significantly more incident than whites ( 4.8% ) , as expected ( p < 0.05 ) . no significant sex difference in the progression of type 2 diabetes was observed ( data not shown ) . among 2,603 participants in the study cohort , there were 144 new diabetes cases ( events ) over an average of 16 years . the sum of person - time ( year ) of the population at risk was 10,949 , 13,531 , 17,653 , and 42,133 person - years for age - groups 1829 , 3039 , 4050 , and total sample , respectively . the person - year incidence rate of the onset of diabetes was 1.6 at age 1829 years , 4.3 at age 3039 years , and 3.9 at age 4050 years per 1,000 person - years . the overall incidence rate was 3.4 per 1,000 person - years in the total sample ( data not shown ) . the prevalence of baseline maternal diabetes at ages 1829 , 3039 , and 4050 years and in total sample was 6.1 , 8.6 , 7.5 , and 7.2% , respectively . the prevalence of maternal diabetes was greater among patients with diabetes than those without diabetes , regardless of age - group ( p < 0.01 ) . further , the prevalence of maternal diabetes occurred 1.5-fold more frequently than paternal diabetes among the different age - groups ( p < 0.01 ) . no significant difference in the prevalence of baseline paternal diabetes between the two groups was detected in all age - groups . at follow - up , maternal diabetes consistently showed much greater prevalence among all age - groups and in the total sample , whereas paternal diabetes showed only more prevalence at age - group 3039 and in the total sample ( p < 0.01 ) ( data not shown ) . prevalence of maternal diabetes at baseline by follow - up age - group and diabetes status . p values were compared between the diabetes groups , adjusted for race , sex , and age at baseline . table 2 shows the results of a multivariable adjusted cox proportional hazards model that included race , sex , race by sex interaction , parental history of diabetes , bmi , map , tg / hdl - c ratio , glucose , and insulin at baseline . after adjusting for age at baseline , race ( black versus white ) showed significant hrs of 2.78 and 1.65 of being young - onset type 2 diabetes after 16 years for age - group 3039 and total sample , respectively ; parental history of diabetes ( yes / no ) displayed hrs of 5.24 , 2.44 , and 2.44 for age - groups 1829 and 4050 and total sample , respectively ; bmi showed hrs of 1.11 , 1.07 , and 1.08 for age - groups 3039 and 4050 and total sample , respectively ; tg / hdl - c ratio showed hrs of 1.07 and 1.04 for age - group 4050 and total sample , respectively ; glucose showed hrs of 1.05 for age - groups 3039 and 4050 and total sample ; and insulin showed a hr of 1.05 for age - group 1829 . baseline predictors of follow - up type 2 diabetes by age - onset status : the bogalusa heart study in terms of discriminative values of different selected baseline cardiometabolic variables ( parental or maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) ( table 3 ) , the predictive models produced c values ranging from 0.706 to 0.742 for onset of type 2 diabetes for these measures , which were relatively similar to each other in magnitude . compared with the traditional model including age , race , sex , and map , the diabetic model that added the selected baseline variable above had a significant increment in c statistic for bmi , insulin , glucose , parental and maternal history of diabetes , and tg / hdl - c ratio , in that order . discriminatory value of selected cardiometabolic risk factor variables in predicting onset of type 2 diabetes : the bogalusa heart study the current study explores the natural history of type 2 diabetes in a biracial community based population of relatively young adults , free from a selection bias , and monitored longitudinally over a period of 16 years . the results show that , after adjusting for race and sex , adiposity ( as depicted by bmi ) , tgs , tg / hdl - c ratio , fasting plasma glucose ( except for age - group 1829 at baseline ) , insulin , homa index , and parental history of diabetes measured at baseline were consistently and significantly different between the nondiabetes and diabetes groups across the age - onset groups and in the total sample size . the incidences of onset of diabetes increased with age when stratifying by race and sex groups ( p for trend 0.01 ) , greater in black versus white females and blacks versus whites in the total cohort ( p < 0.05 ) . in a multivariable cox proportional hazards model adjusted for age , baseline parental history of diabetes and fasting plasma insulin were significantly associated with type 2 diabetes in the 16-year follow - up of young adults in the age - onset group 1829 ; whereas race ( black versus white ) , bmi , and glucose were associated with diabetes in the older age - group with onset , 3039 years ; sex ( female versus male ) , parental history of diabetes , bmi , tg / hdl - c ratio , and glucose were associated with diabetes in age - group 4050 ; and race ( black versus white ) , parental history of diabetes , bmi , tg / hdl - c ratio , and glucose were associated with diabetes in the total sample . in terms of discriminative values of different selected baseline variables ( parental and maternal history of diabetes , bmi , tg / hdl - c ratio , glucose , and insulin ) , the predictive models produced c values ranging from 0.706 to 0.742 for onset of type 2 diabetes for these measures . in the current study , the overall incidence rate of type 2 diabetes was lower than that found previously ( 1,911 ) . nonetheless , the observed incidence rate of type 2 diabetes at ages 1829 ( 1,16,17 ) , 3039 ( 17 ) , and 4050 years ( 5 ) did not differ from other previous findings . the observed incidence rate of young - onset type 2 diabetes increased with age by race - sex group , as might be expected and is consistent with previous observations ( 1,10 ) . further , compared with other race - sex groups , diabetes was less incident among white females ( 17 ) . aside from the traditional risk factors such as anthropometry , parental history of diabetes , and certain physiologic covariates , the differential distribution of a genetic polymorphism in black and white populations , the greater exposure to an adverse environment in early life , and the reduced availability of vitamin d in dark - skinned populations may contribute to the higher incidence of type 2 diabetes in blacks ( 11 ) . having a parent with diabetes contributed strongly to diabetes risk in our present multivariate model , regardless of overall age and other risk factors . the current study , consistent with earlier reports ( 9,18 ) , demonstrated an excess maternal transmission of type 2 diabetes , and importantly , we noted this association was strongest at the young age of onset , 1829 years . indeed , in the current cohort , although parents were also at a younger age , the hr of being type 2 diabetes at age 1829 years was twofold higher than that in other age - groups and in total sample . as in other studies ( 16,17 ) , our observations have indicated that parental ( or maternal ) diabetes , but not glucose or obesity , and fasting plasma insulin measured at the average childhood age at baseline were the independent predictors of young - onset type 2 diabetes at follow - up age of 1829 years . further , fasting plasma glucose , but not insulin or parental ( or maternal ) diabetes , along with black race and obesity at the baseline adolescence age were predictors of younger - onset diabetes at follow - up age of 3039 years ( 17 ) . in addition , environmental exposure ( 21 ) is increasingly involved in the risk for the development of diabetes currently being observed in the epidemic of obesity ( 1,2 ) , and all conventional cardiometabolic risk factors beginning at an early age were predictors of the later ( 4050 years ) age - onset of diabetes . as a result , the simple and feasible composition of elementary office measurements , knowledge of parental diabetes , and childhood glucose and insulin may predict the young onset of type 2 diabetes ( 3,17 ) . although diabetes cases were excluded at participant entry , type 1 and type 2 diabetes could not be distinguished in insulin users at follow - up because of the lack of measurements of glutamic acid decarboxylase antibodies and fasting c - peptide levels , which are used for clinical diagnosis of diabetes types . in summary , these findings indicate that adverse levels of traditional cardiometabolic risk factors , parental ( especially maternal ) diabetes , black race , adiposity , and measures of glucose homeostatis characterize the early natural history of the development of type 2 diabetes . in relatively young to middle age adults , maternal history of type 2 diabetes is an important predictor of diabetes onset , especially at the younger age . the feasible composition of elementary office and insulin measurements with the knowledge of parental history of diabetes should add in the clinical assessment to begin early prevention in children and adolescents to preclude the onset of type 2 diabetes .

the current study was conducted following the approval of the institutional review board of the national cheng kung university hospital . taiwan launched a single - payer national health insurance program on 1 march 1995 . in 2011 , > 99% of the people in taiwan were enrolled in this program ( 13 ) . the national health research institutes transformed these datasets into a research database , entitled nhird . because the identification number of each insured individual was transformed and encrypted , privacy was fully protected . these files provided detailed information on each individual s health care services , including all payments for outpatient visits , hospitalizations , and prescriptions . we identified all incidences of esrd in the individuals undergoing maintenance dialysis from the registry files of catastrophic illnesses in the nhird . because each individual registered in the database of catastrophic illnesses is exempt from any copayment for dialysis therapy , the registry is comprehensive . furthermore , the validity of the registry is assured by the careful monitoring of the national health insurance to prevent any abuse . the registry esrd criteria are as follows : a diagnosis of ckd with an irreversible creatinine level of > 8 mg / dl or a level of > 6 mg / dl with diabetes as a comorbid condition . we allowed 6 months of duration ( between 1 january and 30 june 1997 ) to exclude all prevalent cases . we also excluded cases undergoing maintenance peritoneal dialysis because of the small number of these cases ( n = 769 ) . we defined dialysis of > 90 days as maintenance ( long - term ) dialysis . from 1 july 1997 to 31 december 2005 , there were 52,756 incident cases of esrd under maintenance hemodialysis for > 90 days . among them , 24,243 cases were > 65 years . to estimate the incidence rates of esrd in elderly patients with and without diabetes , we retrospectively searched the database to determine if such a diagnosis had been reported in the discharge summary or outpatient claim for diabetes ( icd-9-cm code 250 ) before the diagnosis of esrd . the diagnosis of diabetes was confirmed if it had been included as one of the diagnoses at discharge or if there had been more than three outpatient claims for diabetes . the proportions of patients with diabetes stratified by age and sex were estimated based on interpolation of data from the following three national surveys conducted in taiwan : the 19992000 elderly nutrition and health survey ( 14 ) and the taiwanese surveys on blood sugar , blood lipids and blood pressure conducted during 2002 and 2007 ( 1517 ) . the 5-year age - specific incidence rates of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis were then calculated with the national population of taiwan as the denominator . cumulative incidence rates between 65 and 84 years of age ( cir6584 ) were also calculated to estimate the occurrence of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis using the following formula:(18,19 ) . to prevent confounding , we further excluded 3,423 cases with malignancy . therefore , a total of 20,820 cases were included in the final analysis . subsequently , the le and eyll in the elderly were estimated after stratification by age , sex , and the presence of diabetes , and lys gained by successful prevention of esrd in elderly patients with diabetes were calculated . the nhird indicates whether an individual is living , and therefore , most deceased cases of esrd can be identified . for cases with missing information on this item , we defined the date of death as 1 month after the date of the last medical visit . because individuals undergoing dialysis typically die within 1 month if no renal replacement therapy is provided , the above assumption seems acceptable . we used the kaplan - meier method to estimate the survival function based on follow - up data from 1 july 1997 to 31 december 2006 . all incident cases were further divided into patients with diabetes and nondiabetic subjects , and le and eyll were analyzed in the 5-year age stratification . because many cohorts with chronic diseases may not have been followed for a sufficient amount of time , we used a semiparametric method for extrapolation ( 20 ) to estimate survival analysis up to 50 years . the extrapolation was mathematically proven to be valid if constant excess hazard can be assumed ( 21 ) . for every individual of the esrd cohort , we first created an age- and sex - matched referent from the life tables of the general population using the monte carlo method . the life tables for the general population were obtained directly from the national vital statistics published by the department of statistics , ministry of the interior , executive yuan , taiwan . the survival curve of the cohort of age- and sex - matched referents was obtained by applying the kaplan - meier method . second , we fitted a simple linear regression line to the logit of the ratio of survival functions between the esrd and referent cohorts to the end of follow - up . finally , the regression line and survival curve of the cohort of referents were used to predict a long - term survival curve beyond the follow - up for the esrd population . to facilitate the computation , we used an isqol ( integration of survival and quality of life ) software program in the r statistical package . the average eyll of an esrd cohort was estimated as the difference in the area between the mean survival curves of the esrd cohort and reference population . it additionally demonstrates the amount of an individual s life that is likely to be saved by a preventive program targeted at those with elevated risk for developing esrd . we first calculated the prevalence of patients with diabetes in 2010 by multiplying the proportion of patients with diabetes from the 2007 taiwanese survey on blood sugar , blood lipids and blood pressure with the 2010 national population of taiwan . then , the number of preventable new esrd cases was estimated from the proportion of patients with diabetes in 2010 by multiplying this number with the excess incidence rate , (= incidence rate of esrd [ patients with diabetes ] incidence rate of esrd [ nondiabetic subjects ] ) , which was then multiplied with the eyll per case of esrd due to diabetes . finally , we summed up the expected lys gained in each age stratum and obtained the total lys that could be saved by successful prevention of esrd in elderly patients with diabetes between 65 and 84 years of age in a single year . the current study was conducted following the approval of the institutional review board of the national cheng kung university hospital . taiwan launched a single - payer national health insurance program on 1 march 1995 . in 2011 , > 99% of the people in taiwan were enrolled in this program ( 13 ) . the national health research institutes transformed these datasets into a research database , entitled nhird . because the identification number of each insured individual was transformed and encrypted , privacy was fully protected . these files provided detailed information on each individual s health care services , including all payments for outpatient visits , hospitalizations , and prescriptions . we identified all incidences of esrd in the individuals undergoing maintenance dialysis from the registry files of catastrophic illnesses in the nhird . because each individual registered in the database of catastrophic illnesses is exempt from any copayment for dialysis therapy , the registry is comprehensive . furthermore , the validity of the registry is assured by the careful monitoring of the national health insurance to prevent any abuse . the registry esrd criteria are as follows : a diagnosis of ckd with an irreversible creatinine level of > 8 mg / dl or a level of > 6 mg / dl with diabetes as a comorbid condition . we allowed 6 months of duration ( between 1 january and 30 june 1997 ) to exclude all prevalent cases . we also excluded cases undergoing maintenance peritoneal dialysis because of the small number of these cases ( n = 769 ) . we defined dialysis of > 90 days as maintenance ( long - term ) dialysis . from 1 july 1997 to 31 december 2005 , there were 52,756 incident cases of esrd under maintenance hemodialysis for > 90 days . among them , 24,243 cases were > 65 years . to estimate the incidence rates of esrd in elderly patients with and without diabetes , we retrospectively searched the database to determine if such a diagnosis had been reported in the discharge summary or outpatient claim for diabetes ( icd-9-cm code 250 ) before the diagnosis of esrd . the diagnosis of diabetes was confirmed if it had been included as one of the diagnoses at discharge or if there had been more than three outpatient claims for diabetes . the proportions of patients with diabetes stratified by age and sex were estimated based on interpolation of data from the following three national surveys conducted in taiwan : the 19992000 elderly nutrition and health survey ( 14 ) and the taiwanese surveys on blood sugar , blood lipids and blood pressure conducted during 2002 and 2007 ( 1517 ) . the 5-year age - specific incidence rates of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis were then calculated with the national population of taiwan as the denominator . cumulative incidence rates between 65 and 84 years of age ( cir6584 ) were also calculated to estimate the occurrence of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis using the following formula:(18,19 ) . to prevent confounding , we further excluded 3,423 cases with malignancy . therefore , a total of 20,820 cases were included in the final analysis . subsequently , the le and eyll in the elderly were estimated after stratification by age , sex , and the presence of diabetes , and lys gained by successful prevention of esrd in elderly patients with diabetes were calculated . the nhird indicates whether an individual is living , and therefore , most deceased cases of esrd can be identified . for cases with missing information on this item , we defined the date of death as 1 month after the date of the last medical visit . because individuals undergoing dialysis typically die within 1 month if no renal replacement therapy is provided , the above assumption seems acceptable . we used the kaplan - meier method to estimate the survival function based on follow - up data from 1 july 1997 to 31 december 2006 . all incident cases were further divided into patients with diabetes and nondiabetic subjects , and le and eyll were analyzed in the 5-year age stratification . because many cohorts with chronic diseases may not have been followed for a sufficient amount of time , we used a semiparametric method for extrapolation ( 20 ) to estimate survival analysis up to 50 years . the extrapolation was mathematically proven to be valid if constant excess hazard can be assumed ( 21 ) . it was composed of three processes ( 20 ) . for every individual of the esrd cohort , we first created an age- and sex - matched referent from the life tables of the general population using the monte carlo method . the life tables for the general population were obtained directly from the national vital statistics published by the department of statistics , ministry of the interior , executive yuan , taiwan . the survival curve of the cohort of age- and sex - matched referents was obtained by applying the kaplan - meier method . second , we fitted a simple linear regression line to the logit of the ratio of survival functions between the esrd and referent cohorts to the end of follow - up . finally , the regression line and survival curve of the cohort of referents were used to predict a long - term survival curve beyond the follow - up for the esrd population . to facilitate the computation , we used an isqol ( integration of survival and quality of life ) software program in the r statistical package . the average eyll of an esrd cohort was estimated as the difference in the area between the mean survival curves of the esrd cohort and reference population . it additionally demonstrates the amount of an individual s life that is likely to be saved by a preventive program targeted at those with elevated risk for developing esrd . we first calculated the prevalence of patients with diabetes in 2010 by multiplying the proportion of patients with diabetes from the 2007 taiwanese survey on blood sugar , blood lipids and blood pressure with the 2010 national population of taiwan . then , the number of preventable new esrd cases was estimated from the proportion of patients with diabetes in 2010 by multiplying this number with the excess incidence rate , (= incidence rate of esrd [ patients with diabetes ] incidence rate of esrd [ nondiabetic subjects ] ) , which was then multiplied with the eyll per case of esrd due to diabetes . finally , we summed up the expected lys gained in each age stratum and obtained the total lys that could be saved by successful prevention of esrd in elderly patients with diabetes between 65 and 84 years of age in a single year . the le and eyll of elderly patients undergoing maintenance hemodialysis are summarized in table 1 . the estimated average eyll of an esrd cohort is the difference between the areas of estimated survival curves for the reference population and the esrd cohort , as shown in fig . elderly patients with esrd exhibited the longest le for those aged 6569 years among both patients with diabetes ( 4.4 0.1 years in males and 4.8 0.2 years in females ) and nondiabetic subjects ( 7.1 0.3 years in males and 7.9 0.3 years in females ) . elderly patients with diabetes generally showed a significantly shorter le than elderly nondiabetic subjects in both sexes . for example , in males aged 6569 years , the average esrd diagnosis age was 66.9 1.5 years among patients with diabetes compared with 67.1 1.4 years among nondiabetic subjects . comparison of le and eyll between elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis mean survival difference between elderly patients with esrd under maintenance hemodialysis and age- and sex - matched reference population after 50 years of extrapolation . a : elderly patients with diabetes . ( a high - quality color representation of this figure is available in the online issue . ) the estimated average eyll was greater in elderly patients with diabetes than in elderly nondiabetic subjects after stratification by sex . for example , table 1 showed that the estimated average eyll for males aged 6569 years was 10.6 0.1 and 7.7 0.3 years ( p < 0.001 ) for patients with diabetes and nondiabetic subjects , respectively . for females aged 6569 years , the estimated average eyll was 12.3 0.2 and 9 0.3 years ( p < 0.001 ) for patients with diabetes and nondiabetic subjects , respectively . we further analyzed the incidence of esrd ( 1/10 person - year ) in elderly patients with diabetes and nondiabetic subjects , as summarized in table 2 . table 2 shows that the incidence rates of esrd undergoing maintenance hemodialysis were approximately three to five times higher in elderly patients with diabetes than in elderly nondiabetic subjects . cir6584 in elderly patients undergoing maintenance hemodialysis during 20022005 was 0.07472 and 0.09282 for diabetic males and females , respectively , but cir6584 was 0.01572 for and 0.01995 for nondiabetic males and females , respectively . in other words , 1 in 14 diabetic males and 1 in 11 diabetic females aged 65 years would develop esrd and be treated with hemodialysis in the next 20 years if he / she did not die of other illnesses . incidence rates of esrd ( 1/10 person - year ) and incident case numbers ( in parentheses ) in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis table 3 displays the number of preventable esrd cases due to diabetes and lys gained by the successful prevention of esrd in elderly patients with diabetes . the estimated prevalence of patients with diabetes in the year 2010 was 215,994 males and 266,991 females between 65 and 84 years of age . there were 676 and 1,080 preventable esrd cases in males and females , respectively , due to diabetes in a year ; thus , there would be 5,430.1 lys and 10,177 lys saved by the successful prevention of esrd in elderly diabetic males and females , respectively , in a year . the number of preventable incident esrd cases due to diabetes and lys gained by successful prevention of esrd in elderly patients with diabetes between 65 and 84 years of age in a single year although previous studies have investigated the impact of diabetes on elderly patients undergoing dialysis ( 9 ) , the current study is the first to estimate the le and eyll ( 22 ) in elderly patients with esrd stratified by age , sex , and the presence of diabetes . we found that the average eyll for the young - old ( between 65 and 75 years of age ) patients with diabetes is quite substantial ( table 1 ) . therefore , attention should be paid to preventive care of esrd in the patients with diabetes . for example , in elderly patients with diabetes who are 7074 years old , the average eyll was 8.1 0.2 and 9.6 0.2 for males and females , respectively , indicating that for this age - group , successfully preventing development of esrd in elderly patients with diabetes would save 8.1 and 9.6 lys for society . thus , the total number of lys gained by successful prevention of esrd in elderly patients with diabetes within a single year would be 5,430.1 and 10,177 lys for males and females , respectively ( table 3 ) . because we did not count the potential lys saved by sound diabetes care in elderly patients with esrd undergoing maintenance dialysis moreover , the above calculation did not include the loss of utility from reduced quality of life or the additional financial burden related to the development of esrd . these facts further stress the value of diabetes care in the elderly and prevention of diabetes - related complications , including esrd . according to a previous report ( 23 ) , the cumulative lifetime risks for esrd were 0.0730.078 and 0.0180.025 for 20-year - old black and white americans , respectively . in the current study , we also calculated the cir6584 in elderly developing esrd that requires maintenance hemodialysis . the results showed that the likelihood of developing esrd for elderly patients with diabetes aged 65 years would be 1 in 14 for males and 1 in 11 for females in the next 20 years , which would be nearly five times the risk for elderly nondiabetic subjects . thus , careful screening and follow - up of renal function accompanied with optimal control of blood glucose , serum lipids , and blood pressure must be considered when treating this population ( 2429 ) . prevention of diabetes - related esrd could possibly be accomplished through comprehensive diabetes care , and efforts to improve public awareness regarding prevention of ckd should also be strengthened ( 30,31 ) . diabetes was reported to be associated with a higher risk of death in individuals with esrd ( 9 ) , which was corroborated in this study . we have further quantified magnitudes of differences in eyll for different age strata ( table 1 ) . however , differences between elderly patients with diabetes and nondiabetic subjects narrowed as the age advanced , and the effects of diabetes seemed to be greater in the young - old individuals . our finding is consistent with a previous report ( 2 ) , which showed that the young - old individuals have a higher risk of mortality , and the oldest old individuals ( > 85 years of age ) have a lower rate of complications . although table 1 shows that the le was better in females than in males , as was reported previously ( 6,32 ) , the eyll was greater in females than in males , which may result from a general longer life expectancy in females ( 33 ) . thus , both male and female elderly patients with diabetes should be paid attention for prevention of esrd ( 24,34 ) . according to the neal thrifty gene hypothesis ( 35 ) , the diabetogenic genes persisted at a high level in times of nutritional deprivation because they contributed to a survival advantage , which might be good for survival of human species . however , as the food shortage is no longer a major threat nowadays , the sedentary lifestyles of modern times aggravate the occurrence of diabetes ( 36 ) . both factors contribute to the development of hyperlipidemia , metabolic syndrome , and diabetes , which further result in an increased premature mortality of cardio- and cerebrovascular diseases , especially among the esrd cohort ( 9 ) . because taiwan has the highest incidence and prevalence of treated esrd ( 5 ) , it is crucial that we identify preventable factors as early as possible in life and control them . as mentioned in the position statement ( 24 ) , all practitioners must emphasize lifestyle modifications on dietary intake and exercise to optimize glucose , lipids , and blood pressure control plus regular screening of urine albumin excretion and serum creatinine for early detection and control of ckd . with the standard of medical care and the ckd prevention program in elderly patients with diabetes with ckd ( 2429 ) , we hoped that diabetes - related esrd could be decreased gradually in the future . moreover , such a control program would generally produce spillover effects on reducing cardio- and cerebrovascular morbidities and mortalities , especially among the elderly ( 37,38 ) . future studies are needed to quantify the magnitudes of effects on different causes of death . although we analyzed the most comprehensive national data currently available in taiwan , there are potential limitations that must be addressed . first , our estimation of le and eyll was dependent upon the assumption of constant excess hazard and premature mortality of esrd . we checked the assumption by fitting the logit transform of the survival ratio between cohorts with esrd and the age- and sex - matched general population , which showed a straight line and fulfilled the assumption . second , the lifetime extrapolation was based on current and prior experiences of survival , including the life tables of the current population . as the dialysis technology and control for comorbidities may improve in the coming years , our results might underestimate the le and overestimate the eyll of elderly patients with diabetes and nondiabetic subjects with esrd . however , because the improved survival of esrd cohorts generally accompanies an increased cost for treatment , the overall saving from prevention is still a worthwhile investment . third , this study did not include elderly patients undergoing maintenance peritoneal dialysis and can not make a general inference for this population . although the bureau of national health insurance has promoted peritoneal dialysis during the last 5 years , hemodialysis seems to be the predominant modality of dialysis therapy in the elderly in taiwan because of a lower requirement for education and learning . because no significant differences on survival for peritoneal dialysis and hemodialysis were found in the previous study ( 22 ) , the current estimates of le and eyll might be a close approximation for peritoneal dialysis among elderly patients with diabetes and nondiabetic subjects . fourth , we did not begin following these subjects when they had been first diagnosed with diabetes . thus , we may underestimate the latency period and overestimate the incidence rate of esrd for elderly patients with diabetes . however , the current study stratified the population at risk into 5-year age groups , which reduces the above effect to some extent . finally , because the nhird does not contain any information related to quality of life , we were not able to calculate the quality - adjusted life expectancy . , we found that successful prevention of esrd in the elderly is worthwhile , especially among elderly patients with diabetes , and may save up to a decade of lys for each diabetic individual . as the incidence of diabetes has increased continually , the lys saved by comprehensive diabetes care in our society would be substantial , and diabetes care deserves special attention , especially in elderly females . future studies should take into account quality of life and cost of active prevention and dialysis to evaluate cost - effectiveness of prevention programs for elderly patients with diabetes .

objectivebecause of the increasing incidence and prevalence of diabetes as a leading cause of end - stage renal disease ( esrd ) in the aging population , we estimated the expected life - years ( lys ) saved from successful prevention of esrd in elderly patients with diabetes.research design and methodswe conducted a population - based cohort study using the national health insurance research database . we identified all incidences of esrd in the individuals > 65 years of age who were receiving maintenance hemodialysis ( n = 24,243 ) from the registry files of catastrophic illnesses in taiwan from 1 july 1997 to 31 december 2005 . we then retrospectively searched the database to determine whether there had been a diagnosis of diabetes in these cases . after the exclusion of individuals with malignancy ( n = 3,423 ) , we extrapolated the survival rates through the end of 2006 using the monte carlo method . using the data of preventable esrd cases due to diabetes and expected years of life lost ( eyll ) in each age stratum , we further estimated the expected lys saved from successful prevention of esrd in elderly patients with diabetes.resultsthe estimated average eyll was 10.65.8 and 12.37.3 years for diabetic males and females , respectively , aged 6579 years . in total , 5,430.1 lys and 10,177 lys could be saved by the successful prevention of esrd in male and female elderly patients with diabetes , respectively , in a single year.conclusionsthe lys saved by successful prevention of esrd in elderly patients with diabetes in a single year are substantial and deserve special attention , especially in elderly females .

RESEARCH DESIGN AND METHODS Study population and data collection Survival in the ESRD population under maintenance hemodialysis Extrapolation of long-term survival for the ESRD population under maintenance hemodialysis Estimation of EYLL Expected LYs gained by diabetes care in the elderly due to successful prevention of ESRD RESULTS CONCLUSIONS

the current study was conducted following the approval of the institutional review board of the national cheng kung university hospital . taiwan launched a single - payer national health insurance program on 1 march 1995 . in 2011 , > 99% of the people in taiwan were enrolled in this program ( 13 ) . the national health research institutes transformed these datasets into a research database , entitled nhird . we identified all incidences of esrd in the individuals undergoing maintenance dialysis from the registry files of catastrophic illnesses in the nhird . because each individual registered in the database of catastrophic illnesses is exempt from any copayment for dialysis therapy , the registry is comprehensive . furthermore , the validity of the registry is assured by the careful monitoring of the national health insurance to prevent any abuse . the registry esrd criteria are as follows : a diagnosis of ckd with an irreversible creatinine level of > 8 mg / dl or a level of > 6 mg / dl with diabetes as a comorbid condition . we also excluded cases undergoing maintenance peritoneal dialysis because of the small number of these cases ( n = 769 ) . from 1 july 1997 to 31 december 2005 , there were 52,756 incident cases of esrd under maintenance hemodialysis for > 90 days . among them , 24,243 cases were > 65 years . to estimate the incidence rates of esrd in elderly patients with and without diabetes , we retrospectively searched the database to determine if such a diagnosis had been reported in the discharge summary or outpatient claim for diabetes ( icd-9-cm code 250 ) before the diagnosis of esrd . the diagnosis of diabetes was confirmed if it had been included as one of the diagnoses at discharge or if there had been more than three outpatient claims for diabetes . the proportions of patients with diabetes stratified by age and sex were estimated based on interpolation of data from the following three national surveys conducted in taiwan : the 19992000 elderly nutrition and health survey ( 14 ) and the taiwanese surveys on blood sugar , blood lipids and blood pressure conducted during 2002 and 2007 ( 1517 ) . the 5-year age - specific incidence rates of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis were then calculated with the national population of taiwan as the denominator . cumulative incidence rates between 65 and 84 years of age ( cir6584 ) were also calculated to estimate the occurrence of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis using the following formula:(18,19 ) . to prevent confounding , we further excluded 3,423 cases with malignancy . subsequently , the le and eyll in the elderly were estimated after stratification by age , sex , and the presence of diabetes , and lys gained by successful prevention of esrd in elderly patients with diabetes were calculated . for cases with missing information on this item , we defined the date of death as 1 month after the date of the last medical visit . we used the kaplan - meier method to estimate the survival function based on follow - up data from 1 july 1997 to 31 december 2006 . all incident cases were further divided into patients with diabetes and nondiabetic subjects , and le and eyll were analyzed in the 5-year age stratification . for every individual of the esrd cohort , we first created an age- and sex - matched referent from the life tables of the general population using the monte carlo method . the life tables for the general population were obtained directly from the national vital statistics published by the department of statistics , ministry of the interior , executive yuan , taiwan . the survival curve of the cohort of age- and sex - matched referents was obtained by applying the kaplan - meier method . second , we fitted a simple linear regression line to the logit of the ratio of survival functions between the esrd and referent cohorts to the end of follow - up . to facilitate the computation , we used an isqol ( integration of survival and quality of life ) software program in the r statistical package . the average eyll of an esrd cohort was estimated as the difference in the area between the mean survival curves of the esrd cohort and reference population . it additionally demonstrates the amount of an individual s life that is likely to be saved by a preventive program targeted at those with elevated risk for developing esrd . we first calculated the prevalence of patients with diabetes in 2010 by multiplying the proportion of patients with diabetes from the 2007 taiwanese survey on blood sugar , blood lipids and blood pressure with the 2010 national population of taiwan . then , the number of preventable new esrd cases was estimated from the proportion of patients with diabetes in 2010 by multiplying this number with the excess incidence rate , (= incidence rate of esrd [ patients with diabetes ] incidence rate of esrd [ nondiabetic subjects ] ) , which was then multiplied with the eyll per case of esrd due to diabetes . finally , we summed up the expected lys gained in each age stratum and obtained the total lys that could be saved by successful prevention of esrd in elderly patients with diabetes between 65 and 84 years of age in a single year . the current study was conducted following the approval of the institutional review board of the national cheng kung university hospital . taiwan launched a single - payer national health insurance program on 1 march 1995 . in 2011 , > 99% of the people in taiwan were enrolled in this program ( 13 ) . the national health research institutes transformed these datasets into a research database , entitled nhird . we identified all incidences of esrd in the individuals undergoing maintenance dialysis from the registry files of catastrophic illnesses in the nhird . because each individual registered in the database of catastrophic illnesses is exempt from any copayment for dialysis therapy , the registry is comprehensive . furthermore , the validity of the registry is assured by the careful monitoring of the national health insurance to prevent any abuse . the registry esrd criteria are as follows : a diagnosis of ckd with an irreversible creatinine level of > 8 mg / dl or a level of > 6 mg / dl with diabetes as a comorbid condition . we also excluded cases undergoing maintenance peritoneal dialysis because of the small number of these cases ( n = 769 ) . from 1 july 1997 to 31 december 2005 , there were 52,756 incident cases of esrd under maintenance hemodialysis for > 90 days . among them , 24,243 cases were > 65 years . to estimate the incidence rates of esrd in elderly patients with and without diabetes , we retrospectively searched the database to determine if such a diagnosis had been reported in the discharge summary or outpatient claim for diabetes ( icd-9-cm code 250 ) before the diagnosis of esrd . the diagnosis of diabetes was confirmed if it had been included as one of the diagnoses at discharge or if there had been more than three outpatient claims for diabetes . the proportions of patients with diabetes stratified by age and sex were estimated based on interpolation of data from the following three national surveys conducted in taiwan : the 19992000 elderly nutrition and health survey ( 14 ) and the taiwanese surveys on blood sugar , blood lipids and blood pressure conducted during 2002 and 2007 ( 1517 ) . the 5-year age - specific incidence rates of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis were then calculated with the national population of taiwan as the denominator . cumulative incidence rates between 65 and 84 years of age ( cir6584 ) were also calculated to estimate the occurrence of esrd in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis using the following formula:(18,19 ) . to prevent confounding , we further excluded 3,423 cases with malignancy . subsequently , the le and eyll in the elderly were estimated after stratification by age , sex , and the presence of diabetes , and lys gained by successful prevention of esrd in elderly patients with diabetes were calculated . for cases with missing information on this item , we defined the date of death as 1 month after the date of the last medical visit . we used the kaplan - meier method to estimate the survival function based on follow - up data from 1 july 1997 to 31 december 2006 . all incident cases were further divided into patients with diabetes and nondiabetic subjects , and le and eyll were analyzed in the 5-year age stratification . for every individual of the esrd cohort , we first created an age- and sex - matched referent from the life tables of the general population using the monte carlo method . the life tables for the general population were obtained directly from the national vital statistics published by the department of statistics , ministry of the interior , executive yuan , taiwan . the survival curve of the cohort of age- and sex - matched referents was obtained by applying the kaplan - meier method . second , we fitted a simple linear regression line to the logit of the ratio of survival functions between the esrd and referent cohorts to the end of follow - up . to facilitate the computation , we used an isqol ( integration of survival and quality of life ) software program in the r statistical package . the average eyll of an esrd cohort was estimated as the difference in the area between the mean survival curves of the esrd cohort and reference population . we first calculated the prevalence of patients with diabetes in 2010 by multiplying the proportion of patients with diabetes from the 2007 taiwanese survey on blood sugar , blood lipids and blood pressure with the 2010 national population of taiwan . then , the number of preventable new esrd cases was estimated from the proportion of patients with diabetes in 2010 by multiplying this number with the excess incidence rate , (= incidence rate of esrd [ patients with diabetes ] incidence rate of esrd [ nondiabetic subjects ] ) , which was then multiplied with the eyll per case of esrd due to diabetes . finally , we summed up the expected lys gained in each age stratum and obtained the total lys that could be saved by successful prevention of esrd in elderly patients with diabetes between 65 and 84 years of age in a single year . the le and eyll of elderly patients undergoing maintenance hemodialysis are summarized in table 1 . the estimated average eyll of an esrd cohort is the difference between the areas of estimated survival curves for the reference population and the esrd cohort , as shown in fig . elderly patients with esrd exhibited the longest le for those aged 6569 years among both patients with diabetes ( 4.4 0.1 years in males and 4.8 0.2 years in females ) and nondiabetic subjects ( 7.1 0.3 years in males and 7.9 0.3 years in females ) . elderly patients with diabetes generally showed a significantly shorter le than elderly nondiabetic subjects in both sexes . for example , in males aged 6569 years , the average esrd diagnosis age was 66.9 1.5 years among patients with diabetes compared with 67.1 1.4 years among nondiabetic subjects . comparison of le and eyll between elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis mean survival difference between elderly patients with esrd under maintenance hemodialysis and age- and sex - matched reference population after 50 years of extrapolation . a : elderly patients with diabetes . the estimated average eyll was greater in elderly patients with diabetes than in elderly nondiabetic subjects after stratification by sex . for example , table 1 showed that the estimated average eyll for males aged 6569 years was 10.6 0.1 and 7.7 0.3 years ( p < 0.001 ) for patients with diabetes and nondiabetic subjects , respectively . for females aged 6569 years , the estimated average eyll was 12.3 0.2 and 9 0.3 years ( p < 0.001 ) for patients with diabetes and nondiabetic subjects , respectively . we further analyzed the incidence of esrd ( 1/10 person - year ) in elderly patients with diabetes and nondiabetic subjects , as summarized in table 2 . table 2 shows that the incidence rates of esrd undergoing maintenance hemodialysis were approximately three to five times higher in elderly patients with diabetes than in elderly nondiabetic subjects . cir6584 in elderly patients undergoing maintenance hemodialysis during 20022005 was 0.07472 and 0.09282 for diabetic males and females , respectively , but cir6584 was 0.01572 for and 0.01995 for nondiabetic males and females , respectively . in other words , 1 in 14 diabetic males and 1 in 11 diabetic females aged 65 years would develop esrd and be treated with hemodialysis in the next 20 years if he / she did not die of other illnesses . incidence rates of esrd ( 1/10 person - year ) and incident case numbers ( in parentheses ) in elderly patients with diabetes and nondiabetic subjects undergoing maintenance hemodialysis table 3 displays the number of preventable esrd cases due to diabetes and lys gained by the successful prevention of esrd in elderly patients with diabetes . the estimated prevalence of patients with diabetes in the year 2010 was 215,994 males and 266,991 females between 65 and 84 years of age . there were 676 and 1,080 preventable esrd cases in males and females , respectively , due to diabetes in a year ; thus , there would be 5,430.1 lys and 10,177 lys saved by the successful prevention of esrd in elderly diabetic males and females , respectively , in a year . the number of preventable incident esrd cases due to diabetes and lys gained by successful prevention of esrd in elderly patients with diabetes between 65 and 84 years of age in a single year although previous studies have investigated the impact of diabetes on elderly patients undergoing dialysis ( 9 ) , the current study is the first to estimate the le and eyll ( 22 ) in elderly patients with esrd stratified by age , sex , and the presence of diabetes . we found that the average eyll for the young - old ( between 65 and 75 years of age ) patients with diabetes is quite substantial ( table 1 ) . therefore , attention should be paid to preventive care of esrd in the patients with diabetes . for example , in elderly patients with diabetes who are 7074 years old , the average eyll was 8.1 0.2 and 9.6 0.2 for males and females , respectively , indicating that for this age - group , successfully preventing development of esrd in elderly patients with diabetes would save 8.1 and 9.6 lys for society . thus , the total number of lys gained by successful prevention of esrd in elderly patients with diabetes within a single year would be 5,430.1 and 10,177 lys for males and females , respectively ( table 3 ) . because we did not count the potential lys saved by sound diabetes care in elderly patients with esrd undergoing maintenance dialysis moreover , the above calculation did not include the loss of utility from reduced quality of life or the additional financial burden related to the development of esrd . these facts further stress the value of diabetes care in the elderly and prevention of diabetes - related complications , including esrd . according to a previous report ( 23 ) , the cumulative lifetime risks for esrd were 0.0730.078 and 0.0180.025 for 20-year - old black and white americans , respectively . in the current study , we also calculated the cir6584 in elderly developing esrd that requires maintenance hemodialysis . the results showed that the likelihood of developing esrd for elderly patients with diabetes aged 65 years would be 1 in 14 for males and 1 in 11 for females in the next 20 years , which would be nearly five times the risk for elderly nondiabetic subjects . diabetes was reported to be associated with a higher risk of death in individuals with esrd ( 9 ) , which was corroborated in this study . however , differences between elderly patients with diabetes and nondiabetic subjects narrowed as the age advanced , and the effects of diabetes seemed to be greater in the young - old individuals . our finding is consistent with a previous report ( 2 ) , which showed that the young - old individuals have a higher risk of mortality , and the oldest old individuals ( > 85 years of age ) have a lower rate of complications . although table 1 shows that the le was better in females than in males , as was reported previously ( 6,32 ) , the eyll was greater in females than in males , which may result from a general longer life expectancy in females ( 33 ) . thus , both male and female elderly patients with diabetes should be paid attention for prevention of esrd ( 24,34 ) . both factors contribute to the development of hyperlipidemia , metabolic syndrome , and diabetes , which further result in an increased premature mortality of cardio- and cerebrovascular diseases , especially among the esrd cohort ( 9 ) . because taiwan has the highest incidence and prevalence of treated esrd ( 5 ) , it is crucial that we identify preventable factors as early as possible in life and control them . as mentioned in the position statement ( 24 ) , all practitioners must emphasize lifestyle modifications on dietary intake and exercise to optimize glucose , lipids , and blood pressure control plus regular screening of urine albumin excretion and serum creatinine for early detection and control of ckd . with the standard of medical care and the ckd prevention program in elderly patients with diabetes with ckd ( 2429 ) , we hoped that diabetes - related esrd could be decreased gradually in the future . first , our estimation of le and eyll was dependent upon the assumption of constant excess hazard and premature mortality of esrd . we checked the assumption by fitting the logit transform of the survival ratio between cohorts with esrd and the age- and sex - matched general population , which showed a straight line and fulfilled the assumption . as the dialysis technology and control for comorbidities may improve in the coming years , our results might underestimate the le and overestimate the eyll of elderly patients with diabetes and nondiabetic subjects with esrd . although the bureau of national health insurance has promoted peritoneal dialysis during the last 5 years , hemodialysis seems to be the predominant modality of dialysis therapy in the elderly in taiwan because of a lower requirement for education and learning . because no significant differences on survival for peritoneal dialysis and hemodialysis were found in the previous study ( 22 ) , the current estimates of le and eyll might be a close approximation for peritoneal dialysis among elderly patients with diabetes and nondiabetic subjects . fourth , we did not begin following these subjects when they had been first diagnosed with diabetes . thus , we may underestimate the latency period and overestimate the incidence rate of esrd for elderly patients with diabetes . finally , because the nhird does not contain any information related to quality of life , we were not able to calculate the quality - adjusted life expectancy . , we found that successful prevention of esrd in the elderly is worthwhile , especially among elderly patients with diabetes , and may save up to a decade of lys for each diabetic individual . as the incidence of diabetes has increased continually , the lys saved by comprehensive diabetes care in our society would be substantial , and diabetes care deserves special attention , especially in elderly females . future studies should take into account quality of life and cost of active prevention and dialysis to evaluate cost - effectiveness of prevention programs for elderly patients with diabetes .

monomethylmercury ( mmhg ) is a toxic and bioaccumulative organometallic compound that poses serious health risks to both humans and wildlife . humans are primarily exposed to mmhg via the consumption of marine fish and shellfish , with over 90% of marine fisheries products originating from estuarine and open ocean areas . many estuarine organisms currently contain elevated mmhg levels , which can cause health problems in humans , but there is considerable uncertainty concerning the relative importance of the sources and exposure pathways of mmhg to estuarine food webs . sediments have long been suggested as the dominant mmhg source to estuarine food webs . estuarine sediments act as an important sink for hg , receiving hg via atmospheric deposition , industrial runoff and riverine input , and provide geochemical conditions that promote biotic methylation . past studies have also reported strong linkages between total hg ( thg ) concentrations in sediments and thg in estuarine forage fish in san francisco bay . in contrast , chen et al . recently documented a strong positive association between pelagic forage fish ( fundulus heteroclittus and menidia menidia ) mmhg concentrations and water column particulate mmhg concentrations , but not with sediments , across multiple estuaries on the northeast coast , u.s . based on this observation , the authors suggested that the mmhg accumulated into water column particulates may be a more dominant mmhg source to pelagic organisms than sediment mmhg . monomethylmercury can enter the water column via diffusion , advection and resuspension from sediments . inflowing fluvial and tidal waters have been suggested as important external mmhg sources to pelagic food webs in the bay of fundy and the hudson river estuary , with coastal sediments being a less important source . at chesapeake bay and long island sound , both sediments and external sources were found to be important mmhg sources to these systems . given these diverse sources , tools that can differentiate mmhg sources and exposure pathways are expected to help highlight the most important biogeochemical processes affecting mmhg in estuarine food webs . studies of the natural abundance of hg isotopes have enhanced understanding of the sources and biogeochemical processing of hg in natural environments . mercury isotopes undergo mass - dependent fractionation ( mdf , reported as hg ) and mass - independent fractionation ( mif , reported as hg and hg ) . while mdf has been documented in various environmentally relevant reactions such as biotic methylation , demethylation , thiol ligand exchange and photochemical reactions , mif in natural systems is thought to occur primarily in odd - mass number isotopes via photochemical reduction and degradation of inorganic hg ( ihg ) and mmhg . recently , hg has been applied as a biological and ecological tracer for understanding processes such as bioaccumulation and trophic transfer as well as transfers of mmhg between ecosystems . the ratio of hg/hg has also become a valuable tool for distinguishing between photochemical reduction of ihg ( hg/hg = 1.00 ) and degradation of mmhg ( hg/hg = 1.21.4 ) . mercury isotope ratios have recently been used to provide insight into the sources and exposure pathways of mmhg to diverse marine food webs . based on these studies , we can make predictions as to how hg isotope ratios might be useful for understanding the sources and biogeochemical processing of mmhg in estuaries . for instance , high positive hg values have been documented in pelagic fish ( > 1 ) compared to coastal fish ( < 1 ) , indicating that mmhg that has been subjected to extensive photochemical degradation in the open ocean water column is the dominant mmhg source to pelagic food webs . in shallow coastal environments it has been suggested that mmhg produced in the sediment , that has undergone relatively little photochemical degradation , enters the base of the food web . in another relevant study , gehrke et al . documented relatively low hg values ( < 1 ) in intertidal forage fish ( i.e. , topsmelt and silverside ) that feed on epibenthic invertebrates across the san francisco bay estuary . fish hg values were found to be consistently higher by 0.6 compared to nearby sediments at multiple sites , which led to the conclusion that the fractionation imparted during production and degradation pathways of mmhg in the sediment or during trophic transfer was responsible for these hg offsets . fish feeding experiments , have now documented an absence of hg isotope fractionation during trophic transfer of mmhg to fish , and therefore the isotopic offset is thought to provide insight into the biogeochemical pathways of mmhg prior to introduction to the food web . we also expect that the isotopic offset between fish and sediments may differ between sites that vary in hg biogeochemical cycling , as well as between different feeding guilds among estuarine organisms . in this study , we investigated the sources and exposure pathways of mmhg in food webs from five estuaries located across the northeastern coast of the u.s . sediments and organisms from these sites have been previously studied for ihg and mmhg concentrations by chen et al . is one of the most productive marine ecosystems in the world and supports valuable commercial and recreational fisheries . in the last century , this area has become severely impacted by an increase in population , industrial activity , and emission of anthropogenic pollutants including hg . here we characterize the hg isotopic compositions of estuarine sediments across sites on the northeast coast to identify hg sources in these regions . the sources and exposure pathways of mmhg were evaluated across food webs consisting of epibenthose ( green crab ; carcinus maenas , killifish ; fundulus heteroclittus ) , filter feeders ( blue mussel ; mytilus edulis , ribbed mussels ; geukensia demissa ) , and consumers ( common eider ; somateria mollissima ) . we determined the hg isotopic composition of the estuarine organisms and estimated the isotopic composition of mmhg to identify the dominant mmhg source and exposure pathway to the estuarine food webs . the mmhg isotopic compositions were compared across sites and between feeding guilds ( i.e. , groups of species that exploit the same resources ) to assess potential spatial and ecological variability in mmhg sources . to our knowledge , this is the first attempt to use hg isotope ratios to compare mmhg sources to varying marine feeding guilds across multiple ecosystems . ( maine ; me , massachusetts ; ma , rhode island , ri ; connecticut , ct , new jersey ; nj ) in the summers of 2008 and 2010 ( supporting information ( si ) figure s1 ) . all sites were adjacent to coastal marsh habitats except for bold point , ri ( bold ) , which was located in an unvegetated area of the providence river estuary . the webhannet estuary in wells , me ( wells ) and buzzards bay , ma ( buzz ) are characterized by sandy beaches and are sparsely populated . both sites receive primarily atmospherically deposited hg from nonpoint sources and the inputs are relatively small compared to the other northeast coast sites . bold is situated in urbanized providence , ri , and receives some atmospheric point source hg from local waste incinerators ( 10% of the total hg budget ) . bold is impacted by local wastewater treatment facilities ( 21% of hg input ) and industrially impacted rivers ( 69% of hg input ) . barn island , ct ( barn ) is located in the southeastern tip of connecticut and also receives hg via local wastewater treatment facilities and industrially impacted rivers the connecticut river ( 59% of hg input ) and the east river ( 25% of hg input ) . mill creek , nj ( mill ) is impacted by the hackensack river , which has been heavily contaminated by landfills , and a hg recovery plant that discharged 30400 tons of hg including elemental hg , mercuric oxide , and other forms of oxidized hg byproducts during its operation between 1929 and 1974 . the estuarine sediments were measured for thg and mmhg concentrations at the university of connecticut department of marine sciences and were reported previously ( n = 9 ) . four types of biota were analyzed for this study : green crabs ( carcinus maenas ) ( n = 6 ) , blue mussels ( mytilus edulis ) ( n = 5 ) or ribbed mussels ( geukensia demissa ) ( n = 2 ) , killifish ( fundulus heteroclittus ) ( n = 9 ) , and eider ( somateria mollissima ) ( n = 5 ) ( referred to as crab , mussel , fish , and bird hereafter ) . the methods for sampling and processing of the sediments and aquatic biota are described in chen et al . the aquatic biota were measured for stable nitrogen and carbon isotopic composition at the stable isotope laboratory , dartmouth college to characterize the trophic positions and feeding guilds . thg and mmhg analyses were conducted for most samples at the trace element analysis laboratory , dartmouth college . additional samples of mussels ( n = 4 ) and crabs ( n = 3 ) were measured for thg concentrations at the university of michigan . these samples were freeze - dried prior to removing the shells , and homogenized in a zirconium grinding mill ( retsch , mixer mill mm 301 ) . the mussels and crabs were measured for thg using atomic absorption spectroscopy ( aas ) following combustion of samples at 800 c using a nippon instruments ma-2000 hg analyzer . standard reference material nist-3133 was used to generate calibration curves and for quality control . standard reference material tort-3 ( n = 4 ) was analyzed as an external standard and agreed within 5% of certified values . samples of bird blood were collected near wells and buzz between january and april 2010 ( si figure s1 ) . the birds were captured using floating mist nets and the blood samples were collected by following the standard tissue collection protocol described in evers et al . the blood samples were drawn nonlethally by venipuncture from either the cutaneous ulnar or tarsal vein using needles and syringes . the samples were sealed on both ends with critocaps , placed in 10 cc plastic vacutainers , and frozen at 25 c prior to the analyses for thg concentrations at the wildlife mercury research lab at the biodiversity research institute , maine , u.s . the thg concentrations were determined using a direct hg analyzer ( dma 80 , milestone incorporated ) . the standard reference materials dolt-4 and dorm-4 were used to generate calibration curves and for quality control . dolt-4 ( n = 5 ) and dorm-3 ( n = 5 ) were analyzed with samples and agreed within 10% of certified values . the thg concentrations of sediments and aquatic biota are reported in dry weight and the aquatic biota represent whole body tissues . the sediment and biota samples were measured for stable hg isotopic composition at the university of michigan . blood samples were thawed and oven - dried at 5060 c in acid washed ceramic boats . samples of aquatic biota were weighed and loaded into ceramic boats with sodium carbonate and aluminum oxide powders . offline two - stage combustion furnace systems were used to release hg ( as hg ) from the samples . the ceramic boats were loaded into the first combustion compartment and heated to 750 c over a 6-h period . released hg was directed to the second combustion compartment ( 1000 c ) via a stream of hg - free oxygen and subsequently oxidized in a trap solution ( 1% kmno4 in 10% trace metal grade h2so4 ) . the solutions containing hg were neutralized with hydroxylamine , reduced back to hg with sncl2 and purged into a new trap solution to remove combustion product matrix components from the sample . procedural blanks were prepared in the same manner as the samples ( but with no sample placed in the ceramic boat ) and measured for thg before and after the transfer steps . the procedural blanks had an average thg of 0.2 0.1 ng ( n = 6 ) . the thg concentrations of the samples as well as the standard reference materials erm ce 464 ( n = 3 ) , tort-2 ( n = 1 ) , and mess-3 ( n = 2 ) were measured before and after the transfer step to monitor the recoveries of thg during the combustion and transfer processes . the recoveries of the combustion and transfer steps of the samples and standard reference materials ranged between 89100% , and 92106% , respectively . stable hg isotope ratios were measured using a nu instruments multicollector inductively coupled plasma mass spectrometer ( mc - icp - ms ) . the trap solutions were neutralized using hydroxylamine . to match the thg concentration of the sample to the standard , the trap solutions containing the sample were diluted to between 1 and 5 ng / g using the same neutralized trap solution matrix . mercury was introduced to the mc - icp - ms as hg by reducing hg in solution with sncl2 , and separating hg using a frosted glass tip phase separator . on - peak instrumental mass bias was corrected using an internal tl standard ( nist srm 997 ) and by bracketing each sample with nist srm 3133 matched to sample thg concentrations and matrix composition . mdf is reported as hg in permil ( ) referenced to nist srm 3133:1 mif represents the difference between the measured hg value and the value predicted based on mdf and the hg value . the calculation is based on an approximation valid for < 10:23 analytical uncertainty at 2 sd is estimated based on either replicate analysis of a standard solution ( um - almden ) or replicate analyses of standard reference materials . we used erm ce 464 to report analytical uncertainty since it had a larger uncertainty . um - almden ( n = 30 ) had mean values ( 2 sd ) of hg = 0.57 0.06 , hg = 0.04 0.04 , and hg = 0.02 0.05 . standard reference material erm ce 464 ( n = 3 ) had mean values of hg = 0.66 0.08 , hg = 1.91 0.06 , and hg = 2.31 0.09 ; tort-2 ( n = 1 ) had values of hg = 0.10 , hg = 0.57 , and hg = 0.79 ; and mess-3 ( n = 2 ) had mean values of hg = 1.81 0.08 , hg = 0.05 0.05 , and hg = 0.01 0.03 . the thg concentrations of the northeast coast sediments are quite low at buzz ( 5.70 ng / g ) and wells ( 9.43 ng / g ) and higher at the other three sites ( barn , bold , mill ) ( 42.0 to 2961 ng / g ) ( si table s1 ) . due to the low thg values at the buzz and wells sites ( typical of uncontaminated sites ) , we designate wells and buzz as background sites and due to the relatively higher values at bold , barn , and mill we designate them as contaminated sites . the contaminated sites are associated with hg point sources ( see site description ) and have at least 6 times higher thg concentration compared to the background sites . overall , the sediments displayed ranges of hg values between 0.89 and 0.38 , and hg values between 0.04 and 0.19 , and were within the range of values reported previously in other coastal marine sediments . we plotted 1/thg concentration against hg and hg to linearize mixing relationships and to characterize the hg isotopic compositions of the sediments in relation to the hg contamination . we observed a weak negative ( r = 0.26 , p = 0.16 ) and significant positive ( r = 0.77 , p < 0.05 ) relationship between 1/thg concentration and hg and hg , respectively ( figure 1 ) . the hg values of the sediments were distinct between the background and contaminated sites with the contaminated sediments exhibiting uniform hg 0 , and slightly elevated hg in the background sediments . the sediments displayed ranges of negative hg values and were less well correlated with the sediment thg concentrations . plot of 1/thg concentration ( ng / g ) versus hg and hg values of the northeast coast estuary sediments . each site is represented with different colors ; buzz ( black ) , wells ( blue ) , barn ( green ) , bold ( orange ) , and mill ( red ) . the fact that the hg values of the northeast coast sediments are correlated with the thg concentrations suggests that these sediments reflect inputs from multiple hg sources associated with the northeast coast estuaries . when we compared the background sediments to the contaminated sediments , the sites designated as background displayed negative hg values with hg values that were distinctly positive compared to the contaminated sediments . coastal and marine sediments from other studies that were characterized as background , or those that are preanthropogenic in age , display low thg concentrations , negative hg and slightly elevated hg . the background sediments from this study are consistent with the hg and hg values reported in those previous studies . the positive hg values in the background sediments are likely the result of fractionation caused during partial photochemical reduction of ihg , which may occur either through deposition of photochemically reduced ihg from surface water , periodic exposure of surface sediments to sunlight during tidal fluctuation of water levels ( 12 m ) , or deposition of photochemically reduced ihg from rainwater . photochemical reduction of ihg has been used to explain the positive hg values documented in shallow and relatively undisturbed marshes ( 0.66 ) and coastal and intertidal sediments ( 0.08 ) and we suggest that positive hg values may be imparted in background sediments that lack significant inputs from anthropogenic hg sources . in the contaminated sediments , we observed similar ranges of negative hg values but uniform hg 0 , reflecting the dominance of anthropogenic hg sources in these sediments . various industrial hg sources have been characterized by wide ranges of negative hg and zero hg values , and the input of industrial hg sources likely explains the low hg values , high thg concentrations , and wide ranges in hg values observed in the contaminated sediments . the range of hg values for the contaminated sites that are impacted by local industrial hg sources ( barn , bold ; 0.60 to 0.82 ) were also consistent with values for hg used in gold mining in the san francisco bay estuary ( 0.59 to 0.78 ) . thus , the hg isotopic compositions and the thg concentrations of the sediments appear to be consistent with the presence of multiple anthropogenic hg sources in these regions . the thg concentrations of the estuarine biota increase in the order : crabs ( 55.3 24 ng / g ) < fish ( 138 188 ng / g ) < mussels ( 145 52 ng / g ) < bird blood ( 863 724 ng / g ) ( si table s1 ) . the fraction of thg that is in the form of mmhg ( referred to as % mmhg hereafter ) for the aquatic biota increases in the order : mussels ( 57.9 4.5% ) < crabs ( 83.7 3.0% ) < fish ( 91.4 5.9% ) and these values follow the same order as n values ( 7.4 1.0 , 10.3 3.0 , 11.3 3.2 ; si table s1 ) , representing the approximate trophic position . the increasing % mmhg with trophic position ( n ) is consistent with the pattern of mmhg biomagnification documented in many aquatic food webs . we designate the feeding guilds of the estuarine biota based on the c values and the detailed feeding behaviors are shown in si table s1 and chen et al . the n ( 13.2 ) and c ( 17.4 ) values for the birds ( si table s1 ) were estimated from hobson et al . who measured the same species in the arctic . this estimate was adequate for approximating the relative trophic position and feeding guild of these birds , which suggests a slightly higher trophic position compared to the fish measured in this study . across the northeast coast estuarine study sites , we observed wide ranges of hg values but relatively narrow ranges of hg values in the estuarine biota ( si table s1 ) . at each study site the hg and hg values of the estuarine biota displayed an increasing trend with trophic position following the order mussels , crabs , fish , and birds ( figure 2a , b ) . similar trends have previously been attributed to the varying extent of mmhg bioaccumulation with trophic position . we plotted % mmhg against hg and hg of the sediment and aquatic biota ( without the birds ) and observed significant positive relationships with hg ( p < 0.05 ) across all sites ; ( wells ; r = 0.86 , buzz ; r = 0.70 , bold ; r = 0.72 , barn ; r = 0.89 ) and of hg at all sites except bold ; ( wells ; r = 0.68 , p < 0.05 , buzz ; r = 0.71 , p < 0.05 , bold ; r = 0.52 , p = 0.08 , barn ; r = 0.80 , p < 0.05 ) . due to the significant positive relationships observed at most sites , the hg isotopic composition reflecting the bioaccumulated mmhg was estimated using a linear regression of % mmhg against hg and hg . the hg and hg values estimated for mmhg demonstrated relatively narrow ranges across the northeast coast estuarine food webs ; 0.22 to 0.28 and 0.39 to 1.00 , respectively ( figure 2a , b ) . at sites where birds were measured ( in close proximity to wells and buzz ) , the birds ( containing > 98% mmhg ) had similar hg but much higher hg compared to the fish ( containing > 90% mmhg ) measured at the same locations ( bird hg ; 0.59 to 1.39 , fish hg ; 0.34 to 0.23 ) . at bold plot of % mmhg versus hg ( a ) and hg values ( b ) of estuarine sediments and biota . each site is represented by a different color ; buzz ( black ) , wells ( blue ) , barn ( green ) , bold ( orange ) , and mill ( red ) . the solid lines represent the range of estimated mmhg isotopic composition needed to explain sediments , crabs , and fish . the dotted lines represent the range of estimated mmhg isotopic composition needed to explain mussels from bold and birds from buzz and wells . the estimated mmhg and estimated ihg represent the ranges of hg isotopic composition extrapolated for 100% mmhg and 100% ihg , respectively , based on the linear regression of % mmhg vs hg and hg . overall , we find strong evidence for an increasing trend in the hg and hg values of estuarine biota with % mmhg ( trophic position ) in the northeast coast estuarine food webs . this trend has also been documented in other aquatic food webs . while metabolic fractionation was previously proposed as a potential mechanism , recent fish feeding experiments showed that metabolic fractionation does not occur during trophic transfer to fish . instead , the differences in hg isotopic composition between mmhg and ihg and the varying extent of mmhg versus ihg bioaccumulation with trophic position provide a consistent explanation for the hg isotopic composition variability in food webs . thus , the absence of mdf and mif during mmhg trophic transfer implies that the estuarine biota mainly reflect the isotopic composition of mmhg incorporated at the base of the food web . the anomalously high hg values observed in the wells and buzz birds and the bold mussels suggest that these organisms may be accumulating mmhg from an additional source . below we assess the dominant mmhg source and exposure pathways in the northeast coast estuarine food webs using the hg isotopic composition estimated for mmhg and provide an explanation for the hg values found in the birds and the bold mussels . the northeast coast estuarine biota analyzed in this study collectively displayed moderately positive hg values ( 0.21.0 ) ( si table s1 ) . this suggests that the bioaccumulated mmhg was subjected to photochemical degradation prior to incorporation into the estuarine food web . in fish , which have consistently high % mmhg and for which we have isotope data at each site , we found lower hg in the contaminated sites ( 0.56 0.15 ) compared to the uncontaminated sites ( 0.90 0.06 ) . this indicates that the fish at the contaminated sites are either exposed to mmhg that was subjected to a lesser degree of photochemical degradation compared to those found at the uncontaminated sites or mmhg originated from anthropogenic ihg sources which have low hg ( 0 ) . the slope of hg/ hg has been used to distinguish between photochemical degradation and reduction of mmhg versus ihg in natural aquatic ecosystems . we plotted hg against hg for all estuarine biota , and used a york regression to estimate the slope of hg/ hg , which is 1.22 0.07 ( r = 0.90 , p < 0.05 ) . this slope is consistent with the slopes reported in other marine fish ( 1.2 ) exposed to photochemically degraded mmhg from their respective environments . on a hg versus hg diagram we plot values for sediments and biota and the experimentally derived slopes representing the expected changes in the mmhg isotopic composition caused by photochemical degradation of mmhg ( hg = 4.79 0.33 hg at 10 mg / l doc ) ( figure 3 ; dotted lines ) . these slopes are derived from previously published mmhg photochemical degradation experiments that employed aquatic solutions spiked with mmhg and doc , and natural sunlight , and are only rough estimates since experimental conditions were different from those in the natural setting we studied . based on values for fish , we estimate that mmhg is photochemically degraded to varying degrees ranging from 5 to 12% across the northeast coast sites prior to entering the food webs . each site is represented with different colors ; buzz ( black ) , wells ( blue ) , barn ( green ) , bold ( orange ) , and mill ( red ) . the hg values for mmhg prior to photochemical degradation were estimated following the approach of gehrke et al . and sherman and blum and were all higher than the corresponding hg values of thg measured in the sediments ( figure 3 ) . given that the sediment is mainly composed of ihg , we suggest that mmhg that is bioaccumulated is mass dependently fractionated compared to the ihg in the sediment . this has been observed in previous studies and has been attributed to the net effect of microbial hg methylation and demethylation , which has increased hg values prior to photochemical degradation . the degree of the offset in hg between the mmhg ( before photodegradation ) and ihg are somewhat variable across the sites but generally consistent with previous studies . an alternate explanation for this offset in hg between ihg and mmhg is that it is caused by introduction of mmhg from a different source than the local sediment , which has higher hg ( e.g. , methylation in upstream marshes ) . the proportion of mmhg photochemical degradation estimated here is comparable with many shallow coastal regions and high turbidity lakes , but much lower than open ocean environments . the simplest explanation for the source of the mmhg to which estuarine biota are exposed is that it is derived from the ihg in sediments and has subsequently undergone small amounts of photochemical degradation in the water column , perhaps becoming attached to particles and subsequently redeposited to the sediment . in a study of fish and sediment in the gulf of mexico it was observed that the hg isotopic composition of coastal fish was consistent with exposure to sediment - derived mmhg that had undergone small amounts of photochemical degradation , whereas open ocean fish had much higher hg values indicative of much higher degrees of photodegradation . day et al . also found ranges of hg values in the eggs of epipelagic seabirds with the individuals feeding near shallow coastal embayments displaying significantly lower hg values compared to those feeding on open ocean species . the positive offsets in hg between ihg ( in sediment ) and mmhg ( in biota ) are consistent with previous studies suggesting that the local sediments may be the primary site for mmhg production prior to photochemical degradation in the water column and bioaccumulation in the food webs . it has been shown that microbial methylation causes the fractionation of hg , leading to lower hg in the product - mmhg compared to the reactant . a portion of hg that has been methylated could subsequently be microbially demethylated , resulting in higher hg values in the remaining mmhg , and the net effect of these processes provides an explanation for the hg values in the sediments . large positive offsets in hg have been documented in estuarine sediments , and to a lesser extent in rocky streambeds where mmhg production in sediments is less likely to occur . the site - specific variation in the offsets in hg suggests that the relative degree of fractionation due to microbial methylation versus demethylation differs across the northeast coast study sites . recently documented variable methylation rates in sediment across the northeast coast estuarine sites . while our results are consistent with the previous findings that suggested sediments as the primary site for mmhg production , it is possible that mmhg derived from various external sources may also play an important role in the northeast coast estuarine food webs . methylmercury produced within wetlands and river watersheds have been shown to supply significant amounts of mmhg to aquatic ecosystems in the northeast u.s . chen et al . recently documented a lack of relationship between mmhg concentrations in sediments and in the water column across the same sites of the northeast coast estuaries that we studied and suggested that sediments may be a minor contributor to mmhg found in water column . given that the mmhg flux via sedimentation exceeds that due to resuspension at our study sites , it is possible that external mmhg sources deposited in the sediment via settling particles may provide a viable alternative explanation for the observed offsets in hg between ihg ( sediment ) and the bioaccumulated mmhg . it is difficult to make a clear differentiation between in situ mass dependent fractionation of sediment - produced mmhg versus mixing of external mmhg sources in this study . further investigation will be required to characterize the isotopic composition of various external mmhg sources and biogeochemical processes affecting mmhg production in sediments . in summary , our results are consistent with either the production of mmhg from ihg in sediments or derivation of mmhg externally with deposition to the sediments . this mmhg is then subjected to a small amount of photodegradation in the water column before entering the food web and being passed to the various organisms at the study sites ( with one exception described below ) . the use of hg isotope ratios in this study provides additional insight into the sources and exposure pathways of mmhg studied by chen et al . in the same northeast coast estuarine food webs . chen et al . documented a strong positive association between pelagic fish ( silverside , killifish ) mmhg concentrations and water column particulate mmhg concentrations , but not with sediment mmhg concentrations . mussels and crabs did not show significant correlations with sediment and water column concentrations of mmhg . based on these observations , it was suggested that while sediments may be the main repository for hg , sediment mmhg concentrations are not an accurate predictor for mmhg bioaccumulation in estuarine biota . the authors also argued that the mmhg exposure via the water column may be the result of either complex interactions between water and sediment mmhg or input of external mmhg sources from offshore . given that the estuarine biota studied demonstrate different feeding behaviors and variable mmhg uptake routes , the consistent mmhg isotopic composition observed in various feeding guilds from each study location suggest that there may be extensive mmhg cycling between the sediment and water column via deposition and resuspension cycles without significant changes in the hg isotopic compositions of the mmhg . in other words , even if the fish derive mmhg primarily from the water column , the mmhg in the water column may be isotopically similar to the small proportion of mmhg in the sediments . while it appears that most aquatic organisms in this study acquire sediment - associated mmhg via trophic transfer , the high positive hg values found in the wells and buzz birds and the bold mussels suggest that these organisms are exposed to a different ( or additional ) mmhg source that has considerably higher hg values ( figure 2 ) . considering the mussels first , the main difference between the bold mussels and the mussels sampled in the other northeast coast estuaries is that while the other mussels have a hg isotopic composition consistent with a mixture of ihg and mmhg from the sediment , this pattern was observed only for hg , but not hg in the bold mussels ( figure 2 ) . thus , we infer that the bold mussels may be exposed to an additional mmhg source , possibly from the water column , due to their active filter feeding mechanism . the c values confirm that they feed on both pelagic and benthic resources , which is clearly different from the crabs and fish that depend dominantly on benthic and epibenthic resources closely associated with the sediments . we have identified two different scenarios that might explain how the bold mussels are exposed to hg with anomalously high positive hg values . first , to achieve high positive hg values , the sediment - associated mmhg would have to be subjected to additional nonphotochemical degradation and accompanying fractionation , causing the remaining mmhg to shift to higher hg values . we speculate that resuspension of sediment - associated mmhg followed by microbial demethylation in the water column could produce mmhg with high positive hg that is available for uptake by the mussels . bold is characterized by the highest tss and particulate mmhg concentrations , and this region may be particularly susceptible to additional biogeochemical processing of hg in the water column . second , it is also possible that the bold mussels were exposed to a second , and possibly unrelated external mmhg source that either has high hg values inherited from an anthropogenic source or has undergone extensive microbial demethylation prior to being released into the estuary . bold is surrounded by an unvegetated area and diverse point sources of hg , which may expose this site to extensive runoff of industrial hg sources . moreover , based on recent evidence that suggests inflowing fluvial mmhg can serve as an important external mmhg source to pelagic food webs , we presume that exposure to an externally derived mmhg source may be a viable alternate explanation for the high hg in the bold mussels . in the birds , we documented similar hg but much higher hg compared to fish from the same sites , which are the highest trophic level aquatic organisms measured in this study ( figure 2 ) . it is possible that the high hg mmhg is caused by the differences in food sources obtained from their respective sampling habitats compared to those of wells and buzz ( si figure s1 ) or by their selective feeding behavior given that they feed dominantly on benthic invertebrates including mussels . thus , if we assume that the birds analyzed in this study feed primarily on mussels , we might expect a significant positive relationship between % mmhg and hg , and between % mmhg and hg of the sediments , mussels , and birds due to the trophic transfer of mmhg . we plotted % mmhg against the hg and hg values of the sediments , mussels , and birds and observed significant positive relationships at wells ( hg ; r = 0.87 , p < 0.05 , hg = 0.93 , p < 0.05 ) and buzz ( hg ; r = 0.99 , p < 0.05 , hg ; r = 0.97 , p < 0.05 ) . however , the trophic transfer of mmhg from the sediment , to mussels , to birds would indicate that the crabs and fish are exposed to a different mmhg source than the mussels , and this is not what we observed , except at the bold site . moreover , given that the species of bird analyzed in this study have a relatively large feeding habitat ( 60 km ) , the small difference in the feeding habitat probably can not account for the high hg in the birds . instead , we attribute the high hg values in the birds to an internal metabolic fractionation of hg . while metabolic fractionation of hg has not been observed in fish , internal demethylation has been proposed to occur in the liver of many species of bird and this may lead to the fractionation of hg in birds . previous studies have documented 12 higher hg values in birds ( egg contents ) , and in mammals including seals , whales , and human hair compared to their respective diets and attributed this pattern to the kinetic fractionation of hg via internal demethylation . our study demonstrates that the measurement of hg isotope ratios provides new insight into sources and exposure pathways of mmhg in estuarine food webs . past studies that utilized hg isotope ratios to decipher mmhg sources and exposure pathways in coastal food webs have only examined fish , and over smaller coastal regions . we found evidence for multiple hg sources across the northeast coast sediments , and the mmhg associated with the sediments appears to serve as the dominant mmhg source to the estuarine food webs . there has been a long - standing debate over the relative importance of mmhg derived from sediment or from the water column as a source to estuarine organisms . complex biogeochemical processes affecting hg bioavailability between sediment and water column however make it difficult to trace the dominant mmhg sources in the estuarine food webs based on hg concentration alone . this study suggests that mmhg associated with sediments probably acts as the dominant source to many estuarine organisms , but that certain feeding guilds and certain localities are more susceptible to accumulating additional external mmhg sources . while further investigation is necessary to characterize various external mmhg sources , our study demonstrates that the measurement of hg isotope ratios can be a valuable tool for deciphering mmhg sources and exposure pathways in diverse aquatic food webs and assessing the ecological variability of mmhg sources .

we measured mercury ( hg ) isotope ratios in sediments and various estuarine organisms ( green crab , blue mussel , killifish , eider ) to investigate methylmercury ( mmhg ) sources and exposure pathways in five northeast coast ( u.s . ) estuaries . the mass independent hg isotopic compositions ( mif ; 199hg ) of the sediments were linearly correlated with the sediment 1/hg concentrations ( 199hg : r2 = 0.77 , p < 0.05 ) , but the mass dependent isotope compositions ( mdf ; 202hg ) were not ( r2 = 0.26 , p = 0.16 ) , reflecting inputs of anthropogenic hg sources with varying 202hg . the estuarine organisms all display positive 199hg values ( 0.21 to 0.98 ) indicating that mmhg is photodegraded to varying degrees ( 512% ) prior to entry into the food web . the 202hg and 199hg values of most organisms can be explained by a mixture of mmhg and inorganic hg from sediments . at one contaminated site mussels have anomalously high 202hg , indicating exposure to a second pool of mmhg , compared to sediment , crabs and fish . eiders have similar 199hg as killifish but much higher 202hg , suggesting that there is an internal fractionation of 202hg in birds . our study shows that hg isotopes can be used to identify multiple anthropogenic inorganic hg and mmhg sources and determine the degree of photodegradation of mmhg in estuarine food webs .

Introduction Materials and Methods Results and Discussion

many estuarine organisms currently contain elevated mmhg levels , which can cause health problems in humans , but there is considerable uncertainty concerning the relative importance of the sources and exposure pathways of mmhg to estuarine food webs . past studies have also reported strong linkages between total hg ( thg ) concentrations in sediments and thg in estuarine forage fish in san francisco bay . recently documented a strong positive association between pelagic forage fish ( fundulus heteroclittus and menidia menidia ) mmhg concentrations and water column particulate mmhg concentrations , but not with sediments , across multiple estuaries on the northeast coast , u.s . given these diverse sources , tools that can differentiate mmhg sources and exposure pathways are expected to help highlight the most important biogeochemical processes affecting mmhg in estuarine food webs . mercury isotopes undergo mass - dependent fractionation ( mdf , reported as hg ) and mass - independent fractionation ( mif , reported as hg and hg ) . mercury isotope ratios have recently been used to provide insight into the sources and exposure pathways of mmhg to diverse marine food webs . based on these studies , we can make predictions as to how hg isotope ratios might be useful for understanding the sources and biogeochemical processing of mmhg in estuaries . for instance , high positive hg values have been documented in pelagic fish ( > 1 ) compared to coastal fish ( < 1 ) , indicating that mmhg that has been subjected to extensive photochemical degradation in the open ocean water column is the dominant mmhg source to pelagic food webs . in shallow coastal environments it has been suggested that mmhg produced in the sediment , that has undergone relatively little photochemical degradation , enters the base of the food web . fish hg values were found to be consistently higher by 0.6 compared to nearby sediments at multiple sites , which led to the conclusion that the fractionation imparted during production and degradation pathways of mmhg in the sediment or during trophic transfer was responsible for these hg offsets . fish feeding experiments , have now documented an absence of hg isotope fractionation during trophic transfer of mmhg to fish , and therefore the isotopic offset is thought to provide insight into the biogeochemical pathways of mmhg prior to introduction to the food web . in this study , we investigated the sources and exposure pathways of mmhg in food webs from five estuaries located across the northeastern coast of the u.s . here we characterize the hg isotopic compositions of estuarine sediments across sites on the northeast coast to identify hg sources in these regions . the sources and exposure pathways of mmhg were evaluated across food webs consisting of epibenthose ( green crab ; carcinus maenas , killifish ; fundulus heteroclittus ) , filter feeders ( blue mussel ; mytilus edulis , ribbed mussels ; geukensia demissa ) , and consumers ( common eider ; somateria mollissima ) . we determined the hg isotopic composition of the estuarine organisms and estimated the isotopic composition of mmhg to identify the dominant mmhg source and exposure pathway to the estuarine food webs . both sites receive primarily atmospherically deposited hg from nonpoint sources and the inputs are relatively small compared to the other northeast coast sites . four types of biota were analyzed for this study : green crabs ( carcinus maenas ) ( n = 6 ) , blue mussels ( mytilus edulis ) ( n = 5 ) or ribbed mussels ( geukensia demissa ) ( n = 2 ) , killifish ( fundulus heteroclittus ) ( n = 9 ) , and eider ( somateria mollissima ) ( n = 5 ) ( referred to as crab , mussel , fish , and bird hereafter ) . the methods for sampling and processing of the sediments and aquatic biota are described in chen et al . um - almden ( n = 30 ) had mean values ( 2 sd ) of hg = 0.57 0.06 , hg = 0.04 0.04 , and hg = 0.02 0.05 . we plotted 1/thg concentration against hg and hg to linearize mixing relationships and to characterize the hg isotopic compositions of the sediments in relation to the hg contamination . we observed a weak negative ( r = 0.26 , p = 0.16 ) and significant positive ( r = 0.77 , p < 0.05 ) relationship between 1/thg concentration and hg and hg , respectively ( figure 1 ) . the hg values of the sediments were distinct between the background and contaminated sites with the contaminated sediments exhibiting uniform hg 0 , and slightly elevated hg in the background sediments . the sediments displayed ranges of negative hg values and were less well correlated with the sediment thg concentrations . plot of 1/thg concentration ( ng / g ) versus hg and hg values of the northeast coast estuary sediments . the fact that the hg values of the northeast coast sediments are correlated with the thg concentrations suggests that these sediments reflect inputs from multiple hg sources associated with the northeast coast estuaries . photochemical reduction of ihg has been used to explain the positive hg values documented in shallow and relatively undisturbed marshes ( 0.66 ) and coastal and intertidal sediments ( 0.08 ) and we suggest that positive hg values may be imparted in background sediments that lack significant inputs from anthropogenic hg sources . in the contaminated sediments , we observed similar ranges of negative hg values but uniform hg 0 , reflecting the dominance of anthropogenic hg sources in these sediments . the range of hg values for the contaminated sites that are impacted by local industrial hg sources ( barn , bold ; 0.60 to 0.82 ) were also consistent with values for hg used in gold mining in the san francisco bay estuary ( 0.59 to 0.78 ) . thus , the hg isotopic compositions and the thg concentrations of the sediments appear to be consistent with the presence of multiple anthropogenic hg sources in these regions . at each study site the hg and hg values of the estuarine biota displayed an increasing trend with trophic position following the order mussels , crabs , fish , and birds ( figure 2a , b ) . we plotted % mmhg against hg and hg of the sediment and aquatic biota ( without the birds ) and observed significant positive relationships with hg ( p < 0.05 ) across all sites ; ( wells ; r = 0.86 , buzz ; r = 0.70 , bold ; r = 0.72 , barn ; r = 0.89 ) and of hg at all sites except bold ; ( wells ; r = 0.68 , p < 0.05 , buzz ; r = 0.71 , p < 0.05 , bold ; r = 0.52 , p = 0.08 , barn ; r = 0.80 , p < 0.05 ) . the hg and hg values estimated for mmhg demonstrated relatively narrow ranges across the northeast coast estuarine food webs ; 0.22 to 0.28 and 0.39 to 1.00 , respectively ( figure 2a , b ) . at sites where birds were measured ( in close proximity to wells and buzz ) , the birds ( containing > 98% mmhg ) had similar hg but much higher hg compared to the fish ( containing > 90% mmhg ) measured at the same locations ( bird hg ; 0.59 to 1.39 , fish hg ; 0.34 to 0.23 ) . each site is represented by a different color ; buzz ( black ) , wells ( blue ) , barn ( green ) , bold ( orange ) , and mill ( red ) . the estimated mmhg and estimated ihg represent the ranges of hg isotopic composition extrapolated for 100% mmhg and 100% ihg , respectively , based on the linear regression of % mmhg vs hg and hg . overall , we find strong evidence for an increasing trend in the hg and hg values of estuarine biota with % mmhg ( trophic position ) in the northeast coast estuarine food webs . instead , the differences in hg isotopic composition between mmhg and ihg and the varying extent of mmhg versus ihg bioaccumulation with trophic position provide a consistent explanation for the hg isotopic composition variability in food webs . thus , the absence of mdf and mif during mmhg trophic transfer implies that the estuarine biota mainly reflect the isotopic composition of mmhg incorporated at the base of the food web . below we assess the dominant mmhg source and exposure pathways in the northeast coast estuarine food webs using the hg isotopic composition estimated for mmhg and provide an explanation for the hg values found in the birds and the bold mussels . the northeast coast estuarine biota analyzed in this study collectively displayed moderately positive hg values ( 0.21.0 ) ( si table s1 ) . this suggests that the bioaccumulated mmhg was subjected to photochemical degradation prior to incorporation into the estuarine food web . in fish , which have consistently high % mmhg and for which we have isotope data at each site , we found lower hg in the contaminated sites ( 0.56 0.15 ) compared to the uncontaminated sites ( 0.90 0.06 ) . this indicates that the fish at the contaminated sites are either exposed to mmhg that was subjected to a lesser degree of photochemical degradation compared to those found at the uncontaminated sites or mmhg originated from anthropogenic ihg sources which have low hg ( 0 ) . we plotted hg against hg for all estuarine biota , and used a york regression to estimate the slope of hg/ hg , which is 1.22 0.07 ( r = 0.90 , p < 0.05 ) . based on values for fish , we estimate that mmhg is photochemically degraded to varying degrees ranging from 5 to 12% across the northeast coast sites prior to entering the food webs . given that the sediment is mainly composed of ihg , we suggest that mmhg that is bioaccumulated is mass dependently fractionated compared to the ihg in the sediment . an alternate explanation for this offset in hg between ihg and mmhg is that it is caused by introduction of mmhg from a different source than the local sediment , which has higher hg ( e.g. the simplest explanation for the source of the mmhg to which estuarine biota are exposed is that it is derived from the ihg in sediments and has subsequently undergone small amounts of photochemical degradation in the water column , perhaps becoming attached to particles and subsequently redeposited to the sediment . in a study of fish and sediment in the gulf of mexico it was observed that the hg isotopic composition of coastal fish was consistent with exposure to sediment - derived mmhg that had undergone small amounts of photochemical degradation , whereas open ocean fish had much higher hg values indicative of much higher degrees of photodegradation . the positive offsets in hg between ihg ( in sediment ) and mmhg ( in biota ) are consistent with previous studies suggesting that the local sediments may be the primary site for mmhg production prior to photochemical degradation in the water column and bioaccumulation in the food webs . large positive offsets in hg have been documented in estuarine sediments , and to a lesser extent in rocky streambeds where mmhg production in sediments is less likely to occur . while our results are consistent with the previous findings that suggested sediments as the primary site for mmhg production , it is possible that mmhg derived from various external sources may also play an important role in the northeast coast estuarine food webs . recently documented a lack of relationship between mmhg concentrations in sediments and in the water column across the same sites of the northeast coast estuaries that we studied and suggested that sediments may be a minor contributor to mmhg found in water column . given that the mmhg flux via sedimentation exceeds that due to resuspension at our study sites , it is possible that external mmhg sources deposited in the sediment via settling particles may provide a viable alternative explanation for the observed offsets in hg between ihg ( sediment ) and the bioaccumulated mmhg . further investigation will be required to characterize the isotopic composition of various external mmhg sources and biogeochemical processes affecting mmhg production in sediments . in summary , our results are consistent with either the production of mmhg from ihg in sediments or derivation of mmhg externally with deposition to the sediments . this mmhg is then subjected to a small amount of photodegradation in the water column before entering the food web and being passed to the various organisms at the study sites ( with one exception described below ) . the use of hg isotope ratios in this study provides additional insight into the sources and exposure pathways of mmhg studied by chen et al . given that the estuarine biota studied demonstrate different feeding behaviors and variable mmhg uptake routes , the consistent mmhg isotopic composition observed in various feeding guilds from each study location suggest that there may be extensive mmhg cycling between the sediment and water column via deposition and resuspension cycles without significant changes in the hg isotopic compositions of the mmhg . considering the mussels first , the main difference between the bold mussels and the mussels sampled in the other northeast coast estuaries is that while the other mussels have a hg isotopic composition consistent with a mixture of ihg and mmhg from the sediment , this pattern was observed only for hg , but not hg in the bold mussels ( figure 2 ) . the c values confirm that they feed on both pelagic and benthic resources , which is clearly different from the crabs and fish that depend dominantly on benthic and epibenthic resources closely associated with the sediments . second , it is also possible that the bold mussels were exposed to a second , and possibly unrelated external mmhg source that either has high hg values inherited from an anthropogenic source or has undergone extensive microbial demethylation prior to being released into the estuary . thus , if we assume that the birds analyzed in this study feed primarily on mussels , we might expect a significant positive relationship between % mmhg and hg , and between % mmhg and hg of the sediments , mussels , and birds due to the trophic transfer of mmhg . we plotted % mmhg against the hg and hg values of the sediments , mussels , and birds and observed significant positive relationships at wells ( hg ; r = 0.87 , p < 0.05 , hg = 0.93 , p < 0.05 ) and buzz ( hg ; r = 0.99 , p < 0.05 , hg ; r = 0.97 , p < 0.05 ) . however , the trophic transfer of mmhg from the sediment , to mussels , to birds would indicate that the crabs and fish are exposed to a different mmhg source than the mussels , and this is not what we observed , except at the bold site . previous studies have documented 12 higher hg values in birds ( egg contents ) , and in mammals including seals , whales , and human hair compared to their respective diets and attributed this pattern to the kinetic fractionation of hg via internal demethylation . our study demonstrates that the measurement of hg isotope ratios provides new insight into sources and exposure pathways of mmhg in estuarine food webs . past studies that utilized hg isotope ratios to decipher mmhg sources and exposure pathways in coastal food webs have only examined fish , and over smaller coastal regions . we found evidence for multiple hg sources across the northeast coast sediments , and the mmhg associated with the sediments appears to serve as the dominant mmhg source to the estuarine food webs . complex biogeochemical processes affecting hg bioavailability between sediment and water column however make it difficult to trace the dominant mmhg sources in the estuarine food webs based on hg concentration alone . this study suggests that mmhg associated with sediments probably acts as the dominant source to many estuarine organisms , but that certain feeding guilds and certain localities are more susceptible to accumulating additional external mmhg sources . while further investigation is necessary to characterize various external mmhg sources , our study demonstrates that the measurement of hg isotope ratios can be a valuable tool for deciphering mmhg sources and exposure pathways in diverse aquatic food webs and assessing the ecological variability of mmhg sources .

it is the most commonly dislocated joint because of the large size of the humeral head compared to the small size of the glenoid fossa ( 1 ) . the glenohumeral ligaments ( ghls ) , joint capsule , and glenoid labrum are parts of the passive stabilizing mechanisms of the glenohumeral joint . the ghls are localized thickenings of the glenohumeral joint capsule that extend from the anterior and inferior glenoid margin of the joint to the anatomical neck of the humerus . three ligaments have been described : the superior glenohumeral ligament ( sghl ) , the middle glenohumeral ligament ( mghl ) , and the inferior glenohumeral ligament ( ighl ) complex , which are composed of an anterior band , a posterior band , and an axillary recess . the main two functions of the ghls are to avoid superior - inferior translation and to maintain anterior stability ( 2 ) . magnetic resonance ( mr ) imaging of the shoulder without intraarticular contrast medium can be used to observe some anatomical structures , such as the bony components , biceps tendon , and rotator cuff tendons . however , mr arthrography is mostly used for demonstrating abnormal ghls , and it is the most accurate imaging technique established ( 3 ) . the sghl is the most important structure in the anterosuperior part of the glenohumeral joint capsule ( 4 , 5 ) . it plays essential roles in anterosuperior impingement syndrome and in stabilizing the long head of the biceps tendon ( lhbt ) . thus , radiologists should observe the sghl and coexistent pathologies in patients presenting with pain at the anterosuperior section of the shoulder . the purpose of this study was to review the normal mr arthrography anatomy and variants of this important ligament . we also discussed about its basic biomechanics and illustrated examples of injuries involving this ligament . different methods of injections into the glenohumeral joint for diagnostic arthrography have been previously described . these methods involve fluoroscopy , ultrasonography , and computed tomography , as well as techniques other than image - guiding . for sonographic guidance , the use of ionizing radiation and iodinated contrast materials are avoided . although the anterior approach is the most commonly used , the posterior and the rotator interval approaches have recently been preferred ( 6 , 7 , 8) . both the posterior and rotator interval approaches are used at our institution via ultrasonography guidance . in the posterior approach , a transducer is placed over the long axis of the myotendinous junction of the infraspinatus muscle , and it is angled to show the contours of the posterior glenoid rim , posterior glenoid labrum , and posteromedial portion of the humeral head . the needle is introduced in a lateral to medial direction , obliquely from the skin to the glenohumeral joint space and parallel to the long axis of the transducer ( fig . 1 ) ( 7 ) . in the rotator interval approach , the coracoid process and the adjacent superomedial subchondral aspect of the humeral head are observed with a high frequency linear probe in a plane transverse to the joint line . the needle entry point is just superior to the subscapularis tendon and lateral to the coracoid process . the needle is advanced freehand , from medial to lateral , obliquely from the skin surface to the humeral head within the imaging plane of the transducer , until the tip reaches the cartilage of the humeral head ( fig . 2 ) . a direct mr arthrography of the glenohumeral joint after intraarticular injection of diluted gadolinium chelates is the preferred imaging technique for the evaluation of the labroligamentous complex and joint capsule ( 9 , 10 ) . observation of the ghls is difficult when using conventional shoulder mr imaging , because these ligaments may be poorly visualized or may mimic labral lesions ( 9 , 11 ) . ( 12 ) presented a cadaveric study comparing conventional mr imaging to mr arthrography of the same shoulder joint . in this study , the sghl was not identified by conventional mr imaging , in all of the cases ; however , it was identified by direct mr arthrography , in all of the cases . at our institution , we prefer direct mr arthrography for the identification of the labroligamentous complex . at our institution , mr arthrography and conventional mr imaging examinations are performed with a 1.5-t or 3-t mr scanner ( magnetom avanto or magnetom skyra ; siemens healthcare , berlin , germany ) within 15 minutes after the injection . our mr arthrography protocol uses fat - suppressed spin - echo ( se ) t1-weighted images . this is performed in the axial , oblique coronal , and oblique sagittal planes , with surface coils placed around the shoulder joint ( 650/15 , echo train length 8 mm , section thickness 3 mm , spacing 0.3 mm , field of view [ fov ] 16 cm , matrix 256 250 , 3 signals acquired ) . in our clinical practice , a fat - suppressed three dimensional ( 3d ) volumetric interpolated breath - hold examination ( vibe ) sequence ( repetition time / echo time 13.2/4.7 msec , flip angle 11 , 160 160 mm fov , matrix 512 512 , one slab of 112 slices with a slice thickness of 0.6 mm , and one acquisition ) is also added to the shoulder mr arthrography scanning protocol , after the t1-weighted se fat - suppressed sequences are acquired . fat - suppressed 3d vibe sequence imaging is a relatively new spoiled gradient - echo protocol that provides fast low angle images . the use of 3d vibe sequences produces thinner image slices with the thickness of 0.6 mm , which enhances effectiveness of the imaging . it also provides good contrast between the glenoid and surrounding soft tissues in mr arthrography of the glenohumeral joint . anatomical knowledge of the labroligamentous structures is crucial to the diagnosis of abnormalities revealed by mr arthrography . the glenoid labrum deepens and increases the surface area of the articulation ( 13 ) . it also serves as a primary anchoring structure for the ghls , the capsule , and the lhbt ( 3 ) . superiorly , the glenoid labrum blends with the sghl and the lhbt ; and anteriorly , it blends with the anterior band of the ighl ( 14 ) . the rotator interval consists of the following structures , in the order from outside to inside : the coracohumeral ligament ( chl ) , the interval capsule , and the sghl . the chl arises from the dorsolateral aspect of the base of the coracoid process and courses through the rotator interval to blend with fibers from the sghl . its fibers then attach laterally to the greater tuberosity , fusing with the anterior fibers of the supraspinatus tendon insertion and the medial bicipital sheath , before fusing with the superior fibers of the subscapularis tendon insertion ( 13 ) . the rotator interval may be thought of as a roof over the intraarticular portion of the lhbt ( 3 ) which arises from the anterosuperior labrum ( 13 , 14 ) . the chl and sghl are composed of a sling - like band surrounding the lhbt proximal to the bicipital sulcus ( 13 ) . it originates from the supraglenoid tubercle anterior to the lhbt attachment , and inserts into the fovea capitis line superior to the lesser tuberosity of the bicipital sulcus ( 13 , 15 ) . the sghl is identified in 97 - 98% of the patients through arthroscopic and arthrographic series ( 16 ) . macroscopically , the sghl is u - shaped and this shape lends support to the lhbt in the superior aspect of the bicipital sulcus ( 13 ) . the sghl runs parallel to the coracoid process and nearly perpendicular to the mghl ( 1 , 3 , 13 , 14 , 16 ) . using mr arthrography , the sghl is best visualized in the sagittal oblique and axial planes ( fig . the sghl is visualized as a low signal intensity intraarticular structure parallel to the coracoid process . sagittal oblique mr arthrography shows that the sghl is located underneath the coracoid process and chl . a normal foramen exists between the sghl and the mghl , allowing communication of the glenohumeral joint cavity through the subscapularis recess ( 1 , 3 , 17 , 18 ) . the sghl usually originates from the supraglenoid tubercle , just anterior to the lhbt attachment . however , its origin may vary ( posterior supraglenoid tubercle , superior labrum , lhbt , mghl , or some combination ) ( figs . 4 , 5 , 6 , 7 ) ( 17 , 19 ) . although the sghl may vary in shape and size ( fig . 8) , it usually presents as a thick structure when the mghl is absent or undeveloped ( figs . it may be absent in arthroscopic and mr arthrographic series , in 2 - 3% of the patients ( 16 ) . in a recent study by kask et al . ( 20 ) , the sghl was found in all 27 shoulder joint specimens investigated . a longitudinal split or duplicate ligament may reflect a normal variant or a longitudinal tear of the sghl ( fig . magnetic resonance arthrography of the glenohumeral joint is the most accurate imaging technique established , for identifying pathologies of the sghl and the associated structures . in a retrospective study comparing mr arthrographic findings with surgical findings , chandnani et al . ( 21 ) determined that mr arthrography had a sensitivity of 100% , a specificity of 94% , and an accuracy of 94% in the diagnosis of sghl tears . a torn sghl may be observed in mr arthrography images as increased signal intensity that looks like disruptions and thick , wavy , or irregular structures ( 19 , 22 ) . this complex anatomical structure consists of the sghl and the chl , and it is also in direct contact with the distal fibers of the subscapularis and the supraspinatus tendons . based on this complex relationship , it has been suggested that the sghl is the major stabilizing component in the anterosuperior portion of the glenohumeral joint capsule ( 23 , 24 , 25 , 26 , 27 ) . therefore , tears in this ligament may lead to medial subluxation or dislocation of the lhbt . recently , schaeffeler et al . ( 28 ) showed that the prevalence of isolated sghl tears is 29% . biceps pulley lesions can occur due to acute trauma , degenerative changes , repetitive microtrauma , congenital rotator interval defects , or injuries associated with tear in a rotator cuff ( 13 , 24 , 28 , 29 , 30 ) . these lesions are thought to be responsible for medial subluxation of the lhbt , and they may result in loss of function in the shoulder and pain in the anterior shoulder ( 24 , 25 , 31 ) . the frequency of lesions on isolated biceps pulley is 29 - 74% ( 24 , 25 , 28 ) . a recent study by braun et al . ( 25 ) prospectively analyzed the prevalence of pulley lesions , which was reported as 32% , in a consecutive series of patients undergoing arthroscopic shoulder surgery . in this study , weishaupt et al . ( 23 ) reported discontinuity of the sghl in biceps pulley lesions . the lesions of the biceps pulley , the lhbt , the subscapularis , and the supraspinatus tendon are often closely associated with each other ( fig . the instability pattern of the lhbt and the presence of tears on the rotator cuff tendons adjacent to the rotator interval can be used to diagnose biceps pulley lesions . ( 31 ) investigated the rotator interval in a series of 116 patients with isolated lesions of the supraspinatus tendon . lesion in the biceps pulley and millimetric tears on the superior border of the subscapularis tendon occurred in 19 of 116 shoulders . a recent arthrographic study by schaeffeler et al . ( 28 ) concluded that the presence of associated tendinopathy of the lhbt in oblique sagittal mr arthrography images has additional value for the diagnosis of a pulley lesion . biceps pulley lesions are difficult to diagnose by routine arthroscopic examination , so they are called " hidden lesions " . these injuries have been arthroscopically classified by bennett ( 32 ) , and this arthroscopic classification system is based on lesions involving the sghl , the chl , and the subscapularis tendon ; however , it is not applicable for mr arthrography ( 28 ) . habermeyer et al . ( 24 ) subsequently classified biceps pulley lesions into four different patterns , which shows the importance of the sghl in the lateral rotator interval ( 28 , 31 ) . according to the habermeyer classification system , group 1 lesions represent the isolated tears in sghl , with the subscapularis and supraspinatus tendons intact without biceps tendon instability ( fig . group 2 lesions involve a biceps pulley lesion in association with a partial tear in the articular - side supraspinatus tendon and a mild medial subluxation of the lhbt ( fig . group 3 lesions involve a biceps pulley lesion in association with a partial tear on articular - side subscapularis tendon and a subluxation of the lhbt ( fig . group 4 lesions involve a biceps pulley lesion in association with partial tears on articular - side of both the supraspinatus and subscapularis tendons and a medial dislocation of the lhbt ( fig . the diseases associated with biceps pulley lesions include internal anterosuperior impingement , instability of the lhbt , biceps tendinopathy , superior labrum anterior and posterior lesions ( slap ) , and adhesive capsulitis ( 13 ) . internal anterosuperior impingement was first described by gerber and sebesta ( 33 ) as a form of intraarticular impingement responsible for unexplained anterior shoulder pain . habermeyer et al . ( 24 ) subsequently described this disease as an impingement of the lhbt by the anterosuperior glenoid rim . in an arthroscopic study , habermeyer et al . ( 24 ) found partial tears on articular side of subscapularis tendon in 71.8% of the patients with anterosuperior impingement . superior labrum anterior and posterior lesions may be associated with lesions of the rotator interval and biceps pulley complex , and this was clearly described by braun et al . this original classification system is still the most widely accepted one , and six additional classifications have been developed . these new classifications represent the combination of slap lesions , with extension into different areas of the glenoid labrum and other adjacent capsuloligamentous structures . according to the new classifications , a type 10 lesion consists of a superior labral tear with extension into the rotator interval complex ( fig . otherwise , in patients with shoulder instability , the extensive anterior and anterosuperior labral tears can extend into the sghl and mghl ( figs . magnetic resonance arthrography of the glenohumeral joint is sensitive to susceptibility artifacts caused by inadvertently injected intraarticular air bubbles . with advanced sequences , such as fat - suppressed vibe , therefore , small air bubbles are usually distributed at the anterosuperior part of the joint cavity , next to the sghl or chl and the horizontal part of the lhbt ( fig . air bubbles may occasionally mimic intraarticular loose bodies , chondrocalcinosis of hyaline cartilage , and inflammatory adhesions of the joint capsule . knowledge of the intraarticular arrangement of gas bubbles would help in avoiding misinterpretation ( 36 ) . the haemorrhage resolves over time , but the hemosiderin particles may accumulate in the synovium . concordantly , the vibe sequence is a sensitive technique that will reveal the hemosiderin particles in the synovium and joint space , as well as intra - articular air bubbles ( fig . different methods of injections into the glenohumeral joint for diagnostic arthrography have been previously described . these methods involve fluoroscopy , ultrasonography , and computed tomography , as well as techniques other than image - guiding . for sonographic guidance , the use of ionizing radiation and iodinated contrast materials are avoided . although the anterior approach is the most commonly used , the posterior and the rotator interval approaches have recently been preferred ( 6 , 7 , 8) . both the posterior and rotator interval approaches are used at our institution via ultrasonography guidance . in the posterior approach , a transducer is placed over the long axis of the myotendinous junction of the infraspinatus muscle , and it is angled to show the contours of the posterior glenoid rim , posterior glenoid labrum , and posteromedial portion of the humeral head . the needle is introduced in a lateral to medial direction , obliquely from the skin to the glenohumeral joint space and parallel to the long axis of the transducer ( fig . 1 ) ( 7 ) . in the rotator interval approach , the coracoid process and the adjacent superomedial subchondral aspect of the humeral head are observed with a high frequency linear probe in a plane transverse to the joint line . the needle entry point is just superior to the subscapularis tendon and lateral to the coracoid process . the needle is advanced freehand , from medial to lateral , obliquely from the skin surface to the humeral head within the imaging plane of the transducer , until the tip reaches the cartilage of the humeral head ( fig . a direct mr arthrography of the glenohumeral joint after intraarticular injection of diluted gadolinium chelates is the preferred imaging technique for the evaluation of the labroligamentous complex and joint capsule ( 9 , 10 ) . observation of the ghls is difficult when using conventional shoulder mr imaging , because these ligaments may be poorly visualized or may mimic labral lesions ( 9 , 11 ) . ( 12 ) presented a cadaveric study comparing conventional mr imaging to mr arthrography of the same shoulder joint . in this study , the sghl was not identified by conventional mr imaging , in all of the cases ; however , it was identified by direct mr arthrography , in all of the cases . at our institution , we prefer direct mr arthrography for the identification of the labroligamentous complex . at our institution , mr arthrography and conventional mr imaging examinations are performed with a 1.5-t or 3-t mr scanner ( magnetom avanto or magnetom skyra ; siemens healthcare , berlin , germany ) within 15 minutes after the injection . our mr arthrography protocol uses fat - suppressed spin - echo ( se ) t1-weighted images . this is performed in the axial , oblique coronal , and oblique sagittal planes , with surface coils placed around the shoulder joint ( 650/15 , echo train length 8 mm , section thickness 3 mm , spacing 0.3 mm , field of view [ fov ] 16 cm , matrix 256 250 , 3 signals acquired ) . in our clinical practice , a fat - suppressed three dimensional ( 3d ) volumetric interpolated breath - hold examination ( vibe ) sequence ( repetition time / echo time 13.2/4.7 msec , flip angle 11 , 160 160 mm fov , matrix 512 512 , one slab of 112 slices with a slice thickness of 0.6 mm , and one acquisition ) is also added to the shoulder mr arthrography scanning protocol , after the t1-weighted se fat - suppressed sequences are acquired . fat - suppressed 3d vibe sequence imaging is a relatively new spoiled gradient - echo protocol that provides fast low angle images . the use of 3d vibe sequences produces thinner image slices with the thickness of 0.6 mm , which enhances effectiveness of the imaging . it also provides good contrast between the glenoid and surrounding soft tissues in mr arthrography of the glenohumeral joint . anatomical knowledge of the labroligamentous structures is crucial to the diagnosis of abnormalities revealed by mr arthrography . the glenoid labrum deepens and increases the surface area of the articulation ( 13 ) . it also serves as a primary anchoring structure for the ghls , the capsule , and the lhbt ( 3 ) . superiorly , the glenoid labrum blends with the sghl and the lhbt ; and anteriorly , it blends with the anterior band of the ighl ( 14 ) . the rotator interval consists of the following structures , in the order from outside to inside : the coracohumeral ligament ( chl ) , the interval capsule , and the sghl . the chl arises from the dorsolateral aspect of the base of the coracoid process and courses through the rotator interval to blend with fibers from the sghl . its fibers then attach laterally to the greater tuberosity , fusing with the anterior fibers of the supraspinatus tendon insertion and the medial bicipital sheath , before fusing with the superior fibers of the subscapularis tendon insertion ( 13 ) . the rotator interval may be thought of as a roof over the intraarticular portion of the lhbt ( 3 ) which arises from the anterosuperior labrum ( 13 , 14 ) . the chl and sghl are composed of a sling - like band surrounding the lhbt proximal to the bicipital sulcus ( 13 ) . it originates from the supraglenoid tubercle anterior to the lhbt attachment , and inserts into the fovea capitis line superior to the lesser tuberosity of the bicipital sulcus ( 13 , 15 ) . the sghl is identified in 97 - 98% of the patients through arthroscopic and arthrographic series ( 16 ) . macroscopically , the sghl is u - shaped and this shape lends support to the lhbt in the superior aspect of the bicipital sulcus ( 13 ) . the sghl runs parallel to the coracoid process and nearly perpendicular to the mghl ( 1 , 3 , 13 , 14 , 16 ) . using mr arthrography , the sghl is best visualized in the sagittal oblique and axial planes ( fig . the sghl is visualized as a low signal intensity intraarticular structure parallel to the coracoid process . sagittal oblique mr arthrography shows that the sghl is located underneath the coracoid process and chl . a normal foramen exists between the sghl and the mghl , allowing communication of the glenohumeral joint cavity through the subscapularis recess ( 1 , 3 , 17 , 18 ) . the sghl usually originates from the supraglenoid tubercle , just anterior to the lhbt attachment . however , its origin may vary ( posterior supraglenoid tubercle , superior labrum , lhbt , mghl , or some combination ) ( figs . 4 , 5 , 6 , 7 ) ( 17 , 19 ) . although the sghl may vary in shape and size ( fig . 8) , it usually presents as a thick structure when the mghl is absent or undeveloped ( figs . it may be absent in arthroscopic and mr arthrographic series , in 2 - 3% of the patients ( 16 ) . in a recent study by kask et al . ( 20 ) , the sghl was found in all 27 shoulder joint specimens investigated . a longitudinal split or duplicate ligament may reflect a normal variant or a longitudinal tear of the sghl ( fig . magnetic resonance arthrography of the glenohumeral joint is the most accurate imaging technique established , for identifying pathologies of the sghl and the associated structures . in a retrospective study comparing mr arthrographic findings with surgical findings , chandnani et al . ( 21 ) determined that mr arthrography had a sensitivity of 100% , a specificity of 94% , and an accuracy of 94% in the diagnosis of sghl tears . a torn sghl may be observed in mr arthrography images as increased signal intensity that looks like disruptions and thick , wavy , or irregular structures ( 19 , 22 ) . the biceps pulley is an important component of the rotator interval . this complex anatomical structure consists of the sghl and the chl , and it is also in direct contact with the distal fibers of the subscapularis and the supraspinatus tendons . based on this complex relationship , it has been suggested that the sghl is the major stabilizing component in the anterosuperior portion of the glenohumeral joint capsule ( 23 , 24 , 25 , 26 , 27 ) . therefore , tears in this ligament may lead to medial subluxation or dislocation of the lhbt . recently , schaeffeler et al . ( 28 ) showed that the prevalence of isolated sghl tears is 29% . biceps pulley lesions can occur due to acute trauma , degenerative changes , repetitive microtrauma , congenital rotator interval defects , or injuries associated with tear in a rotator cuff ( 13 , 24 , 28 , 29 , 30 ) . these lesions are thought to be responsible for medial subluxation of the lhbt , and they may result in loss of function in the shoulder and pain in the anterior shoulder ( 24 , 25 , 31 ) . the frequency of lesions on isolated biceps pulley is 29 - 74% ( 24 , 25 , 28 ) . a recent study by braun et al . ( 25 ) prospectively analyzed the prevalence of pulley lesions , which was reported as 32% , in a consecutive series of patients undergoing arthroscopic shoulder surgery . in this study , the lesions of the biceps pulley , the lhbt , the subscapularis , and the supraspinatus tendon are often closely associated with each other ( fig . the instability pattern of the lhbt and the presence of tears on the rotator cuff tendons adjacent to the rotator interval can be used to diagnose biceps pulley lesions . ( 31 ) investigated the rotator interval in a series of 116 patients with isolated lesions of the supraspinatus tendon . lesion in the biceps pulley and millimetric tears on the superior border of the subscapularis tendon occurred in 19 of 116 shoulders . a recent arthrographic study by schaeffeler et al . ( 28 ) concluded that the presence of associated tendinopathy of the lhbt in oblique sagittal mr arthrography images has additional value for the diagnosis of a pulley lesion . biceps pulley lesions are difficult to diagnose by routine arthroscopic examination , so they are called " hidden lesions " . these injuries have been arthroscopically classified by bennett ( 32 ) , and this arthroscopic classification system is based on lesions involving the sghl , the chl , and the subscapularis tendon ; however , it is not applicable for mr arthrography ( 28 ) . habermeyer et al . ( 24 ) subsequently classified biceps pulley lesions into four different patterns , which shows the importance of the sghl in the lateral rotator interval ( 28 , 31 ) . according to the habermeyer classification system , group 1 lesions represent the isolated tears in sghl , with the subscapularis and supraspinatus tendons intact without biceps tendon instability ( fig . group 2 lesions involve a biceps pulley lesion in association with a partial tear in the articular - side supraspinatus tendon and a mild medial subluxation of the lhbt ( fig . group 3 lesions involve a biceps pulley lesion in association with a partial tear on articular - side subscapularis tendon and a subluxation of the lhbt ( fig . group 4 lesions involve a biceps pulley lesion in association with partial tears on articular - side of both the supraspinatus and subscapularis tendons and a medial dislocation of the lhbt ( fig . the diseases associated with biceps pulley lesions include internal anterosuperior impingement , instability of the lhbt , biceps tendinopathy , superior labrum anterior and posterior lesions ( slap ) , and adhesive capsulitis ( 13 ) . the sghl can be thicker in the patients with adhesive capsulitis ( fig . internal anterosuperior impingement was first described by gerber and sebesta ( 33 ) as a form of intraarticular impingement responsible for unexplained anterior shoulder pain . habermeyer et al . ( 24 ) subsequently described this disease as an impingement of the lhbt by the anterosuperior glenoid rim . in an arthroscopic study , habermeyer et al . ( 24 ) found partial tears on articular side of subscapularis tendon in 71.8% of the patients with anterosuperior impingement . superior labrum anterior and posterior lesions may be associated with lesions of the rotator interval and biceps pulley complex , and this was clearly described by braun et al . this original classification system is still the most widely accepted one , and six additional classifications have been developed . these new classifications represent the combination of slap lesions , with extension into different areas of the glenoid labrum and other adjacent capsuloligamentous structures . according to the new classifications , a type 10 lesion consists of a superior labral tear with extension into the rotator interval complex ( fig . otherwise , in patients with shoulder instability , the extensive anterior and anterosuperior labral tears can extend into the sghl and mghl ( figs . magnetic resonance arthrography of the glenohumeral joint is sensitive to susceptibility artifacts caused by inadvertently injected intraarticular air bubbles . with advanced sequences , such as fat - suppressed vibe , therefore , small air bubbles are usually distributed at the anterosuperior part of the joint cavity , next to the sghl or chl and the horizontal part of the lhbt ( fig . air bubbles may occasionally mimic intraarticular loose bodies , chondrocalcinosis of hyaline cartilage , and inflammatory adhesions of the joint capsule . knowledge of the intraarticular arrangement of gas bubbles would help in avoiding misinterpretation ( 36 ) . the haemorrhage resolves over time , but the hemosiderin particles may accumulate in the synovium . concordantly , the vibe sequence is a sensitive technique that will reveal the hemosiderin particles in the synovium and joint space , as well as intra - articular air bubbles ( fig . the sghl is known to be difficult to evaluate based on anatomical structures identified in arthrographic and arthroscopic examinations . the sghl can be ruptured as an isolated form and it can also be damaged with anterosuperior impingement syndrome , biceps tendon instability , slap lesions , and anterior instability of the glenohumeral joint . in addition to routine mr arthrography sequences , advanced mr arthrographic sequences such as vibe may greatly help in the diagnosis of the sghl and in identifying its morphology and the associated pathologies .

the purpose of this review was to demonstrate magnetic resonance ( mr ) arthrography findings of anatomy , variants , and pathologic conditions of the superior glenohumeral ligament ( sghl ) . this review also demonstrates the applicability of a new mr arthrography sequence in the anterosuperior portion of the glenohumeral joint . the sghl is a very important anatomical structure in the rotator interval that is responsible for stabilizing the long head of the biceps tendon . therefore , a torn sghl can result in pain and instability . observation of the sghl is difficult when using conventional mr imaging , because the ligament may be poorly visualized . shoulder mr arthrography is the most accurately established imaging technique for identifying pathologies of the sghl and associated structures . the use of three dimensional ( 3d ) volumetric interpolated breath - hold examination ( vibe ) sequences produces thinner image slices and enables a higher in - plane resolution than conventional mr arthrography sequences . therefore , shoulder mr arthrography using 3d vibe sequences may contribute to evaluating of the smaller intraarticular structures such as the sghl .

INTRODUCTION Arthrography Procedure Imaging Techniques Normal Anatomy of the Superior Glenohumeral Ligament Congenital Variants of the Superior Glenohumeral Ligament Pathologies of the Superior Glenohumeral Ligament Diagnostic Pitfalls CONCLUSIONS

it is the most commonly dislocated joint because of the large size of the humeral head compared to the small size of the glenoid fossa ( 1 ) . the glenohumeral ligaments ( ghls ) , joint capsule , and glenoid labrum are parts of the passive stabilizing mechanisms of the glenohumeral joint . the ghls are localized thickenings of the glenohumeral joint capsule that extend from the anterior and inferior glenoid margin of the joint to the anatomical neck of the humerus . three ligaments have been described : the superior glenohumeral ligament ( sghl ) , the middle glenohumeral ligament ( mghl ) , and the inferior glenohumeral ligament ( ighl ) complex , which are composed of an anterior band , a posterior band , and an axillary recess . magnetic resonance ( mr ) imaging of the shoulder without intraarticular contrast medium can be used to observe some anatomical structures , such as the bony components , biceps tendon , and rotator cuff tendons . however , mr arthrography is mostly used for demonstrating abnormal ghls , and it is the most accurate imaging technique established ( 3 ) . the sghl is the most important structure in the anterosuperior part of the glenohumeral joint capsule ( 4 , 5 ) . it plays essential roles in anterosuperior impingement syndrome and in stabilizing the long head of the biceps tendon ( lhbt ) . thus , radiologists should observe the sghl and coexistent pathologies in patients presenting with pain at the anterosuperior section of the shoulder . the purpose of this study was to review the normal mr arthrography anatomy and variants of this important ligament . although the anterior approach is the most commonly used , the posterior and the rotator interval approaches have recently been preferred ( 6 , 7 , 8) . in the posterior approach , a transducer is placed over the long axis of the myotendinous junction of the infraspinatus muscle , and it is angled to show the contours of the posterior glenoid rim , posterior glenoid labrum , and posteromedial portion of the humeral head . the needle is introduced in a lateral to medial direction , obliquely from the skin to the glenohumeral joint space and parallel to the long axis of the transducer ( fig . in the rotator interval approach , the coracoid process and the adjacent superomedial subchondral aspect of the humeral head are observed with a high frequency linear probe in a plane transverse to the joint line . a direct mr arthrography of the glenohumeral joint after intraarticular injection of diluted gadolinium chelates is the preferred imaging technique for the evaluation of the labroligamentous complex and joint capsule ( 9 , 10 ) . observation of the ghls is difficult when using conventional shoulder mr imaging , because these ligaments may be poorly visualized or may mimic labral lesions ( 9 , 11 ) . ( 12 ) presented a cadaveric study comparing conventional mr imaging to mr arthrography of the same shoulder joint . in this study , the sghl was not identified by conventional mr imaging , in all of the cases ; however , it was identified by direct mr arthrography , in all of the cases . at our institution , we prefer direct mr arthrography for the identification of the labroligamentous complex . at our institution , mr arthrography and conventional mr imaging examinations are performed with a 1.5-t or 3-t mr scanner ( magnetom avanto or magnetom skyra ; siemens healthcare , berlin , germany ) within 15 minutes after the injection . in our clinical practice , a fat - suppressed three dimensional ( 3d ) volumetric interpolated breath - hold examination ( vibe ) sequence ( repetition time / echo time 13.2/4.7 msec , flip angle 11 , 160 160 mm fov , matrix 512 512 , one slab of 112 slices with a slice thickness of 0.6 mm , and one acquisition ) is also added to the shoulder mr arthrography scanning protocol , after the t1-weighted se fat - suppressed sequences are acquired . the use of 3d vibe sequences produces thinner image slices with the thickness of 0.6 mm , which enhances effectiveness of the imaging . it also provides good contrast between the glenoid and surrounding soft tissues in mr arthrography of the glenohumeral joint . superiorly , the glenoid labrum blends with the sghl and the lhbt ; and anteriorly , it blends with the anterior band of the ighl ( 14 ) . the rotator interval consists of the following structures , in the order from outside to inside : the coracohumeral ligament ( chl ) , the interval capsule , and the sghl . the chl arises from the dorsolateral aspect of the base of the coracoid process and courses through the rotator interval to blend with fibers from the sghl . the rotator interval may be thought of as a roof over the intraarticular portion of the lhbt ( 3 ) which arises from the anterosuperior labrum ( 13 , 14 ) . the sghl is identified in 97 - 98% of the patients through arthroscopic and arthrographic series ( 16 ) . macroscopically , the sghl is u - shaped and this shape lends support to the lhbt in the superior aspect of the bicipital sulcus ( 13 ) . using mr arthrography , the sghl is best visualized in the sagittal oblique and axial planes ( fig . sagittal oblique mr arthrography shows that the sghl is located underneath the coracoid process and chl . a normal foramen exists between the sghl and the mghl , allowing communication of the glenohumeral joint cavity through the subscapularis recess ( 1 , 3 , 17 , 18 ) . it may be absent in arthroscopic and mr arthrographic series , in 2 - 3% of the patients ( 16 ) . a longitudinal split or duplicate ligament may reflect a normal variant or a longitudinal tear of the sghl ( fig . magnetic resonance arthrography of the glenohumeral joint is the most accurate imaging technique established , for identifying pathologies of the sghl and the associated structures . ( 21 ) determined that mr arthrography had a sensitivity of 100% , a specificity of 94% , and an accuracy of 94% in the diagnosis of sghl tears . a torn sghl may be observed in mr arthrography images as increased signal intensity that looks like disruptions and thick , wavy , or irregular structures ( 19 , 22 ) . this complex anatomical structure consists of the sghl and the chl , and it is also in direct contact with the distal fibers of the subscapularis and the supraspinatus tendons . based on this complex relationship , it has been suggested that the sghl is the major stabilizing component in the anterosuperior portion of the glenohumeral joint capsule ( 23 , 24 , 25 , 26 , 27 ) . therefore , tears in this ligament may lead to medial subluxation or dislocation of the lhbt . these lesions are thought to be responsible for medial subluxation of the lhbt , and they may result in loss of function in the shoulder and pain in the anterior shoulder ( 24 , 25 , 31 ) . the lesions of the biceps pulley , the lhbt , the subscapularis , and the supraspinatus tendon are often closely associated with each other ( fig . the instability pattern of the lhbt and the presence of tears on the rotator cuff tendons adjacent to the rotator interval can be used to diagnose biceps pulley lesions . ( 31 ) investigated the rotator interval in a series of 116 patients with isolated lesions of the supraspinatus tendon . lesion in the biceps pulley and millimetric tears on the superior border of the subscapularis tendon occurred in 19 of 116 shoulders . ( 28 ) concluded that the presence of associated tendinopathy of the lhbt in oblique sagittal mr arthrography images has additional value for the diagnosis of a pulley lesion . these injuries have been arthroscopically classified by bennett ( 32 ) , and this arthroscopic classification system is based on lesions involving the sghl , the chl , and the subscapularis tendon ; however , it is not applicable for mr arthrography ( 28 ) . ( 24 ) subsequently classified biceps pulley lesions into four different patterns , which shows the importance of the sghl in the lateral rotator interval ( 28 , 31 ) . superior labrum anterior and posterior lesions may be associated with lesions of the rotator interval and biceps pulley complex , and this was clearly described by braun et al . according to the new classifications , a type 10 lesion consists of a superior labral tear with extension into the rotator interval complex ( fig . otherwise , in patients with shoulder instability , the extensive anterior and anterosuperior labral tears can extend into the sghl and mghl ( figs . magnetic resonance arthrography of the glenohumeral joint is sensitive to susceptibility artifacts caused by inadvertently injected intraarticular air bubbles . with advanced sequences , such as fat - suppressed vibe , therefore , small air bubbles are usually distributed at the anterosuperior part of the joint cavity , next to the sghl or chl and the horizontal part of the lhbt ( fig . although the anterior approach is the most commonly used , the posterior and the rotator interval approaches have recently been preferred ( 6 , 7 , 8) . in the posterior approach , a transducer is placed over the long axis of the myotendinous junction of the infraspinatus muscle , and it is angled to show the contours of the posterior glenoid rim , posterior glenoid labrum , and posteromedial portion of the humeral head . the needle is introduced in a lateral to medial direction , obliquely from the skin to the glenohumeral joint space and parallel to the long axis of the transducer ( fig . in the rotator interval approach , the coracoid process and the adjacent superomedial subchondral aspect of the humeral head are observed with a high frequency linear probe in a plane transverse to the joint line . a direct mr arthrography of the glenohumeral joint after intraarticular injection of diluted gadolinium chelates is the preferred imaging technique for the evaluation of the labroligamentous complex and joint capsule ( 9 , 10 ) . observation of the ghls is difficult when using conventional shoulder mr imaging , because these ligaments may be poorly visualized or may mimic labral lesions ( 9 , 11 ) . ( 12 ) presented a cadaveric study comparing conventional mr imaging to mr arthrography of the same shoulder joint . in this study , the sghl was not identified by conventional mr imaging , in all of the cases ; however , it was identified by direct mr arthrography , in all of the cases . at our institution , mr arthrography and conventional mr imaging examinations are performed with a 1.5-t or 3-t mr scanner ( magnetom avanto or magnetom skyra ; siemens healthcare , berlin , germany ) within 15 minutes after the injection . in our clinical practice , a fat - suppressed three dimensional ( 3d ) volumetric interpolated breath - hold examination ( vibe ) sequence ( repetition time / echo time 13.2/4.7 msec , flip angle 11 , 160 160 mm fov , matrix 512 512 , one slab of 112 slices with a slice thickness of 0.6 mm , and one acquisition ) is also added to the shoulder mr arthrography scanning protocol , after the t1-weighted se fat - suppressed sequences are acquired . the use of 3d vibe sequences produces thinner image slices with the thickness of 0.6 mm , which enhances effectiveness of the imaging . it also provides good contrast between the glenoid and surrounding soft tissues in mr arthrography of the glenohumeral joint . anatomical knowledge of the labroligamentous structures is crucial to the diagnosis of abnormalities revealed by mr arthrography . superiorly , the glenoid labrum blends with the sghl and the lhbt ; and anteriorly , it blends with the anterior band of the ighl ( 14 ) . the rotator interval consists of the following structures , in the order from outside to inside : the coracohumeral ligament ( chl ) , the interval capsule , and the sghl . the chl arises from the dorsolateral aspect of the base of the coracoid process and courses through the rotator interval to blend with fibers from the sghl . its fibers then attach laterally to the greater tuberosity , fusing with the anterior fibers of the supraspinatus tendon insertion and the medial bicipital sheath , before fusing with the superior fibers of the subscapularis tendon insertion ( 13 ) . the rotator interval may be thought of as a roof over the intraarticular portion of the lhbt ( 3 ) which arises from the anterosuperior labrum ( 13 , 14 ) . it originates from the supraglenoid tubercle anterior to the lhbt attachment , and inserts into the fovea capitis line superior to the lesser tuberosity of the bicipital sulcus ( 13 , 15 ) . the sghl is identified in 97 - 98% of the patients through arthroscopic and arthrographic series ( 16 ) . macroscopically , the sghl is u - shaped and this shape lends support to the lhbt in the superior aspect of the bicipital sulcus ( 13 ) . using mr arthrography , the sghl is best visualized in the sagittal oblique and axial planes ( fig . sagittal oblique mr arthrography shows that the sghl is located underneath the coracoid process and chl . a normal foramen exists between the sghl and the mghl , allowing communication of the glenohumeral joint cavity through the subscapularis recess ( 1 , 3 , 17 , 18 ) . a longitudinal split or duplicate ligament may reflect a normal variant or a longitudinal tear of the sghl ( fig . magnetic resonance arthrography of the glenohumeral joint is the most accurate imaging technique established , for identifying pathologies of the sghl and the associated structures . ( 21 ) determined that mr arthrography had a sensitivity of 100% , a specificity of 94% , and an accuracy of 94% in the diagnosis of sghl tears . a torn sghl may be observed in mr arthrography images as increased signal intensity that looks like disruptions and thick , wavy , or irregular structures ( 19 , 22 ) . the biceps pulley is an important component of the rotator interval . this complex anatomical structure consists of the sghl and the chl , and it is also in direct contact with the distal fibers of the subscapularis and the supraspinatus tendons . based on this complex relationship , it has been suggested that the sghl is the major stabilizing component in the anterosuperior portion of the glenohumeral joint capsule ( 23 , 24 , 25 , 26 , 27 ) . therefore , tears in this ligament may lead to medial subluxation or dislocation of the lhbt . these lesions are thought to be responsible for medial subluxation of the lhbt , and they may result in loss of function in the shoulder and pain in the anterior shoulder ( 24 , 25 , 31 ) . in this study , the lesions of the biceps pulley , the lhbt , the subscapularis , and the supraspinatus tendon are often closely associated with each other ( fig . the instability pattern of the lhbt and the presence of tears on the rotator cuff tendons adjacent to the rotator interval can be used to diagnose biceps pulley lesions . ( 31 ) investigated the rotator interval in a series of 116 patients with isolated lesions of the supraspinatus tendon . lesion in the biceps pulley and millimetric tears on the superior border of the subscapularis tendon occurred in 19 of 116 shoulders . ( 28 ) concluded that the presence of associated tendinopathy of the lhbt in oblique sagittal mr arthrography images has additional value for the diagnosis of a pulley lesion . these injuries have been arthroscopically classified by bennett ( 32 ) , and this arthroscopic classification system is based on lesions involving the sghl , the chl , and the subscapularis tendon ; however , it is not applicable for mr arthrography ( 28 ) . ( 24 ) subsequently classified biceps pulley lesions into four different patterns , which shows the importance of the sghl in the lateral rotator interval ( 28 , 31 ) . the sghl can be thicker in the patients with adhesive capsulitis ( fig . ( 24 ) subsequently described this disease as an impingement of the lhbt by the anterosuperior glenoid rim . superior labrum anterior and posterior lesions may be associated with lesions of the rotator interval and biceps pulley complex , and this was clearly described by braun et al . according to the new classifications , a type 10 lesion consists of a superior labral tear with extension into the rotator interval complex ( fig . otherwise , in patients with shoulder instability , the extensive anterior and anterosuperior labral tears can extend into the sghl and mghl ( figs . magnetic resonance arthrography of the glenohumeral joint is sensitive to susceptibility artifacts caused by inadvertently injected intraarticular air bubbles . with advanced sequences , such as fat - suppressed vibe , therefore , small air bubbles are usually distributed at the anterosuperior part of the joint cavity , next to the sghl or chl and the horizontal part of the lhbt ( fig . the sghl is known to be difficult to evaluate based on anatomical structures identified in arthrographic and arthroscopic examinations . the sghl can be ruptured as an isolated form and it can also be damaged with anterosuperior impingement syndrome , biceps tendon instability , slap lesions , and anterior instability of the glenohumeral joint . in addition to routine mr arthrography sequences , advanced mr arthrographic sequences such as vibe may greatly help in the diagnosis of the sghl and in identifying its morphology and the associated pathologies .

the term andropause has been used to describe syndromes in aging males including various clinical symptoms encompassing somatic , psychological , and sexual dimensions , as well as endocrine dysfunction , partly related to an age - related decline in androgen and even androgen deficiency.13 it is also associated with dysthymia4 and an increase in depressive symptoms.5,6 exogenous testosterone replacement may be beneficial for treatment - resistant depression associated with androgen deficiency.6,7 androgen deficiency is associated with premature death , erectile dysfunction ( ed ) , metabolic syndrome , and cardiovascular disease.2 symptoms of andropause and androgen deficiency overlap with common psychiatric disorders such as major depressive disorder ( mdd).8 in one study conducted in a male climacteric clinic in japan , patients with mdd had significantly more severe symptoms of aging as assessed by the aging males symptoms ( ams ) scale than did patients without mdd ; the estimated prevalence of mdd approached 50%.9 sato et al , in 2007 , reported that the prevalence of mdd was 42% in an andrology clinic and recommended routine screening for mdd.10 although the gold standard for the diagnosis of androgen deficiency is the measurement of serum free or bioavailable testosterone , such data are not available for every male suspected of suffering from decreased testicular function.11 in order to assess aging males symptoms , a valuable and easily applicable screening tool should be considered . in the present study , first , the validity and reliability of the ams scale had been established in taiwan.12 second , the internal structure of the ams scale across other countries has been sufficiently similar to conclude that the scale measures the same phenomenon in varying contexts.13 third , the ams scale was developed in response to the lack of a standardized scale14 such as the androgen deficiency in aging males questionnaire . fourth , it can measure the severity of symptoms of aging over time in different dimensions as well as their impact on health - related quality of life ( qol).15 due to the complex nature of the phenomenon and the limited number of studies in the male psychiatric population , we investigated the relationship between aging males symptoms and emotional distress by using the ams scale and the hospital anxiety and depression scale ( hads ) , and examined whether sexual dysfunction could be a differentiating characteristic for symptoms of aging among middle - aged or older male psychiatric outpatients . the sample was previously recruited for validation of the chinese version of the ams scale , and a more detailed description has been published elsewhere.12 one hundred seventy - six taiwanese men , 4080 years of age , attending the psychiatric outpatient service of chang gung memorial hospital at linkou ( a convenience sample ) participated in the study . all participants reported neurotic symptoms such as anxiety , depression , insomnia , or somatic complaints . the participants had received general psychiatric evaluations during their initial visits and had no psychotic , bipolar , or cognitive disorders and no history of psychoactive substance abuse . the age groups were defined as middle - aged ( 4049 years ) , pre - elderly ( 5064 years ) , and elderly ( 65 years ) . the interspousal age gap ( iag ) was defined as the absolute age difference between the participants and their spouses . three iag groups were defined : age - matched ( iag = 02 years ) , moderate age gap ( iag = 35 years ) , and large age gap ( iag 6 years ) . body mass index ( bmi ) groups were defined as normal ( bmi < 24.0 kg / m ) , overweight ( bmi = 24.026.9 kg / m ) , and obese ( bmi > 27.0 kg / m ) . education groups were defined by years of education : primary school and under ( 06 years ) , high school ( 712 years ) , and college or university and above ( > 12 years ) . this 17-item self - administered questionnaire is the best studied and validated questionnaire to help assess the severity of symptoms of aging males and the qol in men > 40 years of age.13,16 the scale has been validated internationally with a standard translation of the chinese version.12 each item is rated on a five - point likert scale , with a response of 5 representing extremely severe and 1 representing none . the total score ( ams - t ) measures the overall severity of symptoms of aging and the qol . the psychological score ( ams - psy ) , the sum of items 68 , 11 , and 13 , assesses the psychological dimension . the somatovegetative score ( ams - som ) , the sum of items 15 , 9 , and 10 , assesses the somatic dimension . the sexual score ( ams - sex ) , the sum of the remaining five items , assesses the sexual dimension . the ams scale is moderately correlated with the beck depression inventory9,17 and the hads.12 with a cut - off value of 27,18 the ams - t has a sensitivity of 29% and a specificity of 97% in predicting androgen deficiency.19 erectile dysfunction ( ed ) is considered if a response to item 15 of the ams scale ( impaired sexual potency ) is rated at 4 or 5.20 the hads , a 14-item self - administered questionnaire comprised of a seven - item anxiety scale ( hads - a ) and a seven - item depression scale ( hads - d ) scored on a four - point likert scale ( 03 ) , is designed to provide a simple yet reliable screening tool for depression and anxiety in various clinical settings , with good internally consistent reliability for anxiety and depression ( cronbach s = 0.80 and 0.76 , respectively),2123 and with good sensitivity and specificity ( approximately 0.8 ) for identifying psychiatric cases.24,25 the total score ( hads - t ) measures affective disturbance ; anxiety is defined as a hads - a 11 ; depression is defined as a hads - d 11.23 four severity grades of psychological distress are distinguished as follows : none ( hads - t < 8) , little ( hads - t = 810 ) , moderate ( hads - t = 1115 ) , and severe ( hads - t 16).22 to determine the effect of anxiety and depression , control was defined as a hads - a < 11 and a hads - d < 11 , anxiety as a hads - a 11 and a hads - d < 11 , depression as a hads - a < 11 and a hads - d 11 , and mixed anxiety and depression as both hads - a 11 and hads - d 11 . statistical analyses were performed using r version 2.14.1 for windows ( r foundation for statistical computing , vienna , austria).26 student s t - test was used to test for continuous variables by two groups . a one - way analysis of variance was used to test for differences among groups . a post hoc tukey hsd test was used to discern subgroup differences . fisher s exact test was used to test for an association between two categorical variables . kendall s correlation coefficients were derived with significance and multiplicity adjusted by holm s method . the independent variables were the hads - t , age , the iag , bmi , marriage , employment , and education . the sample was previously recruited for validation of the chinese version of the ams scale , and a more detailed description has been published elsewhere.12 one hundred seventy - six taiwanese men , 4080 years of age , attending the psychiatric outpatient service of chang gung memorial hospital at linkou ( a convenience sample ) participated in the study . all participants reported neurotic symptoms such as anxiety , depression , insomnia , or somatic complaints . the participants had received general psychiatric evaluations during their initial visits and had no psychotic , bipolar , or cognitive disorders and no history of psychoactive substance abuse . the age groups were defined as middle - aged ( 4049 years ) , pre - elderly ( 5064 years ) , and elderly ( 65 years ) . the interspousal age gap ( iag ) was defined as the absolute age difference between the participants and their spouses . three iag groups were defined : age - matched ( iag = 02 years ) , moderate age gap ( iag = 35 years ) , and large age gap ( iag 6 years ) . body mass index ( bmi ) groups were defined as normal ( bmi < 24.0 kg / m ) , overweight ( bmi = 24.026.9 kg / m ) , and obese ( bmi > 27.0 kg / m ) . education groups were defined by years of education : primary school and under ( 06 years ) , high school ( 712 years ) , and college or university and above ( > 12 years ) . this 17-item self - administered questionnaire is the best studied and validated questionnaire to help assess the severity of symptoms of aging males and the qol in men > 40 years of age.13,16 the scale has been validated internationally with a standard translation of the chinese version.12 each item is rated on a five - point likert scale , with a response of 5 representing extremely severe and 1 representing none . the total score ( ams - t ) measures the overall severity of symptoms of aging and the qol . the psychological score ( ams - psy ) , the sum of items 68 , 11 , and 13 , assesses the psychological dimension . the somatovegetative score ( ams - som ) , the sum of items 15 , 9 , and 10 , assesses the somatic dimension . the sexual score ( ams - sex ) the ams scale is moderately correlated with the beck depression inventory9,17 and the hads.12 with a cut - off value of 27,18 the ams - t has a sensitivity of 29% and a specificity of 97% in predicting androgen deficiency.19 erectile dysfunction ( ed ) is considered if a response to item 15 of the ams scale ( impaired sexual potency ) is rated at 4 or 5.20 this 17-item self - administered questionnaire is the best studied and validated questionnaire to help assess the severity of symptoms of aging males and the qol in men > 40 years of age.13,16 the scale has been validated internationally with a standard translation of the chinese version.12 each item is rated on a five - point likert scale , with a response of 5 representing extremely severe and 1 representing none . the total score ( ams - t ) measures the overall severity of symptoms of aging and the qol . the psychological score ( ams - psy ) , the sum of items 68 , 11 , and 13 , assesses the psychological dimension . the somatovegetative score ( ams - som ) , the sum of items 15 , 9 , and 10 , assesses the somatic dimension . the sexual score ( ams - sex ) the ams scale is moderately correlated with the beck depression inventory9,17 and the hads.12 with a cut - off value of 27,18 the ams - t has a sensitivity of 29% and a specificity of 97% in predicting androgen deficiency.19 erectile dysfunction ( ed ) is considered if a response to item 15 of the ams scale ( impaired sexual potency ) is rated at 4 or 5.20 the hads , a 14-item self - administered questionnaire comprised of a seven - item anxiety scale ( hads - a ) and a seven - item depression scale ( hads - d ) scored on a four - point likert scale ( 03 ) , is designed to provide a simple yet reliable screening tool for depression and anxiety in various clinical settings , with good internally consistent reliability for anxiety and depression ( cronbach s = 0.80 and 0.76 , respectively),2123 and with good sensitivity and specificity ( approximately 0.8 ) for identifying psychiatric cases.24,25 the total score ( hads - t ) measures affective disturbance ; anxiety is defined as a hads - a 11 ; depression is defined as a hads - d 11.23 four severity grades of psychological distress are distinguished as follows : none ( hads - t < 8) , little ( hads - t = 810 ) , moderate ( hads - t = 1115 ) , and severe ( hads - t 16).22 to determine the effect of anxiety and depression , control was defined as a hads - a < 11 and a hads - d < 11 , anxiety as a hads - a 11 and a hads - d < 11 , depression as a hads - a < 11 and a hads - d 11 , and mixed anxiety and depression as both hads - a 11 and hads - d 11 . statistical analyses were performed using r version 2.14.1 for windows ( r foundation for statistical computing , vienna , austria).26 student s t - test was used to test for continuous variables by two groups . a one - way analysis of variance was used to test for differences among groups . a post hoc tukey hsd test was used to discern subgroup differences . fisher s exact test was used to test for an association between two categorical variables . kendall s correlation coefficients were derived with significance and multiplicity adjusted by holm s method . the independent variables were the hads - t , age , the iag , bmi , marriage , employment , and education . the mean age of subjects was 54.3 years ( standard deviation [ sd ] = 10.7 ) . age was negatively correlated with the hads - a ( = 0.16 ) , and positively correlated with the ams - sex ( = 0.22 ) . there were mostly weak - to - moderately positive correlations ( = 0.210.55 ) among responses to the hads and ams scales ( table 2 ) . table 3 shows the results of the one - way analysis of variance among groups in terms of depression and anxiety . there were 103 ( 58.5% ) controls , 18 ( 10.2% ) subjects with depression , 26 ( 14.8% ) with anxiety , and 29 ( 16.5% ) with mixed anxiety and depression . there were significant differences in the iag , ams - sex , and all sexual symptoms except impaired sexual potency among groups with depression and anxiety . a post hoc tukey hsd test showed that the depression group had a significantly higher iag than the control and anxiety groups . the group with anxiety only had a significantly higher score on item 12 ( past peak ) than did the control group , while the groups with depression and mixed anxiety and depression had significantly higher scores on items 12 , 16 ( fewer morning erections ) , and 17 ( disturbed libido ) than did the control group . the group with depression had a significantly higher score on item 14 ( decrease in beard growth ) than did the other 3 groups . there was significant positive association between age and ed ( for every sd [ 10.7 years ] , the odds ratio for ed was 2.51 [ 95% confidence interval 1.584.00 ] ) . subjects with ed were significantly older ( 61.0 11.0 years versus 51.7 9.4 years , t = 5.635 , p < 0.0001 ) ; had higher spousal ages ( 57.0 9.9 years versus 48.4 9.2 years , t = 5.226 , p < 0.0001 ) ; were less employed ( 40% versus 66.7% , p = 0.0020 ) ; and scored higher on the ams - t ( 47.7 11.1 versus 36.8 9.7 , t = 6.419 , p < 0.0001 ) , ams - psy ( 13.4 4.8 versus 11.2 4.3 , t = 2.958 , p = 0.0036 ) , ams - som ( 18.3 5.0 versus 15.7 4.5 , t = 3.333 , p = 0.0011 ) , and ams - sex ( 16.0 2.8 versus 9.9 3.0 , t = 12.523 , p < 0.0001 ) compared with the 126 participants without ed . subjects with ed had a trend towards a higher hads - d than those without ed ( 8.8 5.4 versus 7.3 4.4 , t = 1.922 , p = 0.0563 ) . subjects with ed were not significantly different from those without ed in terms of iag , hads scores , marital status , bmi , and education ( table 4 ) . after adjusting for demographic factors including age , bmi , hads - d , and employment , age was negatively correlated with the hads - a ( = 0.16 ) , and positively correlated with the ams - sex ( = 0.22 ) . there were mostly weak - to - moderately positive correlations ( = 0.210.55 ) among responses to the hads and ams scales ( table 2 ) . table 3 shows the results of the one - way analysis of variance among groups in terms of depression and anxiety . there were 103 ( 58.5% ) controls , 18 ( 10.2% ) subjects with depression , 26 ( 14.8% ) with anxiety , and 29 ( 16.5% ) with mixed anxiety and depression . there were significant differences in the iag , ams - sex , and all sexual symptoms except impaired sexual potency among groups with depression and anxiety . a post hoc tukey hsd test showed that the depression group had a significantly higher iag than the control and anxiety groups . the group with anxiety only had a significantly higher score on item 12 ( past peak ) than did the control group , while the groups with depression and mixed anxiety and depression had significantly higher scores on items 12 , 16 ( fewer morning erections ) , and 17 ( disturbed libido ) than did the control group . the group with depression had a significantly higher score on item 14 ( decrease in beard growth ) than did the other 3 groups . there was significant positive association between age and ed ( for every sd [ 10.7 years ] , the odds ratio for ed was 2.51 [ 95% confidence interval 1.584.00 ] ) . subjects with ed were significantly older ( 61.0 11.0 years versus 51.7 9.4 years , t = 5.635 , p < 0.0001 ) ; had higher spousal ages ( 57.0 9.9 years versus 48.4 9.2 years , t = 5.226 , p < 0.0001 ) ; were less employed ( 40% versus 66.7% , p = 0.0020 ) ; and scored higher on the ams - t ( 47.7 11.1 versus 36.8 9.7 , t = 6.419 , p < 0.0001 ) , ams - psy ( 13.4 4.8 versus 11.2 4.3 , t = 2.958 , p = 0.0036 ) , ams - som ( 18.3 5.0 versus 15.7 4.5 , t = 3.333 , p = 0.0011 ) , and ams - sex ( 16.0 2.8 versus 9.9 3.0 , t = 12.523 , p < 0.0001 ) compared with the 126 participants without ed . subjects with ed had a trend towards a higher hads - d than those without ed ( 8.8 5.4 versus 7.3 4.4 , subjects with ed were not significantly different from those without ed in terms of iag , hads scores , marital status , bmi , and education ( table 4 ) . after adjusting for demographic factors including age , bmi , hads - d , and employment , in the present study , anxiety and depression were associated with more severe symptoms of aging and a poorer qol , yet they exerted different effects on sexual symptoms . it is noteworthy that impaired sexual potency was the only sexual symptom not significantly associated with anxiety and depression . anxiety was associated only with past peak , while depression was associated with all sexual symptoms except impaired sexual potency . the severity of both anxiety and depression was moderately correlated with past peak , while age was not , suggesting that this symptom was related more to psychological health than to aging . the other four sexual symptoms were weakly correlated with severity of depression but not with anxiety , suggesting that depression may play a more important role in sexual dysfunction than does anxiety . there was a weak negative correlation between age and severity of anxiety , and no correlation between age and severity of depression , which is in contrast to a previous report of a weak negative correlation between age and anxiety , and a curvilinear relationship between age and depression.27 age was positively correlated with the severity of sexual symptoms , impaired sexual potency , and fewer morning erections , but was not correlated with past peak and disturbed libido , paralleling the findings associated with age - related sexual dysfunction . a large interspousal age gap was associated with depression and its severity , but not with anxiety . such an age gap may place more of a psychological burden on men based on differences in sexual function , age identity,28 health status , and social life . in one study of men with symptomatic benign prostatic hyperplasia , iag was associated with increased symptoms and burden on the partner.29 erectile dysfunction has a multifaceted relationship with psychological , neurologic , hormonal , and vascular problems,2 and is linked to metabolic syndrome and cardiovascular diseases.30 the prevalence of ed in asian countries is estimated to be 7%15% ( 4049 years of age ) and 39%49% ( 6070 years of age).31 several studies have shown a high prevalence of ed among psychiatric patients.32,33 age is one of the important risk factors for ed . other risk factors include less than good overall health , diabetes mellitus , cardiovascular disease , genitourinary disease , psychiatric disorders , other chronic conditions , smoking , and hormonal factors.30 schneider et al reported that significantly more individuals in clinical samples had ed than did a general population sample ( 28.9%30.8% versus 15% ) , and subjects with ed were older , had lower testosterone levels , and more anxiety than those without ed.20 the point prevalence of ed in our sample was 28.4% , and subjects with ed were significantly older , more likely to be unemployed , and had significantly higher ams scores than did those without ed . the response to the hads was not significantly different between subjects with and without ed . as shown in our logistic regression model , there was only a trend towards an association between ed and depression but not for anxiety . the present cross - sectional study is one of the few papers to investigate relationships between symptoms of aging and emotional distress among male psychiatric outpatients with neurotic complaints , but it does have several methodological limitations . first , we did not have detailed medical and lifestyle assessments of the participants , and not all participants underwent blood tests for testosterone levels due to some economic and technical constraints . second , we did not record potential confounding factors such as income , marriage duration , partner relationship , and psychosocial stressors , all of which may contribute to depression and anxiety . third , self - administered questionnaires do not necessarily infer any specific diagnosis , and they are confounded by the current state of mind and personality traits . nevertheless , they are efficient tools and provide some insight in phenomenological investigation . in addition , the ams scale was not specific for the diagnosis of androgen deficiency , and we did not use a validated assessment of ed . furthermore , the results may not be applied to the community at large due to the small sample size and hospital - based population . anxiety , depression , and ed were associated with more severe symptoms of aging and a poorer qol in males . impaired sexual potency a high prevalence of ed highlighted the importance of routine screening in male psychiatric outpatients who present with symptoms of aging . mental health clinicians should be aware of the relationship between symptoms of aging and psychological health , and request further investigation when warranted .

backgroundandropause and psychiatric disorders are associated with various symptoms in aging males and are part of the differential diagnosis of depression and anxiety . this study was designed to investigate the relationship between symptoms of aging , anxiety , and depression , and to determine if sexual dysfunction could be a differentiating characteristic in the psychiatric outpatient clinic.methodsone hundred seventy - six male psychiatric outpatients participated in the study and completed self - reported measures assessing symptoms of aging , depression , and anxiety . symptoms of aging were assessed by the aging males symptoms scale . anxiety and depression were measured by the hospital anxiety and depression scale . erectile dysfunction was considered if a response to item 15 on the aging males symptoms scale ( impaired sexual potency ) was rated with 4 or 5 points . affective disturbance was assessed by the total scores of the hospital anxiety and depression scale.resultsage was correlated with less anxiety and more sexual symptoms . anxiety and depression were associated with more severe symptoms of aging , and depression was associated with more sexual symptoms than was anxiety . impaired sexual potency was the only sexual symptom not significantly associated with depression and anxiety . depression was associated with an interspousal age gap of 6 years . the point prevalence of erectile dysfunction was 28.4% , and age and affective disturbance were associated with the risk of erectile dysfunction.conclusionimpaired sexual potency should raise the suspicion of androgen deficiency rather than depression and anxiety among middle - aged or older male psychiatric outpatients .

Introduction Methods Population Main outcome measures Aging males symptoms scale Hospital anxiety and depression scale Statistical analysis Results Correlation between age, BMI, HADS, and AMS Groups of depression and anxiety Erectile dysfunction Discussion Conclusion

the term andropause has been used to describe syndromes in aging males including various clinical symptoms encompassing somatic , psychological , and sexual dimensions , as well as endocrine dysfunction , partly related to an age - related decline in androgen and even androgen deficiency.13 it is also associated with dysthymia4 and an increase in depressive symptoms.5,6 exogenous testosterone replacement may be beneficial for treatment - resistant depression associated with androgen deficiency.6,7 androgen deficiency is associated with premature death , erectile dysfunction ( ed ) , metabolic syndrome , and cardiovascular disease.2 symptoms of andropause and androgen deficiency overlap with common psychiatric disorders such as major depressive disorder ( mdd).8 in one study conducted in a male climacteric clinic in japan , patients with mdd had significantly more severe symptoms of aging as assessed by the aging males symptoms ( ams ) scale than did patients without mdd ; the estimated prevalence of mdd approached 50%.9 sato et al , in 2007 , reported that the prevalence of mdd was 42% in an andrology clinic and recommended routine screening for mdd.10 although the gold standard for the diagnosis of androgen deficiency is the measurement of serum free or bioavailable testosterone , such data are not available for every male suspected of suffering from decreased testicular function.11 in order to assess aging males symptoms , a valuable and easily applicable screening tool should be considered . in the present study , first , the validity and reliability of the ams scale had been established in taiwan.12 second , the internal structure of the ams scale across other countries has been sufficiently similar to conclude that the scale measures the same phenomenon in varying contexts.13 third , the ams scale was developed in response to the lack of a standardized scale14 such as the androgen deficiency in aging males questionnaire . fourth , it can measure the severity of symptoms of aging over time in different dimensions as well as their impact on health - related quality of life ( qol).15 due to the complex nature of the phenomenon and the limited number of studies in the male psychiatric population , we investigated the relationship between aging males symptoms and emotional distress by using the ams scale and the hospital anxiety and depression scale ( hads ) , and examined whether sexual dysfunction could be a differentiating characteristic for symptoms of aging among middle - aged or older male psychiatric outpatients . the sample was previously recruited for validation of the chinese version of the ams scale , and a more detailed description has been published elsewhere.12 one hundred seventy - six taiwanese men , 4080 years of age , attending the psychiatric outpatient service of chang gung memorial hospital at linkou ( a convenience sample ) participated in the study . all participants reported neurotic symptoms such as anxiety , depression , insomnia , or somatic complaints . the age groups were defined as middle - aged ( 4049 years ) , pre - elderly ( 5064 years ) , and elderly ( 65 years ) . the interspousal age gap ( iag ) was defined as the absolute age difference between the participants and their spouses . three iag groups were defined : age - matched ( iag = 02 years ) , moderate age gap ( iag = 35 years ) , and large age gap ( iag 6 years ) . education groups were defined by years of education : primary school and under ( 06 years ) , high school ( 712 years ) , and college or university and above ( > 12 years ) . this 17-item self - administered questionnaire is the best studied and validated questionnaire to help assess the severity of symptoms of aging males and the qol in men > 40 years of age.13,16 the scale has been validated internationally with a standard translation of the chinese version.12 each item is rated on a five - point likert scale , with a response of 5 representing extremely severe and 1 representing none . the total score ( ams - t ) measures the overall severity of symptoms of aging and the qol . the psychological score ( ams - psy ) , the sum of items 68 , 11 , and 13 , assesses the psychological dimension . the ams scale is moderately correlated with the beck depression inventory9,17 and the hads.12 with a cut - off value of 27,18 the ams - t has a sensitivity of 29% and a specificity of 97% in predicting androgen deficiency.19 erectile dysfunction ( ed ) is considered if a response to item 15 of the ams scale ( impaired sexual potency ) is rated at 4 or 5.20 the hads , a 14-item self - administered questionnaire comprised of a seven - item anxiety scale ( hads - a ) and a seven - item depression scale ( hads - d ) scored on a four - point likert scale ( 03 ) , is designed to provide a simple yet reliable screening tool for depression and anxiety in various clinical settings , with good internally consistent reliability for anxiety and depression ( cronbach s = 0.80 and 0.76 , respectively),2123 and with good sensitivity and specificity ( approximately 0.8 ) for identifying psychiatric cases.24,25 the total score ( hads - t ) measures affective disturbance ; anxiety is defined as a hads - a 11 ; depression is defined as a hads - d 11.23 four severity grades of psychological distress are distinguished as follows : none ( hads - t < 8) , little ( hads - t = 810 ) , moderate ( hads - t = 1115 ) , and severe ( hads - t 16).22 to determine the effect of anxiety and depression , control was defined as a hads - a < 11 and a hads - d < 11 , anxiety as a hads - a 11 and a hads - d < 11 , depression as a hads - a < 11 and a hads - d 11 , and mixed anxiety and depression as both hads - a 11 and hads - d 11 . the sample was previously recruited for validation of the chinese version of the ams scale , and a more detailed description has been published elsewhere.12 one hundred seventy - six taiwanese men , 4080 years of age , attending the psychiatric outpatient service of chang gung memorial hospital at linkou ( a convenience sample ) participated in the study . all participants reported neurotic symptoms such as anxiety , depression , insomnia , or somatic complaints . the age groups were defined as middle - aged ( 4049 years ) , pre - elderly ( 5064 years ) , and elderly ( 65 years ) . the interspousal age gap ( iag ) was defined as the absolute age difference between the participants and their spouses . three iag groups were defined : age - matched ( iag = 02 years ) , moderate age gap ( iag = 35 years ) , and large age gap ( iag 6 years ) . this 17-item self - administered questionnaire is the best studied and validated questionnaire to help assess the severity of symptoms of aging males and the qol in men > 40 years of age.13,16 the scale has been validated internationally with a standard translation of the chinese version.12 each item is rated on a five - point likert scale , with a response of 5 representing extremely severe and 1 representing none . the total score ( ams - t ) measures the overall severity of symptoms of aging and the qol . the sexual score ( ams - sex ) the ams scale is moderately correlated with the beck depression inventory9,17 and the hads.12 with a cut - off value of 27,18 the ams - t has a sensitivity of 29% and a specificity of 97% in predicting androgen deficiency.19 erectile dysfunction ( ed ) is considered if a response to item 15 of the ams scale ( impaired sexual potency ) is rated at 4 or 5.20 this 17-item self - administered questionnaire is the best studied and validated questionnaire to help assess the severity of symptoms of aging males and the qol in men > 40 years of age.13,16 the scale has been validated internationally with a standard translation of the chinese version.12 each item is rated on a five - point likert scale , with a response of 5 representing extremely severe and 1 representing none . the total score ( ams - t ) measures the overall severity of symptoms of aging and the qol . the sexual score ( ams - sex ) the ams scale is moderately correlated with the beck depression inventory9,17 and the hads.12 with a cut - off value of 27,18 the ams - t has a sensitivity of 29% and a specificity of 97% in predicting androgen deficiency.19 erectile dysfunction ( ed ) is considered if a response to item 15 of the ams scale ( impaired sexual potency ) is rated at 4 or 5.20 the hads , a 14-item self - administered questionnaire comprised of a seven - item anxiety scale ( hads - a ) and a seven - item depression scale ( hads - d ) scored on a four - point likert scale ( 03 ) , is designed to provide a simple yet reliable screening tool for depression and anxiety in various clinical settings , with good internally consistent reliability for anxiety and depression ( cronbach s = 0.80 and 0.76 , respectively),2123 and with good sensitivity and specificity ( approximately 0.8 ) for identifying psychiatric cases.24,25 the total score ( hads - t ) measures affective disturbance ; anxiety is defined as a hads - a 11 ; depression is defined as a hads - d 11.23 four severity grades of psychological distress are distinguished as follows : none ( hads - t < 8) , little ( hads - t = 810 ) , moderate ( hads - t = 1115 ) , and severe ( hads - t 16).22 to determine the effect of anxiety and depression , control was defined as a hads - a < 11 and a hads - d < 11 , anxiety as a hads - a 11 and a hads - d < 11 , depression as a hads - a < 11 and a hads - d 11 , and mixed anxiety and depression as both hads - a 11 and hads - d 11 . age was negatively correlated with the hads - a ( = 0.16 ) , and positively correlated with the ams - sex ( = 0.22 ) . table 3 shows the results of the one - way analysis of variance among groups in terms of depression and anxiety . there were 103 ( 58.5% ) controls , 18 ( 10.2% ) subjects with depression , 26 ( 14.8% ) with anxiety , and 29 ( 16.5% ) with mixed anxiety and depression . there were significant differences in the iag , ams - sex , and all sexual symptoms except impaired sexual potency among groups with depression and anxiety . the group with anxiety only had a significantly higher score on item 12 ( past peak ) than did the control group , while the groups with depression and mixed anxiety and depression had significantly higher scores on items 12 , 16 ( fewer morning erections ) , and 17 ( disturbed libido ) than did the control group . there was significant positive association between age and ed ( for every sd [ 10.7 years ] , the odds ratio for ed was 2.51 [ 95% confidence interval 1.584.00 ] ) . subjects with ed were significantly older ( 61.0 11.0 years versus 51.7 9.4 years , t = 5.635 , p < 0.0001 ) ; had higher spousal ages ( 57.0 9.9 years versus 48.4 9.2 years , t = 5.226 , p < 0.0001 ) ; were less employed ( 40% versus 66.7% , p = 0.0020 ) ; and scored higher on the ams - t ( 47.7 11.1 versus 36.8 9.7 , t = 6.419 , p < 0.0001 ) , ams - psy ( 13.4 4.8 versus 11.2 4.3 , t = 2.958 , p = 0.0036 ) , ams - som ( 18.3 5.0 versus 15.7 4.5 , t = 3.333 , p = 0.0011 ) , and ams - sex ( 16.0 2.8 versus 9.9 3.0 , t = 12.523 , p < 0.0001 ) compared with the 126 participants without ed . subjects with ed were not significantly different from those without ed in terms of iag , hads scores , marital status , bmi , and education ( table 4 ) . after adjusting for demographic factors including age , bmi , hads - d , and employment , age was negatively correlated with the hads - a ( = 0.16 ) , and positively correlated with the ams - sex ( = 0.22 ) . table 3 shows the results of the one - way analysis of variance among groups in terms of depression and anxiety . there were 103 ( 58.5% ) controls , 18 ( 10.2% ) subjects with depression , 26 ( 14.8% ) with anxiety , and 29 ( 16.5% ) with mixed anxiety and depression . there were significant differences in the iag , ams - sex , and all sexual symptoms except impaired sexual potency among groups with depression and anxiety . the group with anxiety only had a significantly higher score on item 12 ( past peak ) than did the control group , while the groups with depression and mixed anxiety and depression had significantly higher scores on items 12 , 16 ( fewer morning erections ) , and 17 ( disturbed libido ) than did the control group . the group with depression had a significantly higher score on item 14 ( decrease in beard growth ) than did the other 3 groups . there was significant positive association between age and ed ( for every sd [ 10.7 years ] , the odds ratio for ed was 2.51 [ 95% confidence interval 1.584.00 ] ) . subjects with ed were significantly older ( 61.0 11.0 years versus 51.7 9.4 years , t = 5.635 , p < 0.0001 ) ; had higher spousal ages ( 57.0 9.9 years versus 48.4 9.2 years , t = 5.226 , p < 0.0001 ) ; were less employed ( 40% versus 66.7% , p = 0.0020 ) ; and scored higher on the ams - t ( 47.7 11.1 versus 36.8 9.7 , t = 6.419 , p < 0.0001 ) , ams - psy ( 13.4 4.8 versus 11.2 4.3 , t = 2.958 , p = 0.0036 ) , ams - som ( 18.3 5.0 versus 15.7 4.5 , t = 3.333 , p = 0.0011 ) , and ams - sex ( 16.0 2.8 versus 9.9 3.0 , t = 12.523 , p < 0.0001 ) compared with the 126 participants without ed . subjects with ed had a trend towards a higher hads - d than those without ed ( 8.8 5.4 versus 7.3 4.4 , subjects with ed were not significantly different from those without ed in terms of iag , hads scores , marital status , bmi , and education ( table 4 ) . after adjusting for demographic factors including age , bmi , hads - d , and employment , in the present study , anxiety and depression were associated with more severe symptoms of aging and a poorer qol , yet they exerted different effects on sexual symptoms . it is noteworthy that impaired sexual potency was the only sexual symptom not significantly associated with anxiety and depression . anxiety was associated only with past peak , while depression was associated with all sexual symptoms except impaired sexual potency . the severity of both anxiety and depression was moderately correlated with past peak , while age was not , suggesting that this symptom was related more to psychological health than to aging . the other four sexual symptoms were weakly correlated with severity of depression but not with anxiety , suggesting that depression may play a more important role in sexual dysfunction than does anxiety . there was a weak negative correlation between age and severity of anxiety , and no correlation between age and severity of depression , which is in contrast to a previous report of a weak negative correlation between age and anxiety , and a curvilinear relationship between age and depression.27 age was positively correlated with the severity of sexual symptoms , impaired sexual potency , and fewer morning erections , but was not correlated with past peak and disturbed libido , paralleling the findings associated with age - related sexual dysfunction . a large interspousal age gap was associated with depression and its severity , but not with anxiety . such an age gap may place more of a psychological burden on men based on differences in sexual function , age identity,28 health status , and social life . in one study of men with symptomatic benign prostatic hyperplasia , iag was associated with increased symptoms and burden on the partner.29 erectile dysfunction has a multifaceted relationship with psychological , neurologic , hormonal , and vascular problems,2 and is linked to metabolic syndrome and cardiovascular diseases.30 the prevalence of ed in asian countries is estimated to be 7%15% ( 4049 years of age ) and 39%49% ( 6070 years of age).31 several studies have shown a high prevalence of ed among psychiatric patients.32,33 age is one of the important risk factors for ed . other risk factors include less than good overall health , diabetes mellitus , cardiovascular disease , genitourinary disease , psychiatric disorders , other chronic conditions , smoking , and hormonal factors.30 schneider et al reported that significantly more individuals in clinical samples had ed than did a general population sample ( 28.9%30.8% versus 15% ) , and subjects with ed were older , had lower testosterone levels , and more anxiety than those without ed.20 the point prevalence of ed in our sample was 28.4% , and subjects with ed were significantly older , more likely to be unemployed , and had significantly higher ams scores than did those without ed . the response to the hads was not significantly different between subjects with and without ed . the present cross - sectional study is one of the few papers to investigate relationships between symptoms of aging and emotional distress among male psychiatric outpatients with neurotic complaints , but it does have several methodological limitations . first , we did not have detailed medical and lifestyle assessments of the participants , and not all participants underwent blood tests for testosterone levels due to some economic and technical constraints . second , we did not record potential confounding factors such as income , marriage duration , partner relationship , and psychosocial stressors , all of which may contribute to depression and anxiety . third , self - administered questionnaires do not necessarily infer any specific diagnosis , and they are confounded by the current state of mind and personality traits . in addition , the ams scale was not specific for the diagnosis of androgen deficiency , and we did not use a validated assessment of ed . anxiety , depression , and ed were associated with more severe symptoms of aging and a poorer qol in males . impaired sexual potency a high prevalence of ed highlighted the importance of routine screening in male psychiatric outpatients who present with symptoms of aging . mental health clinicians should be aware of the relationship between symptoms of aging and psychological health , and request further investigation when warranted .

insomnia is a common sleep disorder with potentially serious economic , health and quality of life ramifications if left untreated . it is characterized by symptoms of difficulty falling asleep , difficulty staying asleep , waking too early or poor quality or non - restorative sleep ( american psychiatric association 2000 ; american sleep disorders association 2001 ) . epidemiological studies have documented the prevalence of chronic insomnia in 10%15% of the general population ( ford and kamerow 1989 ; ancoli - israel and roth 1999 ; national sleep foundation 2002 , 2003 , 2004 , 2005 ; drake et al 2003 ) . furthermore , as many as half of all americans experience transient episodes of insomnia symptoms due to acute circumstances ( national sleep foundation 2002 , 2003 , 2004 , 2005 ) . consequences of the disease can include functional impairment , increased risk of accidents and reductions in quality of life ( ford and kamerow 1989 ; balter and uhlenhuth 1991 ; rosenthal et al 1993 ; zammit 1999 ) . specific medical conditions associated with insomnia include coronary artery disease , hypertension and musculoskeletal problems ( gislason et al 1993 ; janson et al 2001 ; mallon et al 2002 ) . additionally , numerous studies have established a remarkably high association between insomnia and psychiatric disorders , especially depression , other affective disorders and anxiety ( ford and kamerow 1989 ; benca et al 2004 ) . evidence of a bidirectional interaction between insomnia and other co - morbidities , specifically depression and some pain disorders is beginning to emerge ( moldofsky et al 1975 ; moldofsky and scarisbrick 1976 ) . across several studies lasting up to 35 years , insomnia has been shown to increase the risk for the development of depression by as much as a factor 5 ( ford 1989 ; breslau 1996 ) . meanwhile , pain has been shown to exacerbate sleep difficulties , while poor sleep intensifies the perception of pain ( moldofsky et al 1975 ; moldofsky and scarisbrick 1976 ; drewes et al 1997 ) . while the underlying causes of insomnia are only partially understood , certain risk factors may predispose individuals to this disorder . these risk factors include age , gender , psychiatric or physical illness , the use of stimulants like caffeine and nicotine , some forms of shift work and poor sleep hygiene . other potential contributors , including genetic etiology , are the focus of on - going research . after a hiatus of 22 years , the national institutes of health convened a state of the science conference on chronic insomnia in mid-2005 ( national institutes of health 2005 ) . notably , the conference statement recommended that insomnia be considered as either a primary disorder or co - morbid with other disorders . prior to this assertion , insomnia was typically considered to be a symptom secondary to other medical conditions and , as such , was often undertreated . furthermore in the past few years , several new drugs were approved by the fda for the treatment of insomnia . greater national attention to the disorder and an expanded number of treatment options have resulted in marked changes in the pharmaceutical marketplace . have markedly increased from approximately $ 900 m in 2000 to more than $ 2.1b in 2004 ( iskowitz 2005 ) . most of the currently available hypnotics are best suited to address difficulties with sleep onset ( eg , difficulty falling asleep ) . two of the newest treatment options , eszopiclone and zolpidem - cr , appear to assist patients with sleep maintenance difficulties ( eg , nighttime awakenings ) . in order to minimize the potential for negative events associated with residual daytime sedation , all currently approved insomnia therapies are recommended to be taken prior to bedtime so that a patient can spend a full 8 hours in bed . as information about the health impact of insomnia is more widely disseminated and physicians continue to place more importance on its treatment , the number of patients taking prescription sleep medicine will continue to grow , as will the need for more specialized treatment options . benzodiazepines specifically approved for this purpose included flurazepam ( dalmane ) , temazepam ( restoril ) , triazolam ( halcion ) , quazepam ( doral ) and estazolam ( prosom ) ( eddy and walbroehl 1999 ) . this class of drug targets the gaba receptors and can produce sedative , hypnotic , anxiolytic , anticonvulsive , anesthetic and muscle - relaxant effects ( haefely et al 1992 ) . unfortunately , most benzodiazepines interact with the entire gaba receptor complex , creating several of these effects simultaneously . furthermore , short term use of benzodiazepines is recommended because of concerns about tolerance , dependence and the potential for significant adverse events ( kales et al 1983 ; bixler et al 1987 ; adams and oswald 1989 ; holbrook et al 2000 ) . more recently , treatment options have grown to include non - benzodiazepines and melatonin agonists . the off - label usage of other drug classes ( eg , antidepressants , antihistamines ) to treat insomnia complaints is also common , although there is limited evidence that these agents are safe and effective for use in this manner ( hartmann 1995 ; nowell et al 1999 ; richardson et al 2002 ) . in contrast to the benzodiazepines , the non - benzodiazepines are more selective for specific gaba receptor subunits associated with sedation . these compounds have a rapid onset of action and also rapidly eliminated from the body . thus , they are able to quickly induce sedation with fewer of the next - day effects associated with some longer acting benzodiazepines ( nicholson et al 1983 ; walsh et al 1990 ; scharf et al 1994 ; walsh et al 1998 ; fry et al 2000 ) . market include : zaleplon , zolpidem , zolpidem - cr and eszopiclone , the s - isomer of zopiclone . zopiclone is widely used as a sedative outside the u.s . at present , ramelteon is the only melatonin agonist approved for insomnia therapy . it acts on the mt1 and mt2 receptors which help maintain the circadian rhythms underlying the normal sleep - wake cycle ( brzezinski 1997 ; hirai et al 2003 ) . an important difference between ramelteon and both the benzodiazepines and non - benzodiazepines is that it has not been designated as a scheduled drug by the u.s . drug enforcement agency due to its clinically established , extremely low potential for abuse ( hirai et al 2005 ; griffiths et al 2005 ; france et al 2006 ) . in addition , ramelteon is a completely different way to treat insomnia medically , focusing on the melatonin pathway rather than the gaba pathway . in contrast to other members of this class , two formulations of indiplon were simultaneously developed in order to address two distinct insomnia complaints . indiplon - ir ( immediate release ) was designed to address sleep onset difficulties , both at the beginning of the night and after middle of the night awakenings . the fda gave neurocrine biosciences an approvable letter for indiplon - ir in may 2006 and simultaneously stated that the approval of indiplon - mr would require further discussions and potentially additional development efforts . healthy young male and female humans have demonstrated a maximal concentration ( cmax ) of indiplon at 0.73 and 0.82 hours after dosing and exhibited an elimination half life ( t1/2 ) of 1.97 and 1.71 hours , respectively ( rogowski et al 2002 ) . another study comparing indiplon pk in healthy adult and elderly subjects found that indiplon s peak plasma concentration was reached in 2.3 hours and the elimination half life was 1.5 hours in the younger group , as compared to 2.7 hours and 1.8 hours in the older group ( jochelson et al 2002 ) . differences in pk values due to gender and age are frequently observed with both other hypnotics . maximum concentration , total exposure and half life can be up to 50% greater in women ( rosen et al 1999 ; greenblatt et al 2000 ) . in the elderly , impaired renal and hepatic function can lead to a 3-fold increase in plasma concentrations and a doubling of half - life relative to the values seen younger adults . pk differences due to gender have been associated with metabolization via the hepatic isoform cyp 3a4 , while age differences have been linked to the cytochrome p450 metabolic pathway ( greenblatt et al 1991 , 2000 ) . indiplon is metabolized equally by cyp 3a4 and non - microsomal esterases , potentially explaining its relative lack of gender and age effects as compared to other hypnotics . of the currently available treatments , zaleplon is the only non - benzodiazepine hypnotic of the pyrazolopyrimidine class , the same chemical class as indiplon . compared to indiplon , zaleplon has a somewhat shorter time to peak plasma concentration at approximately one hour as well as a shorter elimination half life of approximately one hour . the pharmacokinetics of zaleplon do not vary significantly with age or gender , however hepatic impairment can lead to a 47-fold increase in plasma concentration , a finding similar to indiplon . while indiplon has not been shown to interact with alcohol , zaleplon potentiates the cns - impairing effects for 12.5 hours after co - administration . finally , zaleplon , like indiplon , shows no development of tolerance during short term nightly administration . a significant difference between these two compounds is that indiplon is 10 times more selective for the gaba - a alpha 1 subunit , which is associated with sedation , compared to the alpha 2 , 3 or 5 subunits . co - administration of indiplon and alcohol does has not been shown to produce any pharmacokinetic or pharmacodynamic ( pd ) interactions ( berkowitz et al 2003 ) . the absence of effect on pk values is expected since alcohol and indiplon are eliminated via different metabolic pathways . however , the absence of impact on pd parameters is noteworthy and unique relative to many other hypnotics . finally , the potential for indiplon to produce tolerance was evaluated in healthy volunteers ( jochelson , chen et al 2003 ) . both pk and pd parameters were essentially unchanged between the first and last day of the two week study at all dose levels ( 1040 mg ) , indicating no development of tolerance following repeat administration of indiplon . the immediate release form ( -ir ) was designed to address sleep onset difficulties , while the modified release form ( -mr ) was developed to help patients stay asleep throughout the entire night . the fda gave neurocrine an approvable letter for indiplon - ir 5 and 10 mg in may 2006 . neurocrine plans to resubmit the application in mid - year 2007 after conducting further analyses and modifications to previous analyses . a supplemental pharmacokinetic / food effect profile was also requested by the fda and has been completed . most of the currently available data on indiplon comes from either abstracts written by researchers involved in studies sponsored by neurocrine or from corporate press releases . additional information from these trials will likely become available as the company begins the market launch of one or both formulations of the drug . indiplon - ir has been studied in both healthy volunteers and in patients meeting the diagnostic and statistical manual of mental disorders ( dsm - iv ) criteria for primary insomnia for at least three months ( american psychiatric association 2000 ) . two studies induced transient insomnia in healthy adults via a 2-hour phase advance and the sleep laboratory first night effect . the impact of indiplon - ir relative to placebo was objectively measures using polysomnography ( psg ) in both of these trials ( roth et al 2003 ; scharf et al 2004 ) . objective latency to persistent sleep ( lps ) and patient reported latency to sleep onset ( lso ) were both significantly improved with all four doses evaluated ( 10 and 20 mg in the 1st study , 15 and 30 mg in the 2nd ) . the first study also found that indiplon - ir produced significant improvements in objective and subjective total sleep time ( tst , stst ) , sleep efficiency and several measures of sleep quality . two placebo - controlled trials studied the effects of indiplon - ir ( 10 and 20 mg ) in adults with primary insomnia . the first trial used psg to measure patient sleep parameters at the beginning of the first , third and fifth weeks of treatment ( walsh et al 2004 ) . relative to placebo , both doses of indiplon significantly reduced lps and patient reports of lso were also significantly improved at weeks 1 and 5 . significant improvements were also seen in tst ( 10 mg only ) and sleep quality ( both doses ) at week 1 . the second study evaluated subjective sleep parameters over the course of three months ( scharf et al 2005 ) . patients in this trial reported that indiplon - ir significantly improved the time to sleep onset , stst , subjective wake time after sleep onset ( swaso ) and sleep quality at all time points . the first was a dose response study comparing the effects of 5 , 10 and 20 mg of indiplon - ir with placebo ( scharf et al 2003 ) . all doses were shown to produce significant improvements in lps , lso , stst and sleep quality . the second study used patient reports to evaluate the effects of 5 and 10 mg of indiplon relative to placebo ( walsh et al 2005 ) . both dose levels produced significant improvements in lso , stst , the subjective number of awakenings ( snaw ) and sleep quality during throughout the 2 week treatment period . additionally , the 10 mg dose group showed significantly improvement in swaso at both time points . again , treatment was continued for up to 6 months for a subset of the patients in this trial ( moscovitch et al 2006 ) . over this extended timeframe , 81% of the study participants randomized to indiplon - ir reported that it improved their sleep ( 72% on 5 mg , 92% on 10 mg ) . indiplon - mr is an extended release formulation that delivers part of its dose initially and then provides a controlled release of the remainder over the course of the night . transient insomnia was induced in healthy adults via the sleep laboratory model of the first night effect and a 2-hour phase advance ( neurocrine biosciences 2004d ) . as compared to placebo , psg measured lps and subjective lso were significantly reduced for both doses studied ( 20 and 30 mg ) . a second study in healthy adults used repeated venipunctures to simulate sleep maintenance difficulties ( jochelson , bozigian et al 2003 ) . in this evaluation , indiplon - mr 40 mg significantly improved lso , stst and sleep quality relative to placebo . the subjective number of awakenings declined non - significantly , likely due to the stimulus created by multiple instances of blood sampling . all other indiplon - mr studies were performed with insomnia sufferers , including three trials using adult patients . the first of these three studies randomized patients to either 30 mg of indiplon - mr or placebo ( jochelson et al 2004 ) . sleep continuity , as measured by stst , swaso and snaw , was significantly improved during the two weeks of treatment with indiplon . the second study lasted for three months and treated the adult patients with either indiplon - mr ( 20 or 30 mg ) or placebo ( neurocrine biosciences 2004b ) . again , measures of sleep maintenance ( stst , subjective total wake time ( stwt ) , swaso and snaw ) were significantly improved for both doses over the length of the trial . participants also reported significant improvements in sleep quality and lso . the third and final trial in adults compared the indiplon - mr dose submitted for fda approval ( 15 mg ) with placebo ( klee et al 2006 ) . patient reports demonstrated significant improvements in stst , swaso , snaw , stwt , lso and sleep quality over the course of the 4 week long study . both adult and elderly insomnia patients participated in a placebo - controlled , cross - over psg study of indiplon - mr 15 mg ( rosenberg et al 2006 ) . active treatment produced significant improvements in all objective measures of sleep maintenance ( wtds , waso and tst ) and sleep onset . notably , at present this is the only study in the public domain that uses psg to objectively evaluate indiplon - mr 15 mg in adult patients the dose submitted to the fda for approval . the first of these was a psg dose - response study comparing indiplon - mr 10 , 20 , 30 and 35 mg with placebo ( walsh et al 2003 ) . in this trial , significant improvements in waso and mean sleep efficiency were seen for indiplon - mr doses of 20 mg and above . relative to placebo , significant improvements in lps and naw were demonstrated by all dose levels of active treatment . elderly chronic insomnia patients with sleep maintenance difficulties participated in a two week trial of indiplon - mr 20 mg ( neurocrine biosciences 2004c ) . at all time points two investigator global rating scales , subjective sleep maintenance endpoints ( stwt , snaw ) , sleep onset and quality were also significantly improved relative to placebo . in another study with elderly patients , indiplon - mr 20 and 30 mg were compared with placebo over a period of 5 weeks ( neurocrine biosciences 2004a ) . psg measured sleep maintenance ( waso , tst , wtds , and sleep efficiency ) and onset ( lps ) were significantly improved at all time points . furthermore , relative to those on placebo , indiplon - mr patients also reported that their sleep quality improved significantly . finally , the indiplon - mr dose submitted to the fda for approval ( 15 mg ) was evaluated in 229 elderly insomnia patients ( lankford et al 2005 ; lydiard et al 2006 ) in a manner similar to that of the adult insomnia patient trial of indiplon - mr 15 mg previously mentioned ( klee et al 2006 ) . as in the adult patient study , the participants in this evaluation reported significant improvement in all sleep measures at all time points . measures evaluated included stst , lso , swaso , snaw , stwt and sleep quality . indiplon - ir has been studied in both healthy volunteers and in patients meeting the diagnostic and statistical manual of mental disorders ( dsm - iv ) criteria for primary insomnia for at least three months ( american psychiatric association 2000 ) . two studies induced transient insomnia in healthy adults via a 2-hour phase advance and the sleep laboratory first night effect . the impact of indiplon - ir relative to placebo was objectively measures using polysomnography ( psg ) in both of these trials ( roth et al 2003 ; scharf et al 2004 ) . objective latency to persistent sleep ( lps ) and patient reported latency to sleep onset ( lso ) were both significantly improved with all four doses evaluated ( 10 and 20 mg in the 1st study , 15 and 30 mg in the 2nd ) . the first study also found that indiplon - ir produced significant improvements in objective and subjective total sleep time ( tst , stst ) , sleep efficiency and several measures of sleep quality . two placebo - controlled trials studied the effects of indiplon - ir ( 10 and 20 mg ) in adults with primary insomnia . the first trial used psg to measure patient sleep parameters at the beginning of the first , third and fifth weeks of treatment ( walsh et al 2004 ) . relative to placebo , both doses of indiplon significantly reduced lps and patient reports of lso were also significantly improved at weeks 1 and 5 . significant improvements were also seen in tst ( 10 mg only ) and sleep quality ( both doses ) at week 1 . the second study evaluated subjective sleep parameters over the course of three months ( scharf et al 2005 ) . patients in this trial reported that indiplon - ir significantly improved the time to sleep onset , stst , subjective wake time after sleep onset ( swaso ) and sleep quality at all time points . the first was a dose response study comparing the effects of 5 , 10 and 20 mg of indiplon - ir with placebo ( scharf et al 2003 ) . all doses were shown to produce significant improvements in lps , lso , stst and sleep quality . the second study used patient reports to evaluate the effects of 5 and 10 mg of indiplon relative to placebo ( walsh et al 2005 ) . both dose levels produced significant improvements in lso , stst , the subjective number of awakenings ( snaw ) and sleep quality during throughout the 2 week treatment period . additionally , the 10 mg dose group showed significantly improvement in swaso at both time points . again , treatment was continued for up to 6 months for a subset of the patients in this trial ( moscovitch et al 2006 ) . over this extended timeframe , 81% of the study participants randomized to indiplon - ir reported that it improved their sleep ( 72% on 5 mg , 92% on 10 mg ) . indiplon - mr is an extended release formulation that delivers part of its dose initially and then provides a controlled release of the remainder over the course of the night . transient insomnia was induced in healthy adults via the sleep laboratory model of the first night effect and a 2-hour phase advance ( neurocrine biosciences 2004d ) . as compared to placebo , psg measured lps and subjective lso were significantly reduced for both doses studied ( 20 and 30 mg ) . a second study in healthy adults used repeated venipunctures to simulate sleep maintenance difficulties ( jochelson , bozigian et al 2003 ) . in this evaluation , indiplon - mr 40 mg the subjective number of awakenings declined non - significantly , likely due to the stimulus created by multiple instances of blood sampling . all other indiplon - mr studies were performed with insomnia sufferers , including three trials using adult patients . the first of these three studies randomized patients to either 30 mg of indiplon - mr or placebo ( jochelson et al 2004 ) . sleep continuity , as measured by stst , swaso and snaw , was significantly improved during the two weeks of treatment with indiplon . the second study lasted for three months and treated the adult patients with either indiplon - mr ( 20 or 30 mg ) or placebo ( neurocrine biosciences 2004b ) . again , measures of sleep maintenance ( stst , subjective total wake time ( stwt ) , swaso and snaw ) were significantly improved for both doses over the length of the trial . the third and final trial in adults compared the indiplon - mr dose submitted for fda approval ( 15 mg ) with placebo ( klee et al 2006 ) . patient reports demonstrated significant improvements in stst , swaso , snaw , stwt , lso and sleep quality over the course of the 4 week long study . both adult and elderly insomnia patients participated in a placebo - controlled , cross - over psg study of indiplon - mr 15 mg ( rosenberg et al 2006 ) . active treatment produced significant improvements in all objective measures of sleep maintenance ( wtds , waso and tst ) and sleep onset . notably , at present this is the only study in the public domain that uses psg to objectively evaluate indiplon - mr 15 mg in adult patients the dose submitted to the fda for approval . the first of these was a psg dose - response study comparing indiplon - mr 10 , 20 , 30 and 35 mg with placebo ( walsh et al 2003 ) . in this trial , significant improvements in waso and mean sleep efficiency were seen for indiplon - mr doses of 20 mg and above . relative to placebo , significant improvements in lps and naw were demonstrated by all dose levels of active treatment . elderly chronic insomnia patients with sleep maintenance difficulties participated in a two week trial of indiplon - mr 20 mg ( neurocrine biosciences 2004c ) . at all time points two investigator global rating scales , subjective sleep maintenance endpoints ( stwt , snaw ) , sleep onset and quality were also significantly improved relative to placebo . in another study with elderly patients , indiplon - mr 20 and 30 mg were compared with placebo over a period of 5 weeks ( neurocrine biosciences 2004a ) . psg measured sleep maintenance ( waso , tst , wtds , and sleep efficiency ) and onset ( lps ) were significantly improved at all time points . furthermore , relative to those on placebo , indiplon - mr patients also reported that their sleep quality improved significantly . finally , the indiplon - mr dose submitted to the fda for approval ( 15 mg ) was evaluated in 229 elderly insomnia patients ( lankford et al 2005 ; lydiard et al 2006 ) in a manner similar to that of the adult insomnia patient trial of indiplon - mr 15 mg previously mentioned ( klee et al 2006 ) . as in the adult patient study , the participants in this evaluation reported significant improvement in all sleep measures at all time points . measures evaluated included stst , lso , swaso , snaw , stwt and sleep quality . overall , few adverse events were produced by either formulation , and those that did occur were principally consistent with the known effects of hypnotics . adverse events were evaluated over the course of a 12 month trial in adult insomnia patients randomized to placebo or indiplon - ir 10 or 20 mg ( black et al 2006 ) . the most frequent adverse events seen at the 20 mg dose level were headaches ( 8.7% ) , uri ( 6.5% and nasopharyngitis ( 5.9% ) . the 10 mg group reported headaches ( 11.8% ) , back pain and somnolence ( both 7.9% ) . over the course of the study no clinically relevant changes in vital signs or ecg parameters occurred . several pharmacodynamic tests were used across multiple trials to measure the next - day effects of indiplon . the visual analog scales of sleepiness ( vas ) was used to measure next day sedation , while the digit symbol substitution test ( dsst ) and symbol copying test ( sct ) were both used to evaluate residual functional impairment . across four trials ( roth et al 2003 ; scharf et al 2003 , 2004 ; walsh et al 2004 ) no changes in any of these next - day measures was observed at any dose level of indiplon - ir studied ( 5 to 30 mg ) . evaluations of indiplon - mr s residual effects have been reported for two trials . only the highest dose evaluated ( 35 mg ) was shown to produce a modest effect on dsst scores ( walsh et al 2003 ; lydiard et al 2006 ) . the potential for the development of tolerance and withdrawal symptoms resulting from indiplon was examined across several studies . a 2-week repeat dosing study in healthy volunteers found no evidence of pk or pd tolerance with indiplon - ir ( jochelson , chen et al 2003 ) . three additional trials in insomnia sufferers incorporated the benzodiazepine withdrawal symptom questionnaire ( walsh et al 2004 , 2005 ; scharf et al 2005 ) . using this scale , no signs of withdrawal were found during treatment that lasted up to 3 months with indiplon - ir 5 to 20 mg . in spite of warnings against doing so co - administration of these compounds often leads to changes in drug pk which may , in turn , result in additive impairment . the interaction of indiplon - ir and alcohol was evaluated to assess the likelihood of these effects ( berkowitz et al 2003 ) . this study found a small , non - additive reduction in performance on pharmacodynamic tests ( dsst and sct ) . studies of healthy volunteers have assessed the potential for drug - drug interactions between indiplon and several other compounds ( corrigan et al 2006 ) . these evaluations have found that indiplon does not impact the pk of either digoxin or theophylline . co - administration of indiplon with antidepressants ( sertraline , paroxetine , venlafaxine , amitriptyline ) does not result in additive effects on pd assessments and appears to have no effect on indiplon s pk values . respiratory depression in individuals with chronic obstructive pulmonary disease ( copd ) and sleep apnea has been observed when individuals from these at - risk group use benzodiazepine hypnotics . unfortunately , sleep apnea and copd are strongly linked with co - morbid insomnia , creating complex treatment trade - offs ( fleetham et al 1982 ; weiss et al 2000 ) . indiplon - mr s respiratory depressant effects were assessed in copd patients to evaluate the risk indiplon presents for this patient subset ( hull et al 2004 ) . relative to placebo , no changes in either the arterial oxygen saturation level or the respiratory disturbance index were produced by single night of indiplon - mr 20 mg . indiplon - mr 30 mg was compared to codeine sulfate 60 mg ( active control ) and placebo in a daytime co2 challenge model ( cohn et al 2004 ) . in contrast , no respiratory effects were demonstrated at any time point following administration of indiplon . existing therapeutics are not appropriate for insomniacs who awaken in the middle of the night and have difficulties returning to sleep because these medications relatively long duration of action can produce significant next day impairments when taken with less than 8 hours available time in bed . the next - day effects of taking indiplon - ir with only 4 hours remaining time in bed were studied in three separate trials . two of these trials also incorporated other hypnotics as comparators . a single - night , cross - over study in healthy adults woke participants after they had spent only 4 hours in bed ( garber et al 2004 ) . volunteers were dosed with either indiplon - ir ( 10 or 20 mg ) , zolpidem 10 mg , zopiclone 7.5 mg or placebo and were then permitted to remain in bed for another 4 hours before being awakened a second time . participants performed the dsst , sct and vas at 4 and 6 hours post - dose to assess the relative next - day residual effects of study treatment . relative to placebo , indiplon - ir was the only drug that did not produce any significant changes on any of these tests . zolpidem produced a significant increase in sleepiness at 4 hours post - dose and significantly increased sedation was seen at both 4 and 6 hours post - dose with zopiclone . healthy elderly volunteers participated in a similar trial comparing the effects of 5 and 10 mg of indiplon - ir , following middle of the night dosing , with those resulting from 3.75 mg of zopiclone and placebo ( neurocrine biosciences 2003 ) . as in the study with young adults , neither dose of indiplon - ir produced any significant changes on the dsst , sct or vas at any time point . participants randomized to zopiclone , however , demonstrated significant changes on the dsst at 4 and 8 hours post - dose and showed a trend toward impairment on the sct at both 4 and 6 hours post - dose . the third study of middle of the night dosing was conducted in adult insomnia patients on an outpatient basis ( roth et al 2005 ) . over the course of 4 weeks , double - blind therapy ( indiplon - ir 10 or 20 mg , placebo ) was self administered on a prn basis as long as at least 4 hours sleep time was available to the patient . furthermore , over the course of the study , the average stst was significantly extended for patients randomized to both doses of indiplon - ir relative to those treated with placebo . vas ratings showed no increase in next day sleepiness at either dose of active treatment . indiplon is a novel sedative - hypnotic that modulates subunits of the gaba receptor complex in order to induce sedation . it has been shown to be well tolerated and effective as an insomnia therapy in randomized , placebo - controlled trials involving more than 7,500 participants . both patient reported and objectively measured sleep parameters have shown consistent improvements without next - day residual sedation or impairment . indiplon was developed in two different formulations to address two different types of insomnia complaint : indiplon - ir ( immediate release ) was designed for sleep onset difficulties , while indiplon - mr ( modified release ) was developed for sleep maintenance insomnia . indiplon - ir shows potential for use on a prn basis , for example , by patients who wake in the middle of the night and are unable to return to sleep without assistance .

indiplon is a novel sedative - hypnotic recently approved for the treatment of insomnia . like other non - benzodiazepine hypnotics , its mechanism of action is to modulate subunits , especially the alpha-1 subunit , of the gaba receptor complex in order to induce sedation . indiplon was developed in two different formulations to address two different types of insomnia complaint : indiplon - ir ( immediate release ) was designed for sleep onset difficulties , while indiplon - mr ( modified release ) was developed for sleep maintenance insomnia . while there are currently few peer reviewed articles about indiplon clinical trial results , the early information that is available seems to indicate that both formulations have been well tolerated and have proven effective at improving both patient reported and objectively measured sleep parameters in both adult and elderly insomnia patients . in may 2006 , the fda indicated that indiplon - ir was approvable and plans are to resubmit the application in 2007 . indiplon - mr was unapprovable and may require further evaluation .

Introduction Current treatment options Pharmacology of indiplon Indiplon in clinical trials Indiplon-IR Indiplon-MR Safety and tolerability of indiplon Summary

furthermore , as many as half of all americans experience transient episodes of insomnia symptoms due to acute circumstances ( national sleep foundation 2002 , 2003 , 2004 , 2005 ) . additionally , numerous studies have established a remarkably high association between insomnia and psychiatric disorders , especially depression , other affective disorders and anxiety ( ford and kamerow 1989 ; benca et al 2004 ) . meanwhile , pain has been shown to exacerbate sleep difficulties , while poor sleep intensifies the perception of pain ( moldofsky et al 1975 ; moldofsky and scarisbrick 1976 ; drewes et al 1997 ) . while the underlying causes of insomnia are only partially understood , certain risk factors may predispose individuals to this disorder . after a hiatus of 22 years , the national institutes of health convened a state of the science conference on chronic insomnia in mid-2005 ( national institutes of health 2005 ) . notably , the conference statement recommended that insomnia be considered as either a primary disorder or co - morbid with other disorders . furthermore in the past few years , several new drugs were approved by the fda for the treatment of insomnia . most of the currently available hypnotics are best suited to address difficulties with sleep onset ( eg , difficulty falling asleep ) . two of the newest treatment options , eszopiclone and zolpidem - cr , appear to assist patients with sleep maintenance difficulties ( eg , nighttime awakenings ) . in order to minimize the potential for negative events associated with residual daytime sedation , all currently approved insomnia therapies are recommended to be taken prior to bedtime so that a patient can spend a full 8 hours in bed . as information about the health impact of insomnia is more widely disseminated and physicians continue to place more importance on its treatment , the number of patients taking prescription sleep medicine will continue to grow , as will the need for more specialized treatment options . unfortunately , most benzodiazepines interact with the entire gaba receptor complex , creating several of these effects simultaneously . more recently , treatment options have grown to include non - benzodiazepines and melatonin agonists . in contrast to the benzodiazepines , the non - benzodiazepines are more selective for specific gaba receptor subunits associated with sedation . these compounds have a rapid onset of action and also rapidly eliminated from the body . thus , they are able to quickly induce sedation with fewer of the next - day effects associated with some longer acting benzodiazepines ( nicholson et al 1983 ; walsh et al 1990 ; scharf et al 1994 ; walsh et al 1998 ; fry et al 2000 ) . at present , ramelteon is the only melatonin agonist approved for insomnia therapy . an important difference between ramelteon and both the benzodiazepines and non - benzodiazepines is that it has not been designated as a scheduled drug by the u.s . in addition , ramelteon is a completely different way to treat insomnia medically , focusing on the melatonin pathway rather than the gaba pathway . in contrast to other members of this class , two formulations of indiplon were simultaneously developed in order to address two distinct insomnia complaints . indiplon - ir ( immediate release ) was designed to address sleep onset difficulties , both at the beginning of the night and after middle of the night awakenings . the fda gave neurocrine biosciences an approvable letter for indiplon - ir in may 2006 and simultaneously stated that the approval of indiplon - mr would require further discussions and potentially additional development efforts . another study comparing indiplon pk in healthy adult and elderly subjects found that indiplon s peak plasma concentration was reached in 2.3 hours and the elimination half life was 1.5 hours in the younger group , as compared to 2.7 hours and 1.8 hours in the older group ( jochelson et al 2002 ) . pk differences due to gender have been associated with metabolization via the hepatic isoform cyp 3a4 , while age differences have been linked to the cytochrome p450 metabolic pathway ( greenblatt et al 1991 , 2000 ) . indiplon is metabolized equally by cyp 3a4 and non - microsomal esterases , potentially explaining its relative lack of gender and age effects as compared to other hypnotics . of the currently available treatments , zaleplon is the only non - benzodiazepine hypnotic of the pyrazolopyrimidine class , the same chemical class as indiplon . a significant difference between these two compounds is that indiplon is 10 times more selective for the gaba - a alpha 1 subunit , which is associated with sedation , compared to the alpha 2 , 3 or 5 subunits . the immediate release form ( -ir ) was designed to address sleep onset difficulties , while the modified release form ( -mr ) was developed to help patients stay asleep throughout the entire night . the fda gave neurocrine an approvable letter for indiplon - ir 5 and 10 mg in may 2006 . neurocrine plans to resubmit the application in mid - year 2007 after conducting further analyses and modifications to previous analyses . a supplemental pharmacokinetic / food effect profile was also requested by the fda and has been completed . additional information from these trials will likely become available as the company begins the market launch of one or both formulations of the drug . indiplon - ir has been studied in both healthy volunteers and in patients meeting the diagnostic and statistical manual of mental disorders ( dsm - iv ) criteria for primary insomnia for at least three months ( american psychiatric association 2000 ) . the impact of indiplon - ir relative to placebo was objectively measures using polysomnography ( psg ) in both of these trials ( roth et al 2003 ; scharf et al 2004 ) . objective latency to persistent sleep ( lps ) and patient reported latency to sleep onset ( lso ) were both significantly improved with all four doses evaluated ( 10 and 20 mg in the 1st study , 15 and 30 mg in the 2nd ) . the first study also found that indiplon - ir produced significant improvements in objective and subjective total sleep time ( tst , stst ) , sleep efficiency and several measures of sleep quality . two placebo - controlled trials studied the effects of indiplon - ir ( 10 and 20 mg ) in adults with primary insomnia . the first trial used psg to measure patient sleep parameters at the beginning of the first , third and fifth weeks of treatment ( walsh et al 2004 ) . patients in this trial reported that indiplon - ir significantly improved the time to sleep onset , stst , subjective wake time after sleep onset ( swaso ) and sleep quality at all time points . the first was a dose response study comparing the effects of 5 , 10 and 20 mg of indiplon - ir with placebo ( scharf et al 2003 ) . again , treatment was continued for up to 6 months for a subset of the patients in this trial ( moscovitch et al 2006 ) . over this extended timeframe , 81% of the study participants randomized to indiplon - ir reported that it improved their sleep ( 72% on 5 mg , 92% on 10 mg ) . indiplon - mr is an extended release formulation that delivers part of its dose initially and then provides a controlled release of the remainder over the course of the night . in this evaluation , indiplon - mr 40 mg significantly improved lso , stst and sleep quality relative to placebo . all other indiplon - mr studies were performed with insomnia sufferers , including three trials using adult patients . the first of these three studies randomized patients to either 30 mg of indiplon - mr or placebo ( jochelson et al 2004 ) . the second study lasted for three months and treated the adult patients with either indiplon - mr ( 20 or 30 mg ) or placebo ( neurocrine biosciences 2004b ) . again , measures of sleep maintenance ( stst , subjective total wake time ( stwt ) , swaso and snaw ) were significantly improved for both doses over the length of the trial . the third and final trial in adults compared the indiplon - mr dose submitted for fda approval ( 15 mg ) with placebo ( klee et al 2006 ) . both adult and elderly insomnia patients participated in a placebo - controlled , cross - over psg study of indiplon - mr 15 mg ( rosenberg et al 2006 ) . active treatment produced significant improvements in all objective measures of sleep maintenance ( wtds , waso and tst ) and sleep onset . notably , at present this is the only study in the public domain that uses psg to objectively evaluate indiplon - mr 15 mg in adult patients the dose submitted to the fda for approval . the first of these was a psg dose - response study comparing indiplon - mr 10 , 20 , 30 and 35 mg with placebo ( walsh et al 2003 ) . in this trial , significant improvements in waso and mean sleep efficiency were seen for indiplon - mr doses of 20 mg and above . elderly chronic insomnia patients with sleep maintenance difficulties participated in a two week trial of indiplon - mr 20 mg ( neurocrine biosciences 2004c ) . at all time points two investigator global rating scales , subjective sleep maintenance endpoints ( stwt , snaw ) , sleep onset and quality were also significantly improved relative to placebo . in another study with elderly patients , indiplon - mr 20 and 30 mg were compared with placebo over a period of 5 weeks ( neurocrine biosciences 2004a ) . psg measured sleep maintenance ( waso , tst , wtds , and sleep efficiency ) and onset ( lps ) were significantly improved at all time points . furthermore , relative to those on placebo , indiplon - mr patients also reported that their sleep quality improved significantly . finally , the indiplon - mr dose submitted to the fda for approval ( 15 mg ) was evaluated in 229 elderly insomnia patients ( lankford et al 2005 ; lydiard et al 2006 ) in a manner similar to that of the adult insomnia patient trial of indiplon - mr 15 mg previously mentioned ( klee et al 2006 ) . indiplon - ir has been studied in both healthy volunteers and in patients meeting the diagnostic and statistical manual of mental disorders ( dsm - iv ) criteria for primary insomnia for at least three months ( american psychiatric association 2000 ) . the impact of indiplon - ir relative to placebo was objectively measures using polysomnography ( psg ) in both of these trials ( roth et al 2003 ; scharf et al 2004 ) . objective latency to persistent sleep ( lps ) and patient reported latency to sleep onset ( lso ) were both significantly improved with all four doses evaluated ( 10 and 20 mg in the 1st study , 15 and 30 mg in the 2nd ) . the first study also found that indiplon - ir produced significant improvements in objective and subjective total sleep time ( tst , stst ) , sleep efficiency and several measures of sleep quality . two placebo - controlled trials studied the effects of indiplon - ir ( 10 and 20 mg ) in adults with primary insomnia . the first trial used psg to measure patient sleep parameters at the beginning of the first , third and fifth weeks of treatment ( walsh et al 2004 ) . patients in this trial reported that indiplon - ir significantly improved the time to sleep onset , stst , subjective wake time after sleep onset ( swaso ) and sleep quality at all time points . the first was a dose response study comparing the effects of 5 , 10 and 20 mg of indiplon - ir with placebo ( scharf et al 2003 ) . again , treatment was continued for up to 6 months for a subset of the patients in this trial ( moscovitch et al 2006 ) . over this extended timeframe , 81% of the study participants randomized to indiplon - ir reported that it improved their sleep ( 72% on 5 mg , 92% on 10 mg ) . indiplon - mr is an extended release formulation that delivers part of its dose initially and then provides a controlled release of the remainder over the course of the night . in this evaluation , indiplon - mr 40 mg the subjective number of awakenings declined non - significantly , likely due to the stimulus created by multiple instances of blood sampling . all other indiplon - mr studies were performed with insomnia sufferers , including three trials using adult patients . the first of these three studies randomized patients to either 30 mg of indiplon - mr or placebo ( jochelson et al 2004 ) . the second study lasted for three months and treated the adult patients with either indiplon - mr ( 20 or 30 mg ) or placebo ( neurocrine biosciences 2004b ) . again , measures of sleep maintenance ( stst , subjective total wake time ( stwt ) , swaso and snaw ) were significantly improved for both doses over the length of the trial . the third and final trial in adults compared the indiplon - mr dose submitted for fda approval ( 15 mg ) with placebo ( klee et al 2006 ) . both adult and elderly insomnia patients participated in a placebo - controlled , cross - over psg study of indiplon - mr 15 mg ( rosenberg et al 2006 ) . active treatment produced significant improvements in all objective measures of sleep maintenance ( wtds , waso and tst ) and sleep onset . notably , at present this is the only study in the public domain that uses psg to objectively evaluate indiplon - mr 15 mg in adult patients the dose submitted to the fda for approval . the first of these was a psg dose - response study comparing indiplon - mr 10 , 20 , 30 and 35 mg with placebo ( walsh et al 2003 ) . in this trial , significant improvements in waso and mean sleep efficiency were seen for indiplon - mr doses of 20 mg and above . elderly chronic insomnia patients with sleep maintenance difficulties participated in a two week trial of indiplon - mr 20 mg ( neurocrine biosciences 2004c ) . at all time points two investigator global rating scales , subjective sleep maintenance endpoints ( stwt , snaw ) , sleep onset and quality were also significantly improved relative to placebo . in another study with elderly patients , indiplon - mr 20 and 30 mg were compared with placebo over a period of 5 weeks ( neurocrine biosciences 2004a ) . psg measured sleep maintenance ( waso , tst , wtds , and sleep efficiency ) and onset ( lps ) were significantly improved at all time points . furthermore , relative to those on placebo , indiplon - mr patients also reported that their sleep quality improved significantly . finally , the indiplon - mr dose submitted to the fda for approval ( 15 mg ) was evaluated in 229 elderly insomnia patients ( lankford et al 2005 ; lydiard et al 2006 ) in a manner similar to that of the adult insomnia patient trial of indiplon - mr 15 mg previously mentioned ( klee et al 2006 ) . adverse events were evaluated over the course of a 12 month trial in adult insomnia patients randomized to placebo or indiplon - ir 10 or 20 mg ( black et al 2006 ) . over the course of the study no clinically relevant changes in vital signs or ecg parameters occurred . the visual analog scales of sleepiness ( vas ) was used to measure next day sedation , while the digit symbol substitution test ( dsst ) and symbol copying test ( sct ) were both used to evaluate residual functional impairment . across four trials ( roth et al 2003 ; scharf et al 2003 , 2004 ; walsh et al 2004 ) no changes in any of these next - day measures was observed at any dose level of indiplon - ir studied ( 5 to 30 mg ) . evaluations of indiplon - mr s residual effects have been reported for two trials . the potential for the development of tolerance and withdrawal symptoms resulting from indiplon was examined across several studies . a 2-week repeat dosing study in healthy volunteers found no evidence of pk or pd tolerance with indiplon - ir ( jochelson , chen et al 2003 ) . using this scale , no signs of withdrawal were found during treatment that lasted up to 3 months with indiplon - ir 5 to 20 mg . the interaction of indiplon - ir and alcohol was evaluated to assess the likelihood of these effects ( berkowitz et al 2003 ) . indiplon - mr s respiratory depressant effects were assessed in copd patients to evaluate the risk indiplon presents for this patient subset ( hull et al 2004 ) . relative to placebo , no changes in either the arterial oxygen saturation level or the respiratory disturbance index were produced by single night of indiplon - mr 20 mg . indiplon - mr 30 mg was compared to codeine sulfate 60 mg ( active control ) and placebo in a daytime co2 challenge model ( cohn et al 2004 ) . existing therapeutics are not appropriate for insomniacs who awaken in the middle of the night and have difficulties returning to sleep because these medications relatively long duration of action can produce significant next day impairments when taken with less than 8 hours available time in bed . the next - day effects of taking indiplon - ir with only 4 hours remaining time in bed were studied in three separate trials . volunteers were dosed with either indiplon - ir ( 10 or 20 mg ) , zolpidem 10 mg , zopiclone 7.5 mg or placebo and were then permitted to remain in bed for another 4 hours before being awakened a second time . relative to placebo , indiplon - ir was the only drug that did not produce any significant changes on any of these tests . healthy elderly volunteers participated in a similar trial comparing the effects of 5 and 10 mg of indiplon - ir , following middle of the night dosing , with those resulting from 3.75 mg of zopiclone and placebo ( neurocrine biosciences 2003 ) . as in the study with young adults , neither dose of indiplon - ir produced any significant changes on the dsst , sct or vas at any time point . the third study of middle of the night dosing was conducted in adult insomnia patients on an outpatient basis ( roth et al 2005 ) . over the course of 4 weeks , double - blind therapy ( indiplon - ir 10 or 20 mg , placebo ) was self administered on a prn basis as long as at least 4 hours sleep time was available to the patient . furthermore , over the course of the study , the average stst was significantly extended for patients randomized to both doses of indiplon - ir relative to those treated with placebo . indiplon is a novel sedative - hypnotic that modulates subunits of the gaba receptor complex in order to induce sedation . it has been shown to be well tolerated and effective as an insomnia therapy in randomized , placebo - controlled trials involving more than 7,500 participants . both patient reported and objectively measured sleep parameters have shown consistent improvements without next - day residual sedation or impairment . indiplon was developed in two different formulations to address two different types of insomnia complaint : indiplon - ir ( immediate release ) was designed for sleep onset difficulties , while indiplon - mr ( modified release ) was developed for sleep maintenance insomnia . indiplon - ir shows potential for use on a prn basis , for example , by patients who wake in the middle of the night and are unable to return to sleep without assistance .

structure - based drug design ( sbdd ) and fragment - based drug design ( fbdd ) play increasingly important roles in drug discovery,(1 ) as more protein structures become available and as the computational tools for exploiting those structures become more capable . ultimately , the success or failure of these efforts rests on the ability to accurately compute proteinligand interaction energies . this is a difficult problem because of the complexity of the molecular structures involved and the very significant challenge of computing energy differences to sufficient accuracy to provide useful predicted binding affinities . there are many approaches to this problem that vary greatly in terms of their accuracy , generality , and efficiency . at one extreme are simulation - based approaches , such as free energy perturbation ( fep).(2 ) fep provides a theoretically rigorous estimate of the free energy change for permuting one ligand into another . in particular , fep addresses the problems of sufficient sampling and the computation of true free energies . even so , this approach is limited by the quality of the force field and by other limitations inherent in classical molecular models . at the other extreme are highly empirical scoring functions , such as are commonly employed in docking and scoring programs . these models are designed to be fast and , therefore , inevitably sacrifice theoretical rigor and accuracy . in recent years , there has been significant progress in the development of fast quantum mechanical methods for computing protein - size molecular systems . these linear - scaling approaches have made quantum calculations for proteinligand complexes tractable , and they have provided an important new tool for computing proteinligand interaction energies . in particular , quantum methods offer the prospect of a much more accurate representation of electronic effects in proteins and ligands . indeed , previous work has shown that there are significant charge transfer and polarization effects in proteinligand complexes that are not captured in classical models.(15 ) in addition , methods have long been available for partitioning quantum energies into pairwise contributions . the pairwise decomposition ( pwd ) method divides the electrostatic interaction energy into self- and cross - components between atoms . pwd has successfully been applied to the investigation of the effect of binding in a series of fluorine - substituted ligands to human carbonic anhydrase ii.(17 ) a receptor - based qsar method , comparative binding energy analysis ( combine ) formalism , was proposed by by ortiz and co - workers . combine obtains descriptors from the intermolecular interactions between the receptor and the ligand , which are calculated by using a pairwise molecular mechanics ( mm ) potential energy function . based on the mm descriptors , qsar models were built by multivariate statistical tools , such as partial least - squares ( pls ) . semiempirical pairwise decomposition , along with combine , have been integrated into a new approach for computing proteinligand interaction energies ( se - combine ) on a residue - by - residue basis.(22 ) this se - combine approach offers the potential to provide new mechanistic insight into the factors governing these interactions as well as to improve overall accuracy . a series of 45 inhibitors ( table 1 ) for protein kinase b ( pkb ) were selected to test the se - combine method . these compounds were chosen for two reasons : first , both structures and affinities are available for many of these ligands . this provides a unique opportunity to compare our computational results to high - quality experimental data for both structure and activity . second , the ligands can be grouped into structurally related classes , in many cases being the product of a fragment - based design . qm - pwd was used to compute all of the pairwise ligandresidue interactions between the 45 ligands and the protein kinase a ( pka)pkb chimera . analysis of the interaction energies , heat maps , and structures show that se - combine provides a predictive model for affinity , a clear indication of the most critical residues for binding , and a meaningful indication of the many structural trade - offs necessary to design potent ligands for pkb . class refers to the particular scaffold , or chemotype family , to which each compound belongs . although compound 40 does not contain a pyrazole , it is included with class 6 compounds as it was originally synthesized with them . compounds with protein data bank ( pdb ) identifications ( protein databank accession number ) are members of the training set . in column with title of , all 15 x - ray structures give their pdb identifications , while the rest of the 30 model - built complexes are labeled by their templates pdb identifications with square brackets . in a series of papers , astex pharmaceuticals has reported 16 x - ray structures of pkapkb chimera complexes as well as the binding affinity data for more than 60 additional ligands ( see pdb i d in figure 1 ) . we aligned these sequences using clustalx ( figure 1).(28 ) this alignment shows that the 16 x - ray sequences are fully conserved except at the n - terminus regions that vary in length and at position 47 of one chimera ( 2uw4 ) . for this paper , residue numbers were assigned according to the sequential number label beneath the aligned sequences in figure 1 . all of the active site residues fall within the conserved range so that the active sites for all 16 x - ray structures are identical , at least with regard to sequence . these 16 well - defined proteinligand complexes were selected as the training set for model building . ( colored in gray ) below the ruler indicates the degree of similarity . the * character indicates positions that have been fully conserved . of the 76 ligands considered , 30 contain hypervalent sulfurs ( e.g. , sulfonamides ) . since the semiempirical hamiltonians employed in this study are not suitable for computing hypervalent species , these compounds were removed from consideration . this left 30 ligands ( table 1 ) for the prediction set and 15 proteinligand complexes for the training set . a histogram of ligand binding affinities is provided for the training and test data sets in figure 2 . this histogram shows that the log(ic50)s exhibit a wide range of values ( from 9 to 3 ) and importantly that both the training and test data sets contain members spanning this range of potencies . it should be noted that for this model , the training set is smaller than the prediction set . this size distribution closely mimics the situation often experienced in drug discovery where one may have access to a small representative sample of experimental structures . histogram of inhibitor binding affinity for the training set ( blue ; 15 ligands from the x - ray structures ) and test set ( red ; 30 ligands ) . it is important to demonstrate that the affinities are not highly correlated with molecular size . the number of heavy atoms range from 7 to 30 atoms , and the molecular weights are spread over 300 amu . the potency shows a poor correlation ( figure 3 ) with the number of heavy atoms ( r = 0.1843 ) and with the molecular weight ( r = 0.1458 ) . the ligand efficiencies ( i.e. , le = pic50/ha ) and fit quality ( fq ) scores were also evaluated . fq scores near 1.0 ( scores can exceed 1 ) indicate near optimal ligand binding , while low scores are indicative of suboptimal binding . the values of fq for the combined training and prediction sets range from 0.4 to 1.0 , regardless of size . ( a ) plot of pic50 versus the number of heavy atom ( ha ) . the 15 x - ray structures , corresponding to the 15 ligands in the training set , were downloaded from the pdb . the reduce program(31 ) was employed to protonate the x - ray structures using the definitions in the het dictionary provided by the pdb . this het dictionary was edited to account for correct , bidentate protonation of the ligand pyrazoles . the generalized amber force field ( gaff)(32 ) was used to represent the ligands , while the standard amino acids were treated using the parm99 force field.(33 ) upon protonation , all protons were minimized , while restraining the heavy atoms , using 500 steps of steepest descent followed by 1000 steps of conjugate gradient minimization using the sander module of amber 8.(34 ) once this initial minimization was complete , all atoms in the complex were subjected to limited minimization with a constraint weight of 10.0 on heavy atoms ( 500 steps of steepest descent followed by 1000 conjugate gradient steps ) . since the prediction set ligands have no x - ray structures available , it was necessary to build models for them . these models were built using an in - house flexible three - dimensional structure alignment program that utilizes the mtk++ library,(35 ) along with a number of additional routines in order align a ligand to a structurally similar template ligand . the 15 ligand poses extracted from the x - ray structures served as templates for this process . each test - set ligand was assigned to one of the 15 ligand templates for alignment , according to maximum similarity . after alignment , the ligands were fitted into the active site of the pkapkb chimera in the same way as their templates . the templates for each of the model - built complexes were shown in table 1 by square brackets with pdb identifications of the templates inside . due to structural variations among ligands these hot spots were allowed to relax in the active site using a restrained amber minimization of 1500 steps ( 500 steepest descent followed by 1000 conjugate gradient ) followed by a full minimization of all atoms in the system ( 500 steepest descent followed by 1000 conjugate gradient steps ) . linear scaling , semiempirical divide and conquer ( d&c ) single - point calculations(10 ) were carried out on each pkapkb complex using the pm3(36 ) and am1(13 ) hamiltonians , as implemented in the divcon 5.x program.(37 ) divcon calculates the quantum mechanical atom - to - atom , pairwise energy decomposition ( qm - pwd)(16 ) and the overall qmscore(15 ) for each complex . a cutoff for the fock matrix of 20 and a d&c buffering scheme of 4.2/2 was employed throughout the simulation . the d&c atom - to - atom pairwise interaction energies were calculated and then converted to residue - based pairwise interaction energies ( i.e. , pairwise interactions between each protein residue and the complete ligand structure ) . in addition to the qm calculations , divcon was used to assign a generalized amber force field ( gaff ) or a parm99 atom - type to each atom and to calculate the molecular mechanical atom - by - atom , pairwise energy decomposition ( mm - pwd ) terms . seven scoring functions ( from sf-1 to sf-7 ) using various combinations of the qm - pwd , the qmscore , and the mm - pwd energy terms were evaluated using pls ( table 2).(38 ) the qm - pwd energy terms consist of eab , eab , and eabcore energy terms.(22)eab contains the exchange between atoms and makes a small negative contribution to the total energy . eab represents electronelectron repulsion and contributes positively to the total energy . finally , eabcore is the corecore repulsion between atoms . the computed molecular mechanics ( mm ) energy terms include dispersion , van der waals ( summation of dispersion and repulsion terms ) , and electrostatics . sf-1 includes only the pairwise energies of the complex in vacuum and is a close approximation to the original version of se - combine,(22 ) while sf-2 , -3 , -4 , -6 , and -7 are extensions of the original se - combine . sf-5 only contains mm energy terms and is analogous to the conventional implementation of combine.(39 ) sf-2 contains pairwise energies of the complex in solvent . sf-3 and -4 include both qm pairwise energies of complex and dispersion energy , while sf-6 and -7 include both qm pairwise energies of complex and van der waals energy terms . the gsolv energy term was originally evaluated via qmscore but was dropped since it was found to contribute little . + sign designates that the energy term is included in the scoring function , while the sign indicates the energy is not included . a descriptor matrix was built where each descriptor contains one column of data from the matrix . descriptors were pruned initially by removing those with near zero values or with standard deviations less than 0.05 . the descriptors were autoscaled ( i.e. , each value was subtracted by the average and divided by the standard deviation for that descriptor ) . after scaling , each set of descriptors has a mean of zero and a standard deviation of one . internal validation was carried out using leave - one - out ( loo ) cross - validation for the training set , and the optimal dimensionality of each model was assigned from its cross - validated predictive ability . the models were evaluated for their predictive quality using a range of standard statistical measures , such as the correlation ( r ) and cross - validated correlation ( q ) coefficients , the standard deviation of error of calculations ( sdec ) , and the standard deviation of error prediction ( sdep ) . sdep was defined as the root - mean - squared error of the dependent variables in a loo scheme . similarly , sdec is calculated for those variables used to build the model or training set . the first map is based on the absolute eab terms from pwd and is referred to as an interaction map . in this heat map , all of the interaction terms are negative , since the repulsive energy terms are excluded . the second map consists of the most important residueligand interaction terms derived from the pls analysis of all the interaction terms ( attractive and repulsive ) . these quantities can be either positive or negative . in both maps , the key protein residues , as defined below , are given on the x - axis , and the ligands are given on the y - axis . this is useful when trying to identify common critical residues across a series of ligands . an example of this type of interaction is the hydrogen - bonding network between residues glu114 and ala116 and the heterocycles found in almost all of the ligands examined in this study . the sar map is scaled based on the variance across the series of ligands and tends to highlight protein residues that are responsible for the changes in potency across a series of ligands . for example , met166 varies greatly between the most potent ligand ( 30 ) and one of the weakest ligands ( 40 ) . compound 40 also has a very poor ( red ) interaction relative to any of the other ligands with the hinge residues mentioned above ( glu114 and ala116).the hinge is located between the n- and c - terminal lobes of the kinase domain.(40 ) it is the canonical catalytic site of phosphorylation by atp . a quick look at 40 explains this result , since it is essentially the only ligand in the series that lacks any hydrogen - bonding partners for these two residues . this does not mean that there is no favorable interaction ; it only means that 10 has an average interaction energy ( relative to the series ) for residues glu114 and ala116 . although the interaction map is good at characterizing attractive interaction energy terms , it does not take into account repulsive energy terms . an algorithm derived from the pls method has been developed by quantumbio to address this . a pls - based score function was defined as : where xlk represents a element from the descriptor matrix , xjmean is the column average , xjstd is the column standard deviation , and bij is the associated pls coefficient . thus the activity , yi , can be rewritten as : therefore , the scores resl(i ) are exactly the contribution of each residue to the binding affinity . the more negative the value of log(ic50 ) , the stronger the binding . a negative resl(i ) value means the residue contributes favorably to the binding affinity , while a positive value of resl(i ) suggests it reduces affinity . qmscore calculations were performed with the pm3 hamiltonian on the modeled ( aligned ) and x - ray structures . a plot of experimental log(ic50 ) , as a function of qmscore , is shown in figure 4 . the qmscore values for all 45 ligands range from 833 to 307 kcal / mol . the regression line for the qmscores from the training set is plotted as a blue solid line in the figure 3 . the qmscores achieve good agreement with the experimental log(ic50 ) values of the training set , and the r is high at 0.84 . data for the prediction set distribute themselves evenly along the regression line of the training set . these results demonstrate that the pm3 calculations can reproduce the overall trends in affinity even without any complex statistical analysis or descriptor selection . the qmscore is composed of gas - phase heat of formation , electrostatic solvation , attractive lennard - jones , solvation entropy , and vibrational entropy energy terms . a multiple linear regression analysis has been performed on these five energy terms in order to the r values of the model - fitting qmscore and the experimental log(ic50 ) for the training set and all 45 ligands are 0.90 and 0.82 , which are improved from that of the directly calculated qmscore ( r = 0.84 for training set and r = 0.76 for all 45 ligands ) . the qmscores of the training set ( x - ray structures ) are labeled as blue diamonds , and the prediction set are red squares . the regression lines were plotted for the training / prediction sets , and their associated r values are included . the descriptor matrices associated with different scoring functions were obtained from the se - combine calculations . for example , the scoring function sf-1 consisted only of qm - pwd energy terms , eab , eab , and eabcore . there are 355 residues in the descriptor matrix , and each residue contributes three descriptors , eab , eab , and eabcore terms . thus sf-1 , has a total of 1065 descriptors derived from the ligandreceptor interaction energies . as mentioned above , near zero descriptors were eliminated , as were descriptors with a standard deviation of less than 0.05 . the number of descriptors was reduced to 732 after pruning , and the remaining descriptors were autoscaled because the eab , eab , and eabcore terms span different ranges . pls models were constructed for each of the scoring functions sf-1 through sf-7 . in each case , the r values gradually increase with every additional latent variable . the q behaves differently , reaching a peak at the optimal number of latent variables . thus , the optimal pls model is determined by the standard deviation of prediction ( sdep ) and by q. the optimal pls models are outlined in table 3 . all of the qm - based pls models explain 97100% of the variance , while the pure mm ( sf-5 ) model explains only 79% variance . the qm models have universally higher q values ( as high as 0.81 ) relative to the mm q of only 0.33 . likewise , sdep values from the qm models are significantly lower than that of the mm model . overall , the pure - qm and mixed - qm / mm optimal models characterize the training set better than the pure - mm model . evaluating two different semiempirical qm hamiltonians reveals the training set of pls results from pm3 are comparable to those of am1 . r and q represent the correlation coefficient of the training set and the loo validation . sdec and sdep are the standard deviation of the calculated and predicted values for the internal validation . sdep and r are similar definitions , while applied to the external set . as an example , the log(ic50 ) values the plot of computed and experimental affinities for this model are given in figure 5 . the correlation for the training set ( r = 0.92 ) and the external test data set ( r = 0.71 ) are good . most of the data points in figure 5 fall between the two yellow - dashed lines , indicating errors of less than 1 order of magnitude . there are two ligands , 6 and 11 , that lie far off the line . a careful examination of these structures suggests that the large deviations may be partly due to errors in building the proteinligand complexes . if these two compounds were omitted , then the r increases from 0.71 to 0.83 . plot of calculated versus experimental log(ic50 ) values from the training ( blue ) and prediction ( red ) sets . the blue and red lines are trend lines for the training and prediction sets , respectively and , their corresponding r values are labeled . three dashed lines define the regions of the plot : the yellow line is the diagonal line and the two red lines are 1 offsets to the diagonal line . the relative contribution of descriptors to the optimal pls model can be determined from the pls coefficients . a pls coefficient plot for sf-1 , using the pm3 hamiltonian , these residues are defined as key residues , all of which are in the active site of the receptor . in particular , the eab energy terms for glu114 , ala116 , val50 , and other residues highlighted in figure 6 contribute heavily to the best pls model . these key active site residues were used to construct the interaction map in figure 7 . pls coefficient plot for optimal model ( lv = 2 ) of scoring function sf-1 . the compounds on the y - axis are arranged with respect to the activity . the binding affinity decreases from top to bottom . the legend designates the magnitude of the unscaled descriptor in ev . after the determination of key active site residues , an intermolecular interaction map is generated based on the interaction energies ( figure 7 ) . the ligands on the y - axis of figure 7 are ordered from most to least potent ( based on ic50 s ) with the most potent at the top of the map . the interaction map in figure 7 highlights the dominant eab terms between the ligands and the receptors . the eab interactions are always attractive , and therefore , must be negative or zero . of course , the more negative values indicate stronger attractive interactions between the residue and the ligand . the interaction maps allow one to easily identify the most attractive and important residueligand interactions . for example , the strong interaction between 30 and lys65 stands out by virtue of the negative interaction energy and the corresponding dark blue color . the interaction map in figure 7 shows two columns of consistently dark - blue data points corresponding to glu114 and ala116 . examination of the complex structures shows that there are multiple cooperative hydrogen bonds between these residues and the heterocyclic moieties found in most of the ligands ( figure 8a ) . of course , this is a well - known , and important , interaction for kinase inhibitors that target the adenosine triphosphate ( atp ) domain . thus , it is reassuring , and validates the methodology , to see that this critical interaction is starkly visible in the interaction map . the exception to this trend in figure 7 is compound 40 ( white cells ) . this is reasonable since 40 , unlike the other ligands , has no such hydrogen - bonding functionality ( figure 8b ) . a look at the structure of 40 , and its complex , shows that 40 is not capable of acting as a hydrogen - bond partner to either glu114 or ala116 . this stands out in the interaction map ( white cells ) and clearly identifies 40 as being structurally unique relative to the other ligands . hydrogen - bond interactions between receptor and ligands : ( a ) ligand 35 hydrogen bond to glu114 and ala116 ; ( b ) ligand 40 has no hydrogen bonds with glu114 and ala116 ; and ( c ) ligand 30 hydrogen bond to asn 164 and asp 177 . this interaction is due to the basic nitrogen in 30 that forms a series of hydrogen bonds with the carboxylate in asp177 and the backbone carbonyl of asn164 ( figure 8c ) . this type of interaction is present in most of the more active ligands but does not appear to be as dominant as might appear , if we were using classical point charges . since the semiempirical hamiltonians utilized in the present study are known to have limitations concerning the treatment of dispersion and other nonbonding interactions(41 ) relative to higher level qm methods , one might be concerned that only purely electrostatic interactions would be seen in the interaction map . but there are examples of favorable interactions that are consistent across the series and would not be expected to be strictly electrostatic . one example is the negative interaction , likely hydrophobic , between val50 and the majority of ligands in this study . ev across all 45 ligands ( figure 7 ) . as can be seen in ( figure 7 ) , there are 9 nonpolar residues ( leu42 , gly43 , gly45 , phe47 , val50 , met113 , ala116 , met166 , and phe320 ) in a total of 21 key residues . most of these nonpolar key residues show consistent negative eab values for each ligand . while in some cases the interactions with these nonpolar residues may be due to polar backbone atoms , such as is the case with ala116 , many of these interactions are primarily nonpolar . taking val50 as a specific example , three ligand complexes ( 31 , 43 , and 28 ) are shown in figure 9 ( the binding affinities increase from a to c ) . as can be seen in figure 9 , the potency increases as the number of nonpolar interactions between the ligand and val50 increases . thus , eab becomes more negative going from 31 to 28 due to these nonpolar interactions . hydrophobic interactions between key residue val50 ( magenta ) and ligands ( light blue ) : ( a ) ligand 31 ; ( b ) ligand 43 ; and ( c ) ligand 28 . the pls - based resl(i ) values for sf-1 ( pm3 hamiltonian ) are plotted as an sar map in figure 10 . this sar map is distinct from the interaction map in figure 7 , in that , the pls coefficients are not driven by the absolute interaction energies but instead are ranked by their contribution to the variance in potency across the series of ligands . for example , residues ala116 and glu114 are consistently dark blue in the interaction map ( figure 7 ) because these interactions are strongly negative and are present in all but one of the ligands . in the case of the sar map ( figure 10 ) , the energies , and corresponding colors , range from unfavorable positive interactions ( red ) to favorable negative interactions ( green ) , reflecting the range of interactions with different ligands . the key residues ( x - axis ) are ordered according to their average scores . scores higher than 0.5 or lower than 0.5 were plotted as 0.5 and 0.5 , respectively . a residue that illustrates use of the sar map is met166 . the interaction between met166 and 30 is highly negative ( dark blue ) , consistent with the high potency of 30 . in contrast , the positive interaction ( orange ) between met166 and 40 is likely a contributing factor to the poor affinity of 40 . once again , this demonstrates that the underlying qm calculations are able to reproduce trends in essentially hydrophobic interactions . by comparison , examination of the complex with 40 shows very little contact between met166 and the ligand trends in affinity across chemotypes ( figure 11b ) . previous reseach has suggested that -stacking interaction could be significant between the side chain of methionine and the phenyl group.(42 ) however , the sar map shows that the interaction of phenyl group of 40 and met166 contributes little to the binding afinity . this is also observed from the interaction map , the eab value between me166 and 40 is only 0.017 kcal / mol . checking the x - ray structure of 2uw8 , the shortest distance of heavy atoms between met166 ( sulfur atom ) and the phenyl group of 40 ( carbon atom from the benzene ring ) is 4.41 , which is considered to be too large to achieve good electronic overlapping between two atoms . residue met166 ( magenta ) interacting with ligands ( light blue ) : ( a ) interacting with ligand 30 ; and ( b ) having limited contacts with ligand 40 . the sar map in figure 10 can also be used to identify broad trends in affinity not only within but across chemotypes . for example , while there is a great deal of variation in the heat maps for the most potent compounds on the left side of the map ( i.e. , met166 through gly48 ) , most of these potent ligands appear to benefit from favorable interactions with residues met113lys174 and tyr62gln170 . upon closer inspection , it becomes apparent that the increased affinity is due to interactions with ligands that contain two aromatic rings ( one being the hinge binding heterocycle ) separated by a third ring . these compounds clearly light up green on the right side of the sar map , and the trend holds over all chemotypes in the study . one more interesting trend apparent on the left side of the sar map is the yellow grids ( between met113lys174 and tyr62gln170 ) , for compounds with the lowest affinity . thus , compounds containing a primary amine in the correct location are clearly visible in the interaction map as blue grids for asp177 and/or glu120 . conversely , the sar map shows that these same residues are colored yellow , in the case of ligands , that lack this basic amine . we have used 15 crystal structures for a series of pkb inhibitors to train an se - combine model . this model was then applied to a larger , external test set of 30 ligands . the qm energies and the combine - optimized models were both reasonably predictive for the training and , most importantly , the test set . the goal of this work was not just to build a model of potency but to use the recently developed pairwise decomposition scheme in divcon to analyze the contributions of individual residues to ligand binding . this was done using two formalisms : first , the absolute eab values from pwd were used to construct a heat map ( interaction map ) , where residues were coded by their absolute pairwise interaction energies . second , a sar map was constructed from the pls pairwise terms that are scaled by variance . an example is the hydrogen - bond network between the ligand heterocyclic cores and the hinge region of the kinase . lights up in the interaction map with all but one ligand ( a ligand that lacks this functionality ) . the second map ( sar map ) highlights residues that contribute the most to discriminating between potent and weak ligands . in this case , the hydrogen - bonding interactions between the ligands and the hinge are less striking because there is less variance across the series of ligands . on the other hand , other interactions that play a major role in the variation of potency across the series both heat maps provide valuable insights into the role of specific residues in conveying activity .

quantum mechanical semiempirical comparative binding energy analysis calculations have been carried out for a series of protein kinase b ( pkb ) inhibitors derived from fragment- and structure - based drug design . these proteinligand complexes were selected because they represent a consistent set of experimental data that includes both crystal structures and affinities . seven scoring functions were evaluated based on both the pm3 and the am1 hamiltonians . the optimal models obtained by partial least - squares analysis of the aligned poses are predictive as measured by a number of standard statistical criteria and by validation with an external data set . an algorithm has been developed that provides residue - based contributions to the overall binding affinity . these residue - based binding contributions can be plotted in heat maps so as to highlight the most important residues for ligand binding . in the case of these pkb inhibitors , the maps show that met166 , thr97 , gly43 , glu114 , ala116 , and val50 , among other residues , play an important role in determining binding affinity . the interaction energy map makes it easy to identify the residues that have the largest absolute effect on ligand binding . the structureactivity relationship ( sar ) map highlights residues that are most critical to discriminating between more and less potent ligands . taken together the interaction energy and the sar maps provide useful insights into drug design that would be difficult to garner in any other way .

Introduction Methods Results and Discussion Conclusion

structure - based drug design ( sbdd ) and fragment - based drug design ( fbdd ) play increasingly important roles in drug discovery,(1 ) as more protein structures become available and as the computational tools for exploiting those structures become more capable . ultimately , the success or failure of these efforts rests on the ability to accurately compute proteinligand interaction energies . this is a difficult problem because of the complexity of the molecular structures involved and the very significant challenge of computing energy differences to sufficient accuracy to provide useful predicted binding affinities . there are many approaches to this problem that vary greatly in terms of their accuracy , generality , and efficiency . at one extreme are simulation - based approaches , such as free energy perturbation ( fep). even so , this approach is limited by the quality of the force field and by other limitations inherent in classical molecular models . in recent years , there has been significant progress in the development of fast quantum mechanical methods for computing protein - size molecular systems . these linear - scaling approaches have made quantum calculations for proteinligand complexes tractable , and they have provided an important new tool for computing proteinligand interaction energies . indeed , previous work has shown that there are significant charge transfer and polarization effects in proteinligand complexes that are not captured in classical models. pwd has successfully been applied to the investigation of the effect of binding in a series of fluorine - substituted ligands to human carbonic anhydrase ii. (17 ) a receptor - based qsar method , comparative binding energy analysis ( combine ) formalism , was proposed by by ortiz and co - workers . based on the mm descriptors , qsar models were built by multivariate statistical tools , such as partial least - squares ( pls ) . semiempirical pairwise decomposition , along with combine , have been integrated into a new approach for computing proteinligand interaction energies ( se - combine ) on a residue - by - residue basis. a series of 45 inhibitors ( table 1 ) for protein kinase b ( pkb ) were selected to test the se - combine method . these compounds were chosen for two reasons : first , both structures and affinities are available for many of these ligands . this provides a unique opportunity to compare our computational results to high - quality experimental data for both structure and activity . second , the ligands can be grouped into structurally related classes , in many cases being the product of a fragment - based design . qm - pwd was used to compute all of the pairwise ligandresidue interactions between the 45 ligands and the protein kinase a ( pka)pkb chimera . analysis of the interaction energies , heat maps , and structures show that se - combine provides a predictive model for affinity , a clear indication of the most critical residues for binding , and a meaningful indication of the many structural trade - offs necessary to design potent ligands for pkb . compounds with protein data bank ( pdb ) identifications ( protein databank accession number ) are members of the training set . in a series of papers , astex pharmaceuticals has reported 16 x - ray structures of pkapkb chimera complexes as well as the binding affinity data for more than 60 additional ligands ( see pdb i d in figure 1 ) . for this paper , residue numbers were assigned according to the sequential number label beneath the aligned sequences in figure 1 . all of the active site residues fall within the conserved range so that the active sites for all 16 x - ray structures are identical , at least with regard to sequence . these 16 well - defined proteinligand complexes were selected as the training set for model building . the * character indicates positions that have been fully conserved . this left 30 ligands ( table 1 ) for the prediction set and 15 proteinligand complexes for the training set . the number of heavy atoms range from 7 to 30 atoms , and the molecular weights are spread over 300 amu . the potency shows a poor correlation ( figure 3 ) with the number of heavy atoms ( r = 0.1843 ) and with the molecular weight ( r = 0.1458 ) . the 15 x - ray structures , corresponding to the 15 ligands in the training set , were downloaded from the pdb . these models were built using an in - house flexible three - dimensional structure alignment program that utilizes the mtk++ library,(35 ) along with a number of additional routines in order align a ligand to a structurally similar template ligand . after alignment , the ligands were fitted into the active site of the pkapkb chimera in the same way as their templates . the templates for each of the model - built complexes were shown in table 1 by square brackets with pdb identifications of the templates inside . due to structural variations among ligands these hot spots were allowed to relax in the active site using a restrained amber minimization of 1500 steps ( 500 steepest descent followed by 1000 conjugate gradient ) followed by a full minimization of all atoms in the system ( 500 steepest descent followed by 1000 conjugate gradient steps ) . linear scaling , semiempirical divide and conquer ( d&c ) single - point calculations(10 ) were carried out on each pkapkb complex using the pm3(36 ) and am1(13 ) hamiltonians , as implemented in the divcon 5.x program. (37 ) divcon calculates the quantum mechanical atom - to - atom , pairwise energy decomposition ( qm - pwd)(16 ) and the overall qmscore(15 ) for each complex . the d&c atom - to - atom pairwise interaction energies were calculated and then converted to residue - based pairwise interaction energies ( i.e. seven scoring functions ( from sf-1 to sf-7 ) using various combinations of the qm - pwd , the qmscore , and the mm - pwd energy terms were evaluated using pls ( table 2). (38 ) the qm - pwd energy terms consist of eab , eab , and eabcore energy terms. the computed molecular mechanics ( mm ) energy terms include dispersion , van der waals ( summation of dispersion and repulsion terms ) , and electrostatics . sf-1 includes only the pairwise energies of the complex in vacuum and is a close approximation to the original version of se - combine,(22 ) while sf-2 , -3 , -4 , -6 , and -7 are extensions of the original se - combine . sf-5 only contains mm energy terms and is analogous to the conventional implementation of combine. (39 ) sf-2 contains pairwise energies of the complex in solvent . internal validation was carried out using leave - one - out ( loo ) cross - validation for the training set , and the optimal dimensionality of each model was assigned from its cross - validated predictive ability . the models were evaluated for their predictive quality using a range of standard statistical measures , such as the correlation ( r ) and cross - validated correlation ( q ) coefficients , the standard deviation of error of calculations ( sdec ) , and the standard deviation of error prediction ( sdep ) . the first map is based on the absolute eab terms from pwd and is referred to as an interaction map . in this heat map , all of the interaction terms are negative , since the repulsive energy terms are excluded . the second map consists of the most important residueligand interaction terms derived from the pls analysis of all the interaction terms ( attractive and repulsive ) . these quantities can be either positive or negative . in both maps , the key protein residues , as defined below , are given on the x - axis , and the ligands are given on the y - axis . this is useful when trying to identify common critical residues across a series of ligands . an example of this type of interaction is the hydrogen - bonding network between residues glu114 and ala116 and the heterocycles found in almost all of the ligands examined in this study . the sar map is scaled based on the variance across the series of ligands and tends to highlight protein residues that are responsible for the changes in potency across a series of ligands . for example , met166 varies greatly between the most potent ligand ( 30 ) and one of the weakest ligands ( 40 ) . this does not mean that there is no favorable interaction ; it only means that 10 has an average interaction energy ( relative to the series ) for residues glu114 and ala116 . although the interaction map is good at characterizing attractive interaction energy terms , it does not take into account repulsive energy terms . an algorithm derived from the pls method has been developed by quantumbio to address this . a pls - based score function was defined as : where xlk represents a element from the descriptor matrix , xjmean is the column average , xjstd is the column standard deviation , and bij is the associated pls coefficient . thus the activity , yi , can be rewritten as : therefore , the scores resl(i ) are exactly the contribution of each residue to the binding affinity . a negative resl(i ) value means the residue contributes favorably to the binding affinity , while a positive value of resl(i ) suggests it reduces affinity . qmscore calculations were performed with the pm3 hamiltonian on the modeled ( aligned ) and x - ray structures . the qmscores achieve good agreement with the experimental log(ic50 ) values of the training set , and the r is high at 0.84 . these results demonstrate that the pm3 calculations can reproduce the overall trends in affinity even without any complex statistical analysis or descriptor selection . a multiple linear regression analysis has been performed on these five energy terms in order to the r values of the model - fitting qmscore and the experimental log(ic50 ) for the training set and all 45 ligands are 0.90 and 0.82 , which are improved from that of the directly calculated qmscore ( r = 0.84 for training set and r = 0.76 for all 45 ligands ) . the qmscores of the training set ( x - ray structures ) are labeled as blue diamonds , and the prediction set are red squares . the descriptor matrices associated with different scoring functions were obtained from the se - combine calculations . for example , the scoring function sf-1 consisted only of qm - pwd energy terms , eab , eab , and eabcore . there are 355 residues in the descriptor matrix , and each residue contributes three descriptors , eab , eab , and eabcore terms . the number of descriptors was reduced to 732 after pruning , and the remaining descriptors were autoscaled because the eab , eab , and eabcore terms span different ranges . pls models were constructed for each of the scoring functions sf-1 through sf-7 . in each case , the r values gradually increase with every additional latent variable . the q behaves differently , reaching a peak at the optimal number of latent variables . thus , the optimal pls model is determined by the standard deviation of prediction ( sdep ) and by q. the optimal pls models are outlined in table 3 . all of the qm - based pls models explain 97100% of the variance , while the pure mm ( sf-5 ) model explains only 79% variance . the qm models have universally higher q values ( as high as 0.81 ) relative to the mm q of only 0.33 . overall , the pure - qm and mixed - qm / mm optimal models characterize the training set better than the pure - mm model . evaluating two different semiempirical qm hamiltonians reveals the training set of pls results from pm3 are comparable to those of am1 . r and q represent the correlation coefficient of the training set and the loo validation . sdep and r are similar definitions , while applied to the external set . as an example , the log(ic50 ) values the plot of computed and experimental affinities for this model are given in figure 5 . the correlation for the training set ( r = 0.92 ) and the external test data set ( r = 0.71 ) are good . most of the data points in figure 5 fall between the two yellow - dashed lines , indicating errors of less than 1 order of magnitude . a careful examination of these structures suggests that the large deviations may be partly due to errors in building the proteinligand complexes . three dashed lines define the regions of the plot : the yellow line is the diagonal line and the two red lines are 1 offsets to the diagonal line . the relative contribution of descriptors to the optimal pls model can be determined from the pls coefficients . a pls coefficient plot for sf-1 , using the pm3 hamiltonian , these residues are defined as key residues , all of which are in the active site of the receptor . in particular , the eab energy terms for glu114 , ala116 , val50 , and other residues highlighted in figure 6 contribute heavily to the best pls model . these key active site residues were used to construct the interaction map in figure 7 . the compounds on the y - axis are arranged with respect to the activity . the binding affinity decreases from top to bottom . after the determination of key active site residues , an intermolecular interaction map is generated based on the interaction energies ( figure 7 ) . the ligands on the y - axis of figure 7 are ordered from most to least potent ( based on ic50 s ) with the most potent at the top of the map . the interaction map in figure 7 highlights the dominant eab terms between the ligands and the receptors . of course , the more negative values indicate stronger attractive interactions between the residue and the ligand . the interaction maps allow one to easily identify the most attractive and important residueligand interactions . for example , the strong interaction between 30 and lys65 stands out by virtue of the negative interaction energy and the corresponding dark blue color . examination of the complex structures shows that there are multiple cooperative hydrogen bonds between these residues and the heterocyclic moieties found in most of the ligands ( figure 8a ) . thus , it is reassuring , and validates the methodology , to see that this critical interaction is starkly visible in the interaction map . this stands out in the interaction map ( white cells ) and clearly identifies 40 as being structurally unique relative to the other ligands . this interaction is due to the basic nitrogen in 30 that forms a series of hydrogen bonds with the carboxylate in asp177 and the backbone carbonyl of asn164 ( figure 8c ) . this type of interaction is present in most of the more active ligands but does not appear to be as dominant as might appear , if we were using classical point charges . since the semiempirical hamiltonians utilized in the present study are known to have limitations concerning the treatment of dispersion and other nonbonding interactions(41 ) relative to higher level qm methods , one might be concerned that only purely electrostatic interactions would be seen in the interaction map . as can be seen in ( figure 7 ) , there are 9 nonpolar residues ( leu42 , gly43 , gly45 , phe47 , val50 , met113 , ala116 , met166 , and phe320 ) in a total of 21 key residues . while in some cases the interactions with these nonpolar residues may be due to polar backbone atoms , such as is the case with ala116 , many of these interactions are primarily nonpolar . as can be seen in figure 9 , the potency increases as the number of nonpolar interactions between the ligand and val50 increases . the pls - based resl(i ) values for sf-1 ( pm3 hamiltonian ) are plotted as an sar map in figure 10 . this sar map is distinct from the interaction map in figure 7 , in that , the pls coefficients are not driven by the absolute interaction energies but instead are ranked by their contribution to the variance in potency across the series of ligands . for example , residues ala116 and glu114 are consistently dark blue in the interaction map ( figure 7 ) because these interactions are strongly negative and are present in all but one of the ligands . in the case of the sar map ( figure 10 ) , the energies , and corresponding colors , range from unfavorable positive interactions ( red ) to favorable negative interactions ( green ) , reflecting the range of interactions with different ligands . a residue that illustrates use of the sar map is met166 . in contrast , the positive interaction ( orange ) between met166 and 40 is likely a contributing factor to the poor affinity of 40 . by comparison , examination of the complex with 40 shows very little contact between met166 and the ligand trends in affinity across chemotypes ( figure 11b ) . (42 ) however , the sar map shows that the interaction of phenyl group of 40 and met166 contributes little to the binding afinity . this is also observed from the interaction map , the eab value between me166 and 40 is only 0.017 kcal / mol . checking the x - ray structure of 2uw8 , the shortest distance of heavy atoms between met166 ( sulfur atom ) and the phenyl group of 40 ( carbon atom from the benzene ring ) is 4.41 , which is considered to be too large to achieve good electronic overlapping between two atoms . the sar map in figure 10 can also be used to identify broad trends in affinity not only within but across chemotypes . for example , while there is a great deal of variation in the heat maps for the most potent compounds on the left side of the map ( i.e. , met166 through gly48 ) , most of these potent ligands appear to benefit from favorable interactions with residues met113lys174 and tyr62gln170 . upon closer inspection , it becomes apparent that the increased affinity is due to interactions with ligands that contain two aromatic rings ( one being the hinge binding heterocycle ) separated by a third ring . these compounds clearly light up green on the right side of the sar map , and the trend holds over all chemotypes in the study . one more interesting trend apparent on the left side of the sar map is the yellow grids ( between met113lys174 and tyr62gln170 ) , for compounds with the lowest affinity . thus , compounds containing a primary amine in the correct location are clearly visible in the interaction map as blue grids for asp177 and/or glu120 . conversely , the sar map shows that these same residues are colored yellow , in the case of ligands , that lack this basic amine . we have used 15 crystal structures for a series of pkb inhibitors to train an se - combine model . the qm energies and the combine - optimized models were both reasonably predictive for the training and , most importantly , the test set . this was done using two formalisms : first , the absolute eab values from pwd were used to construct a heat map ( interaction map ) , where residues were coded by their absolute pairwise interaction energies . an example is the hydrogen - bond network between the ligand heterocyclic cores and the hinge region of the kinase . lights up in the interaction map with all but one ligand ( a ligand that lacks this functionality ) . the second map ( sar map ) highlights residues that contribute the most to discriminating between potent and weak ligands . in this case , the hydrogen - bonding interactions between the ligands and the hinge are less striking because there is less variance across the series of ligands . on the other hand , other interactions that play a major role in the variation of potency across the series both heat maps provide valuable insights into the role of specific residues in conveying activity .

the 1978 alma ata declaration codified the right to primary health care ( phc ) for all . nine years later , african ministers of health convened in bamako , mali , to emphasize the need to strengthen phc on the continent , with a particular focus on maternal and child health . decades later , however , phc remains a lofty goal for many african countries , despite the impact on profile and funding for phc of alma ata , the bamako initiative and their successor initiatives . the alma ata declaration acknowledged the impact of good governance , economic and social development , inequality , health system functioning , inter - sector cohesiveness , and education on health outcomes . recent work by rohde et al . illustrated how these factors continue to influence progress in achieving phc for all . countries that have shown weak gains in life expectancy over the last couple of decades include those affected by conflict , those having a high hiv / aids prevalence , those plagued by poor governance and social inequality , and those experiencing specific adult mortality challenges . the country continues to have low immunization rates and a low life expectancy , despite significant financial and political support for phc since the 1980s . health inequities , pervasive corruption , and the autonomy of nigeria s 36 states have prevented the country from establishing a national framework to support a phc system that works for all . the autonomy of the states and local government authorities ( lgas ) has complicated the building of a cohesive phc system in nigeria . in the nigerian constitution , health is a concomitant responsibility of the three tiers of government : the federal level , 37 states including the federal capital territory , and 774 lgas . according to the national health policy , the federal level is responsible for tertiary care , the state level for secondary care and the lga level for phc . however , details in the policy regarding the roles and responsibilities for each level are unclear . matters have become arguably even less clear since the end of military rule in 1999 . reality on the ground frequently demonstrates how actors within the health system are unable to distinguish the roles and responsibilities between each level of government . there exists a myriad of different departments , directorates and units at each level with overlapping responsibilities . crucially , the way in which the three levels of government should interact has not been elaborated in policy . thus , it can be a challenge for innovations , lessons from experience and planned activities to permeate from one level to the next . for example , how do practitioners who are operating on the ground within states or lgas influence federal level policy makers and other states ? or , how do federal level policy makers ensure that planned activities are implemented across the three tiers of government ? these are the sorts of questions that are crucial if sustained efforts towards coordination of health provision - and concomitant population health gains - are to be attained in the nigerian context . they are particularly salient in the context of northern nigeria , where the coverage of health services ( and thus health outcomes ) lags well behind even the modest achievements of other parts of the country . a systematic survey across three northern nigerian states found that less than one - quarter of women who gave birth in the five years preceding the survey had ever received antenatal care ( anc ) from a trained health professional , and only one in eight of those women had delivered in a health facility . child health status indicators were also poor . only one in twenty children had received the third dose of dpt by the age of one year . a range of initiatives has been established to address the strengthening of health systems in these northern states . these include a range of focused initiatives , addressing specific diseases or program areas . bringing together as a combined programme two such initiatives - the partnership for reviving routine immunization in northern nigeria ( prrinn ) and the maternal , newborn and child health ( mnch ) programme - provided the opportunity to specifically address some of the wider systems issues that constrain health programming in nigeria . prrinn - mnch - with cofunding from the department for international development of the united kingdom ( dfid ) and the government of norway - has since 2007 provided a platform to specifically address the systems linkages required between federal , states and local structures to implement policy and service changes . programs for achieving the laudable goals set forth by alma ata and the bamako initiative have ranged from offering selective phc services to more comprehensive packages . the prrinn - mnch initiative was developed as a programme that sought to build upon the strategies outlined in the bamako initiative by explicitly addressing the broken linkages between phc services at all levels of government . by facilitating better partnerships , the prrinnmnch program supported the government s effort to integrate phc services and strengthen its implementation at the district level , support child immunizations through improved funding mechanisms , and ensure maternal health is addressed at the community level . this paper focuses upon three prrinnmnch supported initiatives that sought to improve linkages between , and the functioning of , the federal , state , and lga levels of health system responsible for phc : bringing phc under one roof ; enhancing access to global alliance for vaccines and immunization ( gavi ) funds ; and strengthening the midwives service scheme ( mss ) . these three examples provide an opportunity to explore how concepts and experience developed at subnational levels were utilized to influence national policy and practice , and the way in which work at subnational levels can contribute to nationally conceived and driven plans for phc . programs for achieving the laudable goals set forth by alma ata and the bamako initiative have ranged from offering selective phc services to more comprehensive packages . the prrinn - mnch initiative was developed as a programme that sought to build upon the strategies outlined in the bamako initiative by explicitly addressing the broken linkages between phc services at all levels of government . by facilitating better partnerships , the prrinnmnch program supported the government s effort to integrate phc services and strengthen its implementation at the district level , support child immunizations through improved funding mechanisms , and ensure maternal health is addressed at the community level . this paper focuses upon three prrinnmnch supported initiatives that sought to improve linkages between , and the functioning of , the federal , state , and lga levels of health system responsible for phc : bringing phc under one roof ; enhancing access to global alliance for vaccines and immunization ( gavi ) funds ; and strengthening the midwives service scheme ( mss ) . these three examples provide an opportunity to explore how concepts and experience developed at subnational levels were utilized to influence national policy and practice , and the way in which work at subnational levels can contribute to nationally conceived and driven plans for phc . this paper analyses the development of policy and programme implementation in these three areas by drawing upon documents from the federal ministry of health in nigeria , prrinn - mnch reports , and discussions - both informal and formal - at a range of national workshops and programme planning meetings . complexity theory provides a way of understanding health systems as complex adaptive systems , and has been increasingly advocated as a tool for health policy development and health systems reform . with complexity theory , health systems are seen as open systems in which different components are interdependent and influence each other in a non - linear fashion . non - linearity and the notion of emergent behavior ( i.e. behavior of a system that is not a property of any of the components of that system but a result of the interactions of the components ) mean that a change in one part of the system can have unpredictable ripple effects in other parts of the system . for example , the world health organization s report systems thinking for health system strengthening , heavily influenced by the ideas of complexity theory , acknowledges non - linearity and interdependence in a proposed framework for health system strengthening . this requires policymakers and health system reformers to adopt a whole system approach in order to ensure changes at one level will not impede changes at another . the complex adaptive systems approach reinforces concepts such as feedback loops ( both positive and negative , that influence the pace and direction of change ) ; path dependence ( processes that have similar starting points can have very dissimilar outcomes resulting from different contexts and histories and different choices ) ; scale - free networks ( incorporating focal points - including key powerful people - that can dominate a structure ) ; and phase transitions ( when critical - tipping - points are reached and initiate change ) . the ideas of complexity theory are closely linked to the drivers of change ( doc ) approach adopted by dfid . the doc approach conceptualizes three interacting components operating within any system and influencing change within that system : structural features / structures - the history of the state ; natural and human resources ; economic and social structures ; demographic changes ; regional issues ; globalization , trade and investment ; and urbanization ; institutions - the informal and formal rules that determine the realm of possible behavior by agents , such as political and public administration processes ; and agents - individuals and organizations pursuing particular interests . examples of agents include the political elite ; civil servants ; political parties ; local government ; the judiciary ; the military ; faith groups ; trade unions ; civil society groups ; the media ; the private sector ; academics ; and donors . the doc analysis and approach is essentially focused on power and the mechanisms through which that power is transacted within society and the health system . the doc approach formed the basis of political economy assessments at federal and state level health systems in nigeria , which led to a deeper understanding of the structural features , the power relations , the institutions ( particularly the informal rules ) and the agents operating in the sector . both complexity theory and the doc approach to political economy any new policy development needs to be understood in the context within which the potential change will be located . this context requires a deep and ongoing understanding of the structures , institutions and agents operating within the whole system . however , complexity theory requires a further understanding or analysis of the changes that a new policy will bring ( especially an appreciation of non - linearity and likely emergent behavior ; and an understanding of likely feedback loops , path dependent bifurcation points , focal points and transition points ) . only then , and in an ongoing fashion as the context and the whole system is dynamic , can policy be developed and implemented . the bamako initiative of 1987 supported strengthening health systems at the district and community - levels in parallel with efforts to decentralize political systems throughout africa . however , roles and responsibilities over phc programs , facilities , human resources , and financing remained unclear - a mishmash of centralization and decentralization . although the federal level is principally responsible for tertiary health care , the national primary health care development agency ( nphcda ) and national programme on immunization were developed at the federal level to maintain some control over phc . for example , the nphcda is intended to ( and does ) carry out supplemental immunization campaigns against vaccine preventable diseases including polio . state ministries of health are also involved in the building and managing of phc centers although , according to the national health policy , ostensibly responsible for managing secondary health care hospitals . the states provide health care services ( including phc services ) through the state ministry of health ( smoh ) , the state hospital management board ( shmb ) , and the state phc development agency ( sphcda ) . several bodies are involved in human resource management at the lga level for phc services : the ministry of local government ( molg ) , the state phc agency , the local government service commission , the public service commission and the lga . the local government service commission is a state level organization that is responsible for all health professionals ( level 7 and above ) while the lgas managed admin , security , and lower level personnel . budget development and release is similarly fragmented . while finance was nominally devolved in block grants to both states and lgas , state governments established joint accounts in which state and lga financial resources were placed . adding to the confusion over financial control , external donors usually direct programmatic funding into federal - level accounts , although ultimately implementation should occur at the local level . for example , large sums of money provided by the millennium development goal ( mdg ) office are given to the federal ministry of health to implement the community - based national health insurance scheme ( nhis ) scheme . the diversification of responsible units at the federal and state levels exacerbates the confusion of responsibilities between tiers . although phc is the responsibility of local governments , the multitude of different agents actually involved in phc services makes coordinated management across the ministry of health technically difficult . thus , several states - starting with enugu and jigawa , and then yobe and zamfara - have been exploring strategies for bringing phc under one roof . this first occurred through the partnership to transform health systems ( paths ) programme from 2002 to 2008 , and then through the prrinn - mnch programme , which began in 2006 . the possibility of the funding that would flow from the federal health act drove policy and legislative changes regarding phc in several states in nigeria . although implementation varies across states , relevant policy and legislative changes generally address three key issues . firstly , health services - particularly phc services - are being integrated , where previously all three tiers of government were involved in implementation . secondly , health services are being decentralized - both through devolution and de - concentration . and thirdly , but not uniformly , through the de - concentration to sub - state bodies ( the names are different in the different states ) , the balance of power in the management of key resources ( especially financial and human resources ) is shifting from the politicians to administrators and managers . it is important to highlight the process adopted through these adjustments and how the outcome of integrated health services ( at least of phc ) was achieved . since both federal government and states can pass legislation , both the paths and the prrinn - mnch programmes worked closely with the states to develop appropriate legislation and accompanying regulations for phc ; to strengthen systems ( especially financial , human resource and information ) ; and to reposition the state level bodies for their new roles and functions . this process was not without challenges and extended over substantial periods of time . at the same time , the states ( with support from the programmes ) lobbied at federal level to gain acceptance and to provide access to materials and support for states willing to embark on this process . draft policy memos and an implementation guide were finalized during the second workshop , were approved by the nphcda board in 2010 and submitted to the highest health policy body in nigeria ( the national council for health ) . as summarized in the official record of the council : council noted the thrust of the national health bill in strengthening primary health care ( phc ) through the creation of phc boards / agencies and the phc development fund . council noted efforts in bringing phc under one roof in line with the provisions of the national health bill . council also noted the importance of enacting relevant state legislation and regulations that will facilitate the implementation of national health bill . council therefore approved the implementation guide on bringing phc under one roof ( phcuor ) as a working document to be used by the three tiers of government and approved that all states establish primary health care boards . the national council for health not only adopted the policy document and implementation guide on bringing phc under one roof but encouraged the 36 states to proceed in implementing this concept ( table 1 ) . at the time of the 2012 workshop , 22 of the 36 states in nigeria were in various stages of implementing bringing phc under one roof . gavi has supported nigeria s immunization system since 2001 to address extremely poor immunization coverage , particularly in the northern states ( http://www.gavialliance.org/country/nigeria/ ) . states were able to access the first tranche of the gavi funds , which were managed at the federal level , following initial engagement with gavi , but several states were unable to retire the monies appropriately . thus in 2007 , prrinn - mnch began at the state level supporting state ministries of health to effectively retire and access ongoing funding tranches from gavi . this work shifted to the federal level where prrinn - mnch assisted the gavi office in nphcda to review the processes and tools for accessing and retiring the funds and participated in developing a set of master trainers who would train others across all states in nigeria . in 2009 , prrinn - mnch supported nphcda to develop financial guidelines for gavi fund management and provided appropriate training on the use of these guidelines for relevant staff at agency headquarters and in its programme states . the nphcda also requested that prrinn - mnch train focal nphcda staff on the use of the financial guidelines and tools in the non - programme states . the subsequent workshop enabled the participants to understand the use of the financial guidelines and equipped them with the capacity to train other relevant staff at the lower levels . the twenty - two participants comparised six senior staff from nphcda headquarters , two representatives of the nphcda offices from each of the six geo - political zones of the federation , and gavi fund accountants from the four prrinn - mnch focal states . this example illustrates how state level experience of an international agency s funds was used to drive changes in how the funds were administered and accessed at federal level . initial work started within the states to ensure that the proposed system would work , and had significant impact on coverage ( figure 1 ) . later this was taken up to federal level where capacity was built to maintain and nationally roll out the new system . nigeria has a high maternal mortality ratio , low attendance for antenatal services , and few births are attended by a skilled birth attendant . thus , to improve access to care for pregnant women , the mss was a federally conceived and driven scheme to place retired and unemployed midwives in health facilities ; later this was extended to community health extension workers ( chews ) to ensure remote communities have access to perinatal services . the nphcda initiated the mss using funds from mdg fund . in each state , four midwives were deployed to each of the selected phc facilities to ensure provision of maternal and child health care services on a 24/7 basis . in each state , a selection of four phc facilities is clustered around the referral general hospital , with the creation of 156 clusters nationwide . six of these clusters are in three prrinn - mnch target states ( katsina , yobe and zamfara ) . this was followed by a second wave ( both midwives and chews ) in late 2010 . specific areas of collaboration between prrinn - mnch and nphcda under the banner of mss have included : i ) participation in the recruitment of midwives for the north - west zone in nigeria ; ii ) sharing of relevant tools for health facility baseline assessment , participatory appraisal and continuous transformation ( including impact , a systems strengthening and management capacity building initiative developed by health partners international : http://www.healthpartners-int.co.uk/ ; http://www.prrinn-mnch.org/index.html ) and assessment of training institutions ; iii ) sharing experience on planning and conducting baseline assessments , development of a monitoring and evaluation framework , and the comprehensive emergency obstetric care ( ceoc ) cluster model used ; iv ) participation in the planning of refresher training of midwives countrywide by nphcda - mss ; v ) developing national integrated supportive supervision ( iss ) tools ( the iss tools have been institutionalized and the iss teams oriented nationwide ) ; vi ) providing in - service training to mss midwives in the three states based on training needs earlier identified during an induction workshop . while there are multiple interventions in the clusters and the states , the increased presence of midwives ( and chews ) is considered a major contribution to increased utilization of mnh services in all the prrinn - mnch supported facilities where mss midwives are deployed ( as compared to baseline data collected between august and october 2008 ; figure 2 ) . overall , more significant increases were noted in anc attendance when compared with intrapartum care ( delivery ) . for example , in furfuri phc , zamfara , the number of anc visits increased from 0 to 455 , and deliveries from 0 to 28 between april and june , 2010 . mss midwives initiated community mobilization activities to increase utilization of mnh services , especially skilled care at delivery . mss has also shown improvement in documentation , including the keeping of registers and summary graphs and statistics in all facilities ( and bar charts in some facilities ) . additionally , all prrinn - mnch supported health facilities where there are mss midwives ( at least 3 - 4 mss midwives per facility ) now provide 24-h intra - partum care . for instance , a night shift was introduced by mss midwives on arrival at baimari maternity phc , yobe . problems experienced by the midwives continue to be largely administrative and systems related : e.g. payment issues , accommodation , poor working environment etc . although there has been progress in most of these areas , they remain critical issues to monitor in support of retention of these important health workers . this third example illustrates how state level practitioners can assist in supporting , and adding value to , nationally conceived and driven plans . in this case the nphcda had no formal jurisdiction but clearly needed support to implement a laudable scheme with promising results . the bamako initiative of 1987 supported strengthening health systems at the district and community - levels in parallel with efforts to decentralize political systems throughout africa . however , roles and responsibilities over phc programs , facilities , human resources , and financing remained unclear - a mishmash of centralization and decentralization . although the federal level is principally responsible for tertiary health care , the national primary health care development agency ( nphcda ) and national programme on immunization were developed at the federal level to maintain some control over phc . for example , the nphcda is intended to ( and does ) carry out supplemental immunization campaigns against vaccine preventable diseases including polio . state ministries of health are also involved in the building and managing of phc centers although , according to the national health policy , ostensibly responsible for managing secondary health care hospitals . the states provide health care services ( including phc services ) through the state ministry of health ( smoh ) , the state hospital management board ( shmb ) , and the state phc development agency ( sphcda ) . several bodies are involved in human resource management at the lga level for phc services : the ministry of local government ( molg ) , the state phc agency , the local government service commission , the public service commission and the lga . the local government service commission is a state level organization that is responsible for all health professionals ( level 7 and above ) while the lgas managed admin , security , and lower level personnel . budget development and release is similarly fragmented . while finance was nominally devolved in block grants to both states and lgas , state governments established joint accounts in which state and lga financial resources were placed . adding to the confusion over financial control , external donors usually direct programmatic funding into federal - level accounts , although ultimately implementation should occur at the local level . for example , large sums of money provided by the millennium development goal ( mdg ) office are given to the federal ministry of health to implement the community - based national health insurance scheme ( nhis ) scheme . the diversification of responsible units at the federal and state levels exacerbates the confusion of responsibilities between tiers . although phc is the responsibility of local governments , the multitude of different agents actually involved in phc services makes coordinated management across the ministry of health technically difficult . thus , several states - starting with enugu and jigawa , and then yobe and zamfara - have been exploring strategies for bringing phc under one roof . this first occurred through the partnership to transform health systems ( paths ) programme from 2002 to 2008 , and then through the prrinn - mnch programme , which began in 2006 . the possibility of the funding that would flow from the federal health act drove policy and legislative changes regarding phc in several states in nigeria . although implementation varies across states , relevant policy and legislative changes generally address three key issues . firstly , health services - particularly phc services - are being integrated , where previously all three tiers of government were involved in implementation . secondly , health services are being decentralized - both through devolution and de - concentration . and thirdly , but not uniformly , through the de - concentration to sub - state bodies ( the names are different in the different states ) , the balance of power in the management of key resources ( especially financial and human resources ) is shifting from the politicians to administrators and managers . it is important to highlight the process adopted through these adjustments and how the outcome of integrated health services ( at least of phc ) was achieved . since both federal government and states can pass legislation , both the paths and the prrinn - mnch programmes worked closely with the states to develop appropriate legislation and accompanying regulations for phc ; to strengthen systems ( especially financial , human resource and information ) ; and to reposition the state level bodies for their new roles and functions . this process was not without challenges and extended over substantial periods of time . at the same time , the states ( with support from the programmes ) lobbied at federal level to gain acceptance and to provide access to materials and support for states willing to embark on this process . draft policy memos and an implementation guide were finalized during the second workshop , were approved by the nphcda board in 2010 and submitted to the highest health policy body in nigeria ( the national council for health ) . as summarized in the official record of the council : council noted the thrust of the national health bill in strengthening primary health care ( phc ) through the creation of phc boards / agencies and the phc development fund . council noted efforts in bringing phc under one roof in line with the provisions of the national health bill . council also noted the importance of enacting relevant state legislation and regulations that will facilitate the implementation of national health bill . council therefore approved the implementation guide on bringing phc under one roof ( phcuor ) as a working document to be used by the three tiers of government and approved that all states establish primary health care boards . the national council for health not only adopted the policy document and implementation guide on bringing phc under one roof but encouraged the 36 states to proceed in implementing this concept ( table 1 ) . at the time of the 2012 workshop , 22 of the 36 states in nigeria were in various stages of implementing bringing phc under one roof . gavi has supported nigeria s immunization system since 2001 to address extremely poor immunization coverage , particularly in the northern states ( http://www.gavialliance.org/country/nigeria/ ) . states were able to access the first tranche of the gavi funds , which were managed at the federal level , following initial engagement with gavi , but several states were unable to retire the monies appropriately . thus in 2007 , prrinn - mnch began at the state level supporting state ministries of health to effectively retire and access ongoing funding tranches from gavi . this work shifted to the federal level where prrinn - mnch assisted the gavi office in nphcda to review the processes and tools for accessing and retiring the funds and participated in developing a set of prrinn - mnch supported nphcda to develop financial guidelines for gavi fund management and provided appropriate training on the use of these guidelines for relevant staff at agency headquarters and in its programme states . the nphcda also requested that prrinn - mnch train focal nphcda staff on the use of the financial guidelines and tools in the non - programme states . the subsequent workshop enabled the participants to understand the use of the financial guidelines and equipped them with the capacity to train other relevant staff at the lower levels . the twenty - two participants comparised six senior staff from nphcda headquarters , two representatives of the nphcda offices from each of the six geo - political zones of the federation , and gavi fund accountants from the four prrinn - mnch focal states . this example illustrates how state level experience of an international agency s funds was used to drive changes in how the funds were administered and accessed at federal level . initial work started within the states to ensure that the proposed system would work , and had significant impact on coverage ( figure 1 ) . later this was taken up to federal level where capacity was built to maintain and nationally roll out the new system . nigeria has a high maternal mortality ratio , low attendance for antenatal services , and few births are attended by a skilled birth attendant . thus , to improve access to care for pregnant women , the mss was a federally conceived and driven scheme to place retired and unemployed midwives in health facilities ; later this was extended to community health extension workers ( chews ) to ensure remote communities have access to perinatal services . the nphcda initiated the mss using funds from mdg fund . in each state , four midwives were deployed to each of the selected phc facilities to ensure provision of maternal and child health care services on a 24/7 basis . in each state , a selection of four phc facilities is clustered around the referral general hospital , with the creation of 156 clusters nationwide . six of these clusters are in three prrinn - mnch target states ( katsina , yobe and zamfara ) . this was followed by a second wave ( both midwives and chews ) in late 2010 . specific areas of collaboration between prrinn - mnch and nphcda under the banner of mss have included : i ) participation in the recruitment of midwives for the north - west zone in nigeria ; ii ) sharing of relevant tools for health facility baseline assessment , participatory appraisal and continuous transformation ( including impact , a systems strengthening and management capacity building initiative developed by health partners international : http://www.healthpartners-int.co.uk/ ; http://www.prrinn-mnch.org/index.html ) and assessment of training institutions ; iii ) sharing experience on planning and conducting baseline assessments , development of a monitoring and evaluation framework , and the comprehensive emergency obstetric care ( ceoc ) cluster model used ; iv ) participation in the planning of refresher training of midwives countrywide by nphcda - mss ; v ) developing national integrated supportive supervision ( iss ) tools ( the iss tools have been institutionalized and the iss teams oriented nationwide ) ; vi ) providing in - service training to mss midwives in the three states based on training needs earlier identified during an induction workshop . while there are multiple interventions in the clusters and the states , the increased presence of midwives ( and chews ) is considered a major contribution to increased utilization of mnh services in all the prrinn - mnch supported facilities where mss midwives are deployed ( as compared to baseline data collected between august and october 2008 ; figure 2 ) . overall , more significant increases were noted in anc attendance when compared with intrapartum care ( delivery ) . for example , in furfuri phc , zamfara , the number of anc visits increased from 0 to 455 , and deliveries from 0 to 28 between april and june , 2010 . mss midwives initiated community mobilization activities to increase utilization of mnh services , especially skilled care at delivery . mss has also shown improvement in documentation , including the keeping of registers and summary graphs and statistics in all facilities ( and bar charts in some facilities ) . additionally , all prrinn - mnch supported health facilities where there are mss midwives ( at least 3 - 4 mss midwives per facility ) now provide 24-h intra - partum care . for instance , a night shift was introduced by mss midwives on arrival at baimari maternity phc , yobe . problems experienced by the midwives continue to be largely administrative and systems related : e.g. payment issues , accommodation , poor working environment etc . although there has been progress in most of these areas , they remain critical issues to monitor in support of retention of these important health workers . this third example illustrates how state level practitioners can assist in supporting , and adding value to , nationally conceived and driven plans . in this case the nphcda had no formal jurisdiction but clearly needed support to implement a laudable scheme with promising results . the bamako initiative of 1987 supported strengthening health systems at the district and community - levels in parallel with efforts to decentralize political systems throughout africa . however , roles and responsibilities over phc programs , facilities , human resources , and financing remained unclear - a mishmash of centralization and decentralization . although the federal level is principally responsible for tertiary health care , the national primary health care development agency ( nphcda ) and national programme on immunization were developed at the federal level to maintain some control over phc . for example , the nphcda is intended to ( and does ) carry out supplemental immunization campaigns against vaccine preventable diseases including polio . state ministries of health are also involved in the building and managing of phc centers although , according to the national health policy , ostensibly responsible for managing secondary health care hospitals . the states provide health care services ( including phc services ) through the state ministry of health ( smoh ) , the state hospital management board ( shmb ) , and the state phc development agency ( sphcda ) . several bodies are involved in human resource management at the lga level for phc services : the ministry of local government ( molg ) , the state phc agency , the local government service commission , the public service commission and the lga . the local government service commission is a state level organization that is responsible for all health professionals ( level 7 and above ) while the lgas managed admin , security , and lower level personnel . budget development and release is similarly fragmented . while finance was nominally devolved in block grants to both states and lgas , state governments established joint accounts in which state and lga financial resources were placed . adding to the confusion over financial control , external donors usually direct programmatic funding into federal - level accounts , although ultimately implementation should occur at the local level . for example , large sums of money provided by the millennium development goal ( mdg ) office are given to the federal ministry of health to implement the community - based national health insurance scheme ( nhis ) scheme . the diversification of responsible units at the federal and state levels exacerbates the confusion of responsibilities between tiers . although phc is the responsibility of local governments , the multitude of different agents actually involved in phc services makes coordinated management across the ministry of health technically difficult . thus , several states - starting with enugu and jigawa , and then yobe and zamfara - have been exploring strategies for bringing phc under one roof . this first occurred through the partnership to transform health systems ( paths ) programme from 2002 to 2008 , and then through the prrinn - mnch programme , which began in 2006 . the possibility of the funding that would flow from the federal health act drove policy and legislative changes regarding phc in several states in nigeria . although implementation varies across states , relevant policy and legislative changes generally address three key issues . firstly , health services - particularly phc services - are being integrated , where previously all three tiers of government were involved in implementation . secondly , health services are being decentralized - both through devolution and de - concentration . and thirdly , but not uniformly , through the de - concentration to sub - state bodies ( the names are different in the different states ) , the balance of power in the management of key resources ( especially financial and human resources ) is shifting from the politicians to administrators and managers . it is important to highlight the process adopted through these adjustments and how the outcome of integrated health services ( at least of phc ) was achieved . since both federal government and states can pass legislation , both the paths and the prrinn - mnch programmes worked closely with the states to develop appropriate legislation and accompanying regulations for phc ; to strengthen systems ( especially financial , human resource and information ) ; and to reposition the state level bodies for their new roles and functions . this process was not without challenges and extended over substantial periods of time . at the same time , the states ( with support from the programmes ) lobbied at federal level to gain acceptance and to provide access to materials and support for states willing to embark on this process . draft policy memos and an implementation guide were finalized during the second workshop , were approved by the nphcda board in 2010 and submitted to the highest health policy body in nigeria ( the national council for health ) . as summarized in the official record of the council : council noted the thrust of the national health bill in strengthening primary health care ( phc ) through the creation of phc boards / agencies and the phc development fund . council noted efforts in bringing phc under one roof in line with the provisions of the national health bill . council also noted the importance of enacting relevant state legislation and regulations that will facilitate the implementation of national health bill . council therefore approved the implementation guide on bringing phc under one roof ( phcuor ) as a working document to be used by the three tiers of government and approved that all states establish primary health care boards . the national council for health not only adopted the policy document and implementation guide on bringing phc under one roof but encouraged the 36 states to proceed in implementing this concept ( table 1 ) . at the time of the 2012 workshop , 22 of the 36 states in nigeria were in various stages of implementing bringing phc under one roof . gavi has supported nigeria s immunization system since 2001 to address extremely poor immunization coverage , particularly in the northern states ( http://www.gavialliance.org/country/nigeria/ ) . states were able to access the first tranche of the gavi funds , which were managed at the federal level , following initial engagement with gavi , but several states were unable to retire the monies appropriately . thus in 2007 , prrinn - mnch began at the state level supporting state ministries of health to effectively retire and access ongoing funding tranches from gavi . this work shifted to the federal level where prrinn - mnch assisted the gavi office in nphcda to review the processes and tools for accessing and retiring the funds and participated in developing a set of master trainers who would train others across all states in nigeria . in 2009 , prrinn - mnch supported nphcda to develop financial guidelines for gavi fund management and provided appropriate training on the use of these guidelines for relevant staff at agency headquarters and in its programme states . the nphcda also requested that prrinn - mnch train focal nphcda staff on the use of the financial guidelines and tools in the non - programme states . the subsequent workshop enabled the participants to understand the use of the financial guidelines and equipped them with the capacity to train other relevant staff at the lower levels . the twenty - two participants comparised six senior staff from nphcda headquarters , two representatives of the nphcda offices from each of the six geo - political zones of the federation , and gavi fund accountants from the four prrinn - mnch focal states . this example illustrates how state level experience of an international agency s funds was used to drive changes in how the funds were administered and accessed at federal level . initial work started within the states to ensure that the proposed system would work , and had significant impact on coverage ( figure 1 ) . later this was taken up to federal level where capacity was built to maintain and nationally roll out the new system . nigeria has a high maternal mortality ratio , low attendance for antenatal services , and few births are attended by a skilled birth attendant . thus , to improve access to care for pregnant women , the mss was a federally conceived and driven scheme to place retired and unemployed midwives in health facilities ; later this was extended to community health extension workers ( chews ) to ensure remote communities have access to perinatal services . the nphcda initiated the mss using funds from mdg fund . in each state , four midwives were deployed to each of the selected phc facilities to ensure provision of maternal and child health care services on a 24/7 basis . in each state , a selection of four phc facilities is clustered around the referral general hospital , with the creation of 156 clusters nationwide . six of these clusters are in three prrinn - mnch target states ( katsina , yobe and zamfara ) . this was followed by a second wave ( both midwives and chews ) in late 2010 . specific areas of collaboration between prrinn - mnch and nphcda under the banner of mss have included : i ) participation in the recruitment of midwives for the north - west zone in nigeria ; ii ) sharing of relevant tools for health facility baseline assessment , participatory appraisal and continuous transformation ( including impact , a systems strengthening and management capacity building initiative developed by health partners international : http://www.healthpartners-int.co.uk/ ; http://www.prrinn-mnch.org/index.html ) and assessment of training institutions ; iii ) sharing experience on planning and conducting baseline assessments , development of a monitoring and evaluation framework , and the comprehensive emergency obstetric care ( ceoc ) cluster model used ; iv ) participation in the planning of refresher training of midwives countrywide by nphcda - mss ; v ) developing national integrated supportive supervision ( iss ) tools ( the iss tools have been institutionalized and the iss teams oriented nationwide ) ; vi ) providing in - service training to mss midwives in the three states based on training needs earlier identified during an induction workshop . while there are multiple interventions in the clusters and the states , the increased presence of midwives ( and chews ) is considered a major contribution to increased utilization of mnh services in all the prrinn - mnch supported facilities where mss midwives are deployed ( as compared to baseline data collected between august and october 2008 ; figure 2 ) . overall , more significant increases were noted in anc attendance when compared with intrapartum care ( delivery ) . for example , in furfuri phc , zamfara , the number of anc visits increased from 0 to 455 , and deliveries from 0 to 28 between april and june , 2010 . mss midwives initiated community mobilization activities to increase utilization of mnh services , especially skilled care at delivery . mss has also shown improvement in documentation , including the keeping of registers and summary graphs and statistics in all facilities ( and bar charts in some facilities ) . additionally , all prrinn - mnch supported health facilities where there are mss midwives ( at least 3 - 4 mss midwives per facility ) now provide 24-h intra - partum care . for instance , a night shift was introduced by mss midwives on arrival at baimari maternity phc , yobe . problems experienced by the midwives continue to be largely administrative and systems related : e.g. payment issues , accommodation , poor working environment etc . although there has been progress in most of these areas , they remain critical issues to monitor in support of retention of these important health workers . this third example illustrates how state level practitioners can assist in supporting , and adding value to , nationally conceived and driven plans . in this case the nphcda had no formal jurisdiction but clearly needed support to implement a laudable scheme with promising results . informed by the whole systems approach of complexity theory , both the paths and prrinn - mnch programmes were either conceived or morphed into broad health systems strengthening programmes , and engaged non - health ministries , departments , and agencies . a key component of complexity theory is allowing - and often encouraging - variation in the system . this is a result of the path dependence principle noted earlier where , from similar starting points , different outcomes can emerge based on different contexts and different decisions at key bifurcation points . thus , faced by similar challenges , some states adopted an integrated phc system ( e.g. yobe and zamfara ) while other adopted an integrated phc and secondary health care system ( e.g. enugu and jigawa ) . this was a combination of history and context ( e.g. jigawa had no state hospital management board ) and of different choices at key moments ( usually by the state governor ) . in one example , key legislative changes in the gunduma or district system in jigawa meant that the previously fragmented management of financial and human resources was transferred to the gunduma health services board from state and lgas . the outcome was that administrative management and procedures were strengthened at the expense of political power and control . improvements in jigawa , as demonstrated by the 2010 national immunization cluster survey data , led to other states exploring variations of the gunduma approach . this illustrates to further features of complex systems : the importance of positive feedback loops ( as initially there was widespread scepticism of the changes in jigawa ) and the influence of phase transitions where a critical tipping point is reached to initiate change . in the second strategy , states encouraged coordination with federal agencies in order to strengthen the system involved in accessing , spending and retiring gavi funds . the whole systems principle meant that all had to be addressed . improved state - level gavi systems led to increased funding in the northern states , which led to the nphcda requesting assistance to strengthen the federal level gavi systems . this illustrates the non - linearity principle . once the federal level nphcda was made aware of the challenges at state level in accessing gavi funds and the changes brought by strengthening state level processes , the nphcda was more amenable to changing processes for accessing the gavi funds . increased frequency of accessing , spending and retiring gavi funds from prrinn - mnch supported states led to nphcda requesting assistance in rolling out the methodology to other states in addition , in one state ( jigawa ) the positive experience with the gavi funds led to the state applying the same methodology to other funds . finally , the tipping point was the realization by the federal level gavi office that states that previously could not access the funds could now readily be assisted to comply with the guidelines . mss also involved both the federal and state levels of government , except here coordination was initiated from the top - down . the federal level nphcda realized that it needed to look creatively for options to redress the problem of poor maternal health indices , hence supporting retired and unemployed midwives to find jobs in underserved states . the interaction between the nphcda planners and the prrinn - mnch has identified tools and approaches that can be shared and experiences that can be built on to improve the mss . each of these examples thus illustrated the influences on services and provision of multiple drivers , some within the health system and some outside of it . in advance , it would have been hard to anticipate all the factors that would prove influential on specific outcomes . rather , a sensitivity to the potential for such influences in real time policy and implementation work served to highlight emerging opportunities - and barriers - and develop appropriate strategies with respect to them . we consider that understanding and utilizing complexity theory and the complex adaptive systems approach has had a profound impact on how to strategize regarding health systems development in northern nigeria . it has introduced flexibility in the implementation of key health systems strengthening initiatives that stands to significantly benefit health care services and improve longer - term health outcomes in this challenging environment . such application of systems thinking is emerging as an important approach to tackling recurrent challenges in health systems development , on issues ranging from disease control programme sustainability and integration and the management of user fees to the resilience of health systems in times of civic crisis .

the partnership for reviving routine immunization in northern nigeria - maternal , newborn and child health initiative supports efforts by the government of nigeria to bridge primary health care ( phc ) policies and services at three levels of government : federal , state and local . the paper suggests that understandings informed by complexity theory and complex adaptive systems have been helpful in shaping policy and programme design across these levels . to illustrate this , three initiatives are explored : bringing phc under one roof , enhancing access to funding provided by the global alliance for vaccines and immunization , and strengthening the midwives service scheme . these initiatives have demonstrated how concepts and experience developed at subnational level can influence national policy and practice , and how work at subnational levels can add value to nationally conceived and nationally driven plans for phc .

Introduction The Partnership for Reviving Routine Immunization in Northern Nigeria - Maternal, Newborn and Child Health Materials and Methods Analysis Bringing primary health care under one roof Enhngaccess to Global Alliance for Vaccines and Immunization funds Strngthening the midwives service scheme Results and Discussion

the 1978 alma ata declaration codified the right to primary health care ( phc ) for all . nine years later , african ministers of health convened in bamako , mali , to emphasize the need to strengthen phc on the continent , with a particular focus on maternal and child health . the alma ata declaration acknowledged the impact of good governance , economic and social development , inequality , health system functioning , inter - sector cohesiveness , and education on health outcomes . health inequities , pervasive corruption , and the autonomy of nigeria s 36 states have prevented the country from establishing a national framework to support a phc system that works for all . in the nigerian constitution , health is a concomitant responsibility of the three tiers of government : the federal level , 37 states including the federal capital territory , and 774 lgas . according to the national health policy , the federal level is responsible for tertiary care , the state level for secondary care and the lga level for phc . crucially , the way in which the three levels of government should interact has not been elaborated in policy . a systematic survey across three northern nigerian states found that less than one - quarter of women who gave birth in the five years preceding the survey had ever received antenatal care ( anc ) from a trained health professional , and only one in eight of those women had delivered in a health facility . bringing together as a combined programme two such initiatives - the partnership for reviving routine immunization in northern nigeria ( prrinn ) and the maternal , newborn and child health ( mnch ) programme - provided the opportunity to specifically address some of the wider systems issues that constrain health programming in nigeria . prrinn - mnch - with cofunding from the department for international development of the united kingdom ( dfid ) and the government of norway - has since 2007 provided a platform to specifically address the systems linkages required between federal , states and local structures to implement policy and service changes . the prrinn - mnch initiative was developed as a programme that sought to build upon the strategies outlined in the bamako initiative by explicitly addressing the broken linkages between phc services at all levels of government . by facilitating better partnerships , the prrinnmnch program supported the government s effort to integrate phc services and strengthen its implementation at the district level , support child immunizations through improved funding mechanisms , and ensure maternal health is addressed at the community level . this paper focuses upon three prrinnmnch supported initiatives that sought to improve linkages between , and the functioning of , the federal , state , and lga levels of health system responsible for phc : bringing phc under one roof ; enhancing access to global alliance for vaccines and immunization ( gavi ) funds ; and strengthening the midwives service scheme ( mss ) . these three examples provide an opportunity to explore how concepts and experience developed at subnational levels were utilized to influence national policy and practice , and the way in which work at subnational levels can contribute to nationally conceived and driven plans for phc . the prrinn - mnch initiative was developed as a programme that sought to build upon the strategies outlined in the bamako initiative by explicitly addressing the broken linkages between phc services at all levels of government . by facilitating better partnerships , the prrinnmnch program supported the government s effort to integrate phc services and strengthen its implementation at the district level , support child immunizations through improved funding mechanisms , and ensure maternal health is addressed at the community level . this paper focuses upon three prrinnmnch supported initiatives that sought to improve linkages between , and the functioning of , the federal , state , and lga levels of health system responsible for phc : bringing phc under one roof ; enhancing access to global alliance for vaccines and immunization ( gavi ) funds ; and strengthening the midwives service scheme ( mss ) . these three examples provide an opportunity to explore how concepts and experience developed at subnational levels were utilized to influence national policy and practice , and the way in which work at subnational levels can contribute to nationally conceived and driven plans for phc . this paper analyses the development of policy and programme implementation in these three areas by drawing upon documents from the federal ministry of health in nigeria , prrinn - mnch reports , and discussions - both informal and formal - at a range of national workshops and programme planning meetings . complexity theory provides a way of understanding health systems as complex adaptive systems , and has been increasingly advocated as a tool for health policy development and health systems reform . with complexity theory , health systems are seen as open systems in which different components are interdependent and influence each other in a non - linear fashion . for example , the world health organization s report systems thinking for health system strengthening , heavily influenced by the ideas of complexity theory , acknowledges non - linearity and interdependence in a proposed framework for health system strengthening . the complex adaptive systems approach reinforces concepts such as feedback loops ( both positive and negative , that influence the pace and direction of change ) ; path dependence ( processes that have similar starting points can have very dissimilar outcomes resulting from different contexts and histories and different choices ) ; scale - free networks ( incorporating focal points - including key powerful people - that can dominate a structure ) ; and phase transitions ( when critical - tipping - points are reached and initiate change ) . both complexity theory and the doc approach to political economy any new policy development needs to be understood in the context within which the potential change will be located . only then , and in an ongoing fashion as the context and the whole system is dynamic , can policy be developed and implemented . although the federal level is principally responsible for tertiary health care , the national primary health care development agency ( nphcda ) and national programme on immunization were developed at the federal level to maintain some control over phc . state ministries of health are also involved in the building and managing of phc centers although , according to the national health policy , ostensibly responsible for managing secondary health care hospitals . the states provide health care services ( including phc services ) through the state ministry of health ( smoh ) , the state hospital management board ( shmb ) , and the state phc development agency ( sphcda ) . several bodies are involved in human resource management at the lga level for phc services : the ministry of local government ( molg ) , the state phc agency , the local government service commission , the public service commission and the lga . while finance was nominally devolved in block grants to both states and lgas , state governments established joint accounts in which state and lga financial resources were placed . for example , large sums of money provided by the millennium development goal ( mdg ) office are given to the federal ministry of health to implement the community - based national health insurance scheme ( nhis ) scheme . thus , several states - starting with enugu and jigawa , and then yobe and zamfara - have been exploring strategies for bringing phc under one roof . this first occurred through the partnership to transform health systems ( paths ) programme from 2002 to 2008 , and then through the prrinn - mnch programme , which began in 2006 . it is important to highlight the process adopted through these adjustments and how the outcome of integrated health services ( at least of phc ) was achieved . as summarized in the official record of the council : council noted the thrust of the national health bill in strengthening primary health care ( phc ) through the creation of phc boards / agencies and the phc development fund . council noted efforts in bringing phc under one roof in line with the provisions of the national health bill . council therefore approved the implementation guide on bringing phc under one roof ( phcuor ) as a working document to be used by the three tiers of government and approved that all states establish primary health care boards . the national council for health not only adopted the policy document and implementation guide on bringing phc under one roof but encouraged the 36 states to proceed in implementing this concept ( table 1 ) . at the time of the 2012 workshop , 22 of the 36 states in nigeria were in various stages of implementing bringing phc under one roof . the twenty - two participants comparised six senior staff from nphcda headquarters , two representatives of the nphcda offices from each of the six geo - political zones of the federation , and gavi fund accountants from the four prrinn - mnch focal states . thus , to improve access to care for pregnant women , the mss was a federally conceived and driven scheme to place retired and unemployed midwives in health facilities ; later this was extended to community health extension workers ( chews ) to ensure remote communities have access to perinatal services . in each state , four midwives were deployed to each of the selected phc facilities to ensure provision of maternal and child health care services on a 24/7 basis . specific areas of collaboration between prrinn - mnch and nphcda under the banner of mss have included : i ) participation in the recruitment of midwives for the north - west zone in nigeria ; ii ) sharing of relevant tools for health facility baseline assessment , participatory appraisal and continuous transformation ( including impact , a systems strengthening and management capacity building initiative developed by health partners international : http://www.healthpartners-int.co.uk/ ; http://www.prrinn-mnch.org/index.html ) and assessment of training institutions ; iii ) sharing experience on planning and conducting baseline assessments , development of a monitoring and evaluation framework , and the comprehensive emergency obstetric care ( ceoc ) cluster model used ; iv ) participation in the planning of refresher training of midwives countrywide by nphcda - mss ; v ) developing national integrated supportive supervision ( iss ) tools ( the iss tools have been institutionalized and the iss teams oriented nationwide ) ; vi ) providing in - service training to mss midwives in the three states based on training needs earlier identified during an induction workshop . problems experienced by the midwives continue to be largely administrative and systems related : e.g. this third example illustrates how state level practitioners can assist in supporting , and adding value to , nationally conceived and driven plans . although the federal level is principally responsible for tertiary health care , the national primary health care development agency ( nphcda ) and national programme on immunization were developed at the federal level to maintain some control over phc . the states provide health care services ( including phc services ) through the state ministry of health ( smoh ) , the state hospital management board ( shmb ) , and the state phc development agency ( sphcda ) . several bodies are involved in human resource management at the lga level for phc services : the ministry of local government ( molg ) , the state phc agency , the local government service commission , the public service commission and the lga . while finance was nominally devolved in block grants to both states and lgas , state governments established joint accounts in which state and lga financial resources were placed . for example , large sums of money provided by the millennium development goal ( mdg ) office are given to the federal ministry of health to implement the community - based national health insurance scheme ( nhis ) scheme . thus , several states - starting with enugu and jigawa , and then yobe and zamfara - have been exploring strategies for bringing phc under one roof . this first occurred through the partnership to transform health systems ( paths ) programme from 2002 to 2008 , and then through the prrinn - mnch programme , which began in 2006 . although implementation varies across states , relevant policy and legislative changes generally address three key issues . it is important to highlight the process adopted through these adjustments and how the outcome of integrated health services ( at least of phc ) was achieved . at the same time , the states ( with support from the programmes ) lobbied at federal level to gain acceptance and to provide access to materials and support for states willing to embark on this process . as summarized in the official record of the council : council noted the thrust of the national health bill in strengthening primary health care ( phc ) through the creation of phc boards / agencies and the phc development fund . council noted efforts in bringing phc under one roof in line with the provisions of the national health bill . council therefore approved the implementation guide on bringing phc under one roof ( phcuor ) as a working document to be used by the three tiers of government and approved that all states establish primary health care boards . the national council for health not only adopted the policy document and implementation guide on bringing phc under one roof but encouraged the 36 states to proceed in implementing this concept ( table 1 ) . at the time of the 2012 workshop , 22 of the 36 states in nigeria were in various stages of implementing bringing phc under one roof . initial work started within the states to ensure that the proposed system would work , and had significant impact on coverage ( figure 1 ) . thus , to improve access to care for pregnant women , the mss was a federally conceived and driven scheme to place retired and unemployed midwives in health facilities ; later this was extended to community health extension workers ( chews ) to ensure remote communities have access to perinatal services . in each state , four midwives were deployed to each of the selected phc facilities to ensure provision of maternal and child health care services on a 24/7 basis . specific areas of collaboration between prrinn - mnch and nphcda under the banner of mss have included : i ) participation in the recruitment of midwives for the north - west zone in nigeria ; ii ) sharing of relevant tools for health facility baseline assessment , participatory appraisal and continuous transformation ( including impact , a systems strengthening and management capacity building initiative developed by health partners international : http://www.healthpartners-int.co.uk/ ; http://www.prrinn-mnch.org/index.html ) and assessment of training institutions ; iii ) sharing experience on planning and conducting baseline assessments , development of a monitoring and evaluation framework , and the comprehensive emergency obstetric care ( ceoc ) cluster model used ; iv ) participation in the planning of refresher training of midwives countrywide by nphcda - mss ; v ) developing national integrated supportive supervision ( iss ) tools ( the iss tools have been institutionalized and the iss teams oriented nationwide ) ; vi ) providing in - service training to mss midwives in the three states based on training needs earlier identified during an induction workshop . problems experienced by the midwives continue to be largely administrative and systems related : e.g. this third example illustrates how state level practitioners can assist in supporting , and adding value to , nationally conceived and driven plans . although the federal level is principally responsible for tertiary health care , the national primary health care development agency ( nphcda ) and national programme on immunization were developed at the federal level to maintain some control over phc . the states provide health care services ( including phc services ) through the state ministry of health ( smoh ) , the state hospital management board ( shmb ) , and the state phc development agency ( sphcda ) . while finance was nominally devolved in block grants to both states and lgas , state governments established joint accounts in which state and lga financial resources were placed . for example , large sums of money provided by the millennium development goal ( mdg ) office are given to the federal ministry of health to implement the community - based national health insurance scheme ( nhis ) scheme . thus , several states - starting with enugu and jigawa , and then yobe and zamfara - have been exploring strategies for bringing phc under one roof . this first occurred through the partnership to transform health systems ( paths ) programme from 2002 to 2008 , and then through the prrinn - mnch programme , which began in 2006 . it is important to highlight the process adopted through these adjustments and how the outcome of integrated health services ( at least of phc ) was achieved . draft policy memos and an implementation guide were finalized during the second workshop , were approved by the nphcda board in 2010 and submitted to the highest health policy body in nigeria ( the national council for health ) . as summarized in the official record of the council : council noted the thrust of the national health bill in strengthening primary health care ( phc ) through the creation of phc boards / agencies and the phc development fund . council noted efforts in bringing phc under one roof in line with the provisions of the national health bill . council therefore approved the implementation guide on bringing phc under one roof ( phcuor ) as a working document to be used by the three tiers of government and approved that all states establish primary health care boards . the national council for health not only adopted the policy document and implementation guide on bringing phc under one roof but encouraged the 36 states to proceed in implementing this concept ( table 1 ) . at the time of the 2012 workshop , 22 of the 36 states in nigeria were in various stages of implementing bringing phc under one roof . later this was taken up to federal level where capacity was built to maintain and nationally roll out the new system . thus , to improve access to care for pregnant women , the mss was a federally conceived and driven scheme to place retired and unemployed midwives in health facilities ; later this was extended to community health extension workers ( chews ) to ensure remote communities have access to perinatal services . in each state , four midwives were deployed to each of the selected phc facilities to ensure provision of maternal and child health care services on a 24/7 basis . specific areas of collaboration between prrinn - mnch and nphcda under the banner of mss have included : i ) participation in the recruitment of midwives for the north - west zone in nigeria ; ii ) sharing of relevant tools for health facility baseline assessment , participatory appraisal and continuous transformation ( including impact , a systems strengthening and management capacity building initiative developed by health partners international : http://www.healthpartners-int.co.uk/ ; http://www.prrinn-mnch.org/index.html ) and assessment of training institutions ; iii ) sharing experience on planning and conducting baseline assessments , development of a monitoring and evaluation framework , and the comprehensive emergency obstetric care ( ceoc ) cluster model used ; iv ) participation in the planning of refresher training of midwives countrywide by nphcda - mss ; v ) developing national integrated supportive supervision ( iss ) tools ( the iss tools have been institutionalized and the iss teams oriented nationwide ) ; vi ) providing in - service training to mss midwives in the three states based on training needs earlier identified during an induction workshop . problems experienced by the midwives continue to be largely administrative and systems related : e.g. this third example illustrates how state level practitioners can assist in supporting , and adding value to , nationally conceived and driven plans . informed by the whole systems approach of complexity theory , both the paths and prrinn - mnch programmes were either conceived or morphed into broad health systems strengthening programmes , and engaged non - health ministries , departments , and agencies . yobe and zamfara ) while other adopted an integrated phc and secondary health care system ( e.g. in one example , key legislative changes in the gunduma or district system in jigawa meant that the previously fragmented management of financial and human resources was transferred to the gunduma health services board from state and lgas . mss also involved both the federal and state levels of government , except here coordination was initiated from the top - down . we consider that understanding and utilizing complexity theory and the complex adaptive systems approach has had a profound impact on how to strategize regarding health systems development in northern nigeria .

aida v4 ( accessible freely at http://www.2aida.org ) is a downloadable program that permits the interactive simulation of plasma insulin and blood glucose ( bg ) profiles for teaching / demonstration / self - learning / research purposes . the software incorporates a compartmental / physiological model describing glucose - insulin interaction in insulin - dependent diabetic patients ( lacking endogenous insulin secretion ) . the graphical interface of the downloadable software allows nonspecialist users to interact with the model . aida v4 permits the effects of insulin dosage and dietary adjustments to be simulated for a typical patient 's bg profile , with the working hypothesis being that patients , relatives , students and health - care professionals ( hcps ) should be able to experience metabolic adjustments without risk of hypoglycaemia . aida v4 also incorporates a knowledge - based system that can suggest changes in insulin dose for users unsure about what to simulate next [ 2 , 3 ] . although a range of other interactive simulation programs of glucose - insulin interaction in diabetes have been described in the literature [ 412 ] , to date , most of these do not seem to have been distributed so widely via the internet or been made particularly widely available . indeed , in a number of cases , it would seem that readers are entirely dependent on the authors ' own descriptions of their prototypes in research articles , since no versions appear to be available for general use by others [ 5 , 7 , 8 , 12 ] . by contrast , with aida , multiple updates and versions of the software have been freely available on the web since 1996 , and before that made available to researchers on diskette [ 1315 ] . this has led to a substantial experience with the software worldwide , with over 426,000 downloads of the program taking place since the program 's original internet launch . the burgeoning popularity of the world wide web means that more people are surfing the net than ever before . this surge in popularity has led people to increasingly consult web sites as sources of bona fide medical information . although there is currently a vast amount of educational material that is available on the internet , it is helpful to have interactive resources which engage students in the learning process . in this respect , it has been found that students learn best through active learning and discovery rather than by simply listening or reading [ 16 , 17 ] . interactive multimedia has changed the teacher / student relationship from one of the sage on the stage to the guide on the side and provides tools for students to collaborate together on projects or to work independently at their own pace and for teachers to present new types of materials [ 1825 ] . in days gone by most computers networks connected dumb terminals with the central mainframe . with the development of personal computers ( pcs ) , the pendulum swung towards distributed computing with significant processing taking place locally on individual 's desktop / notebook pcs . with the massive expansion of the internet , nowadays , a range of applications can operate across the internet , from distant servers . while most internet users do not have dumb terminals - there is a move back to using less and less complex devices for accessing the internet ( e.g. , mobile phones / smartphones , webtv , etc . ) . it is interesting how things seem to have moved almost full circle . with the recent move towards cloud computing , the interest has focused less on a single central server and more on distributed services that can be hosted on a range of servers , with more resources ( bandwidth , central processing units [ cpus ] , memory , and disk space ) commissioned as usage requires . using aida v4 as an example , work has been done to show how it is possible to move from purely static , informational resources on the web to using the internet to provide more interactive and dynamic information about clinically relevant situations in diabetes care . the purpose of the aida online diabetes simulator has not been to provide individual patient bg prediction or medical advice or therapy planning but instead to provide an interactive educational web - based tool to help people with diabetes and their relatives / carers , as well as hcps and students , to better understand how meal ( carbohydrate ) and insulin interactions can affect bg levels ; that is the intended role for self - learning / teaching / demonstration / education purposes . while the downloadable aida software has been widely applied , there may be a number of practical and theoretical limitations to the standalone aida pc software approach . ( i ) people who wish to make use of the program need to download an archive file and then install it on their hard disk . ( ii ) some people might be concerned about the theoretical risk of receiving some sort of virus when downloading an executable file from the internet ( notwithstanding the fact that the aida pc software has repeatedly been shown to be virus free ) . ( iii ) the software requires an ibm - compatible dos / windows pc ( or an apple mac running softwindows or virtual pc emulators or a powerpc macintosh ) . people without access to one of these computers or operating systems can not run the standalone aida v4 downloadable software . ( iv ) the availability of enhancements or upgrades to the pc software can take time to reach end users , since further downloads and local installation are required to obtain the latest version / release . this issue could be addressed with an autoupdate facility , but such functionality is currently not yet available within aida v4 . ( v ) the downloadable software , being dos based , has a user interface that is not totally adherent to the principles of a windows graphical user interface ( gui ) with which end users will be most familiar . for all these reasons , the value of the internet has been recognised for making a diabetes simulator even more widely available than via just a downloadable program . indeed , users might wish to try out the simulator online to see what it is all about , before going to the trouble of downloading and installing a standalone version . therefore , it was hypothesised that an even wider audience might benefit from the aida diabetes - simulation approach if it did not require any local download or installation , could run on a wider range of computers , and would offer a standard windows gui for user interaction . to address these issues , the authors set out to collaborate on the development of a web - based version of the aida diabetes simulator , called aida online , which would be totally internet based and which therefore would be able to operate from any computer , anywhere in the world , provided it had a connection to the internet and a graphical display . the intention was that via such a web - based simulator enhancements and upgrades could be made available instantaneously , worldwide . in fact , it would not even be necessary to have a computer to use aida online , with usage being possible through any internet - enabled device , for example , webtv or a smartphone . recognising that the new target audience may be less computer savvy than aida v4 dos users , it was planned for the input variables for the web - based simulator to be somewhat simplified with pull - down menu options used for ranges of values ( e.g. increased , normal and reduced ) instead of absolute numbers . along similar lines , clickable terms were planned to allow unfamiliar users to see the definitions of various terms via an online glossary and also permit users to see some of the assumed values used in the simulations . in order to make the diabetes simulator available to as many people as possible who could get on the internet , web programmers distinguish client - side and server - side processing . in client - side processing , the browser asks the server to send it the program and then runs it locally . server - side processing , the user types data into a form , and this is sent to a web server , which calculates the results and sends them back as html ( hypertext markup language ) data . the developer only needs to write the program once and connect it to a web server . clearly , such a centralised approach might lead to issues with load on the server , although this has not been a problem to date with the web - based version of aida . haag et al . have suggested that in contrast to conventional computer - assisted instruction ( cai ) programs , web - based training ( wbt ) programs can be considered as four main types : client - based ; remote data and knowledge ; distributed teaching ; and server - based . using this classification , the web - based version of aida based on this , a decision was taken to make use of server - side processing and translate the original aida v4 program source code from pascal into perl ( practical extraction and report language ) and avoid java and javascript . educational models written in perl allow a wide variety of internet browsers to access and run simulations without requiring time - consuming downloading of lengthy files , utilising compilers , or using local disk storage space . perl v5.0 was selected for this work for a variety of reasons , the most compelling being its portability between platforms and its widespread acceptance by web browsers . perl is the language used in cgi ( common gateway interface ) scripts , which are used by web servers to allow for communication with the outside world , and therefore must be recognised by web browsers . furthermore , perl places a minimal load on the user 's system and is compatible with all major web browsers that are currently in use . perl is a high - level , general - purpose , nonproprietary , interpreted , and dynamic shell scripting / programming language that does not require a special linker . it is a relatively simple language where variables and commands are passed to the script and output is sent directly back . in other words , perl can be run from the command line of different platforms like dos or unix and of course via the web . another reason , which was nearly as important , was the ease with which perl could be learned . designed to be a user friendly language and written by one person , perl takes many of the good attributes of languages like c++ , grep , shell scripting ( sh ) , and others to make it user friendly . conversely , java , for example , has been written like c and has a much steeper learning curve for first time users . furthermore , perl can be used without modification on multiple computer platforms . as a scripting language , it does not require compilation in order to be executed . perl is distributed under the gnu general public license and is maintained by a worldwide network of volunteers , a factor which also contributed to its choice for the implementation of aida online , which is itself a free web resource implementation of a freeware pc program . designed to be accessible and usable by anyone who desires to learn more about diabetes , aida online has been developed to provide a windows - compatible easy - to - use web - based interface for the aida v4 diabetes simulator . the method of interaction between the user and the program is an important aspect of the simulator which merited consideration . the simplest method is to utilise the interaction built into html through the form function . this allows input by the user through both text and buttons , allowing a page designer to guide the user through the steps required to fill in all the necessary information to run the program . default values can also be given to input areas to allow for the creation of a simple default example to help lead the user through what needs to be done by them to fill out the form . using a form also lends itself to the next step , which is displaying output . using forms is supported by the use of perl , that can be used to create an output html page on the fly , which will incorporate both text and graphs to provide the required information for the user . aida online has been designed to allow the user to run multiple simulations in order to compare the results of changes made to a patient 's diet and/or insulin regimen . the design plan was for results to be viewed through the use of several different graphs that display the bg and insulin concentrations over a 24 hour period , the results of two different simulations being viewed on graphs at the same time . it was also planned for the graphs to be able to display glucose absorption , renal excretion , peripheral glucose utilization , and net hepatic glucose balance data ( fluxes within the aida v4 model ) . one of the authors , edl , collaborated on this work with two design project students ( dkdw and can ) at north carolina state university and made the aida v4 pascal source code available for translation into perl . under edl 's direction , the existing aida v4 pascal source code was translated , and an online version of aida was developed in order to produce a more accessible version of the simulator for a wider ( different ) audience . the result of this work has become a widely utilised resource for hcps and educators , students , patients , and relatives / carers of people with diabetes . it is freely available to anyone with internet access and has the ability to engage people from a wide variety of educational , social , and cultural backgrounds . the aida online website utilises two divisions of a web server , the html public domain area and the cgi - bin script / program area ( figure 1 ) . the html area hosts all static web pages including most of the forms that post information directly as input to programs stored in the cgi - bin area of the host ( aida online ) web server . this area also hosts the graphics files and temporary numerical data files produced by the simulation engine . this allows the user , if required , to download the information produced by the simulator into local storage . the cgi - bin perl scripts ( programs ) which actually run the simulations are executed in realtime , so that they can output dynamic information ( data and graphs ) which can be viewed immediately through a web browser . the perl scripts have been set up to accept input from the web server ( html form submission ) as well as from static text files stored at the website . this allows the user 's web browser to view and download the output of the aida online scripts . the html output does not actually exist physically as files but rather is created dynamically in realtime , upon demand ( generated on the fly ) . the perl scripts ' other outputs include physical text files that store data used in the generated plots . there are a total of three scripts used to generate aida online simulations ( cases.cgi , intro.cgi , and simulate.cgi ) . two scripts ( cases.cgi and intro.cgi ) are used to process user preferences in order to set up forms for the users ' input . the third script ( simulate.cgi ) actually computes the aida diabetes model and can be considered the simulation engine , generating the simulations . the html area is also used to store all static web pages that provide information , help , and directions to the simulator . for example , users can select their preferred units for bg display ( mmol / l versus mg / dl ) , select the insulin types to be used , and decide whether or not they would like to see the advanced fluxes plots underlying the simulations ( net hepatic glucose balance , peripheral glucose utilization , renal excretion of glucose into the urine , and glucose absorption from the gut ) as part of their simulation runs . as in aida v4 , users can choose from 40 existing , pre - stored scenarios or create a new case from scratch . an abbreviated description of the cases is provided initially on the options web page . however , if the user needs a further description or more details , a link is given to a web page where each of the 40 sample cases is described in full . the case details page allows the user to choose a case and return to a similar customisation page to proceed with a simulation . once the user has selected a case scenario , a cgi - bin perl script is invoked . this script is passed the preferences of the user and the sample case , if one has been chosen . if an existing case is chosen , the script populates an html template form with data extracted from static text files obtained from aida v4 , which are stored on the aida online web server for that individual case scenario ( figure 2(a ) ) . this form would be left blank if the user decides to start a new case . using this form , when the user opts to run the simulation , this html form data and the user 's preferences are passed to the simulation engine , implemented in perl , and which makes use of the same model as that adopted for the downloadable aida v4 simulator to generate the bg and plasma insulin profiles . based on the user 's time of carbohydrate intake and amount , as in aida v4 , the simulator reads prestored values from a static text file effectively a large lookup table containing precomputed carbohydrate gut absorption profiles generated by one of the authors ( edl ) and taken from aida v4 . the time , amount , and type of insulin injections are used to determine which file and what value to extract . this data is then used by the simulator to calculate the bg and plasma insulin levels as well as each of the four fluxes at 15-minute intervals , using an euler integration method , during a simulated 24 hour day , as per the original aida v4 model . an early prototype version of aida online incorporated observed bg data points for example , glucostix measurements as in the downloadable version of aida , but these were not retained when aida online went live on the web , in order to try and reduce the amount of user entered data required to run a simulation and thereby simplify user interaction with aida online . storage data files used by aida online were previously processed and saved in the same location as the perl script . eight of the data files represent the different insulin absorption profiles based on the type of insulin injected and the dosage . four of the eight files represent the active insulin absorption , while the other four pertain to the plasma insulin profiles . a different file represents the glucose absorption data based on the amount of carbohydrates ingested . the data arrays of absorption profiles stored in these files are the product of a precomputation program written by lehmann and deutsch . the storage file was originally written in binary for the dos version of aida which allowed for fast access to the data . however , for aida online , a decision was taken to have the storage files written in an ascii ( american standard code for information interchange ) text file format to accommodate future easier upgrades , if necessary . also , to compensate for any increase in search time , the storage arrays were divided up into nine files so that there would be a faster access rate . ascii text files are used to store precomputed plasma ( _ pa ) and active ( _ act ) insulin profiles as shown below for the four classes of insulin preparations catered for by aida online ; actrapid = short - acting ; nph + lente = intermediate - acting ; and ultralente = long - acting insulin preparations ; car = carbohydrate absorption profiles from the gut . actrapid_act actrapid_pa nph_act nph_pa lente_act lente_pa ultralente_act ultralente_pa car . the amount of carbohydrates ingested in each meal determines the offset ( 080 grams ) for fetching the appropriate absorption profile from the carbohydrate storage file . the data retrieved from the carbohydrate storage , along with the meal times , are used to calculate the glucose absorption profile over the 24-hour period . for each meal , the absorption of glucose into the blood stream from the gut is calculated over a period of several hours after ingestion . the type of insulin determines which of the eight insulin storage files to read , while the dosage determines the offset ( 040 iu ) within that file . there are different forms of insulin injections offered as choices in the web - based simulator , as in pc aida v4 : soluble short - acting insulin preparations ( 10 types ) , intermediate - acting preparations ( 19 types ) , long - acting preparations ( 4 types ) , and a mixture of short- and intermediate - acting biphasic preparations ( 18 types ) . upon completion of the main computation process , over a simulated 24 hour period , the data is stored in six separate files from time 0 to 24 hours in preparation for graphing . aida online makes use of an open access graphical program , called gnuplot , to generate the aida online graphs in real time . the plots are displayed on a line graph with a frequency of 15 minute data points . gnuplot is a portable command - line driven graphing utility for linux and many other platforms . it was originally created to allow scientists and students to visualise mathematical functions and data interactively , but , it has grown to support many noninteractive uses such as web scripting . the six ascii text files created by aida online and stored in the html area contain the x , y values for the graphical plots . once these files have been closed , the simulator makes system calls that pass these files to the gnuplot graphing program , which takes in the x , y coordinates from the ascii text data files and produces a graphic file . in the original aida online version 1 development , gnuplot would produce a .ppm ( portable pixel map ) graphical file . however , web browsers did not support the .ppm file type produced , so the simulator would make another system call to a separate program called ppmtogif to convert the .ppm graphics file into a common .gif ( graphics interchange format ) file supported by web browsers . the .gif file is stored along with the data files in a temporary directory within the html area . this ensures that ( i ) the files can be accessed by the user for download which may assist data retrieval and ( ii ) the files are kept in the directory for approximately 6 hours before being deleted by an automated timed cronjob in order to save server hard disk space . with all the calculations and plot creations completed , the simulator displays its output in html format to the user 's web browser . the html output automatically contains references to the newly created plots and data files for the user 's browser to display . once the simulator has finished its execution , the user also has the opportunity to download any of the plots as well as the actual data files . the aida online website is designed so that multiple simulations can be run in succession . each run of the simulator returns not only the graphs but also a new form already filled in with the data previously submitted . this allows the user to make changes to the input data based on their observations from the simulation graphs . when the user submits the new regimen data , new graphs are generated and a further data form is returned . a key feature of the aida online diabetes simulation approach is the ability to visualise and compare graphical simulation results before and after a change in the regimen . this is achieved by the simulator submitting in the background not only the changed data and preferences but also references to the data files that were produced during the original simulation run . one of the ways in which user preferences are stored and communicated between simulation runs is via the use of hidden fields in the dynamically perl - script generated html code . as shown in figure 2(b ) , a series of parameters and variable values are recorded via this approach . for a subsequent simulation however , this time , the previously stored data files are passed with the new data to the gnuplot graphing program . the present aida online simulator is set up to handle two separate data files at a time . the user can continue to submit changed data , but the new simulation will only contain the present and most recent previous simulation data . aida online was initially developed and run on a suse linux 6.1 ( suse inc , ca , usa ) world wide web server on a dual pentium ii/450 mhz ( intel , ca , usa ) with 256 mb ram and 36 gb dasd ( direct access storage device ) . the web server software used was apache 1.33 ( apache group , ca , usa ) . the apache web server development effort has aimed at creating a robust , commercial - grade , and freely - available source code implementation of an http ( hypertext transfer protocol ) web server . the apache web server used on the linux platform utilises a multithreading algorithm that allows multiple simultaneous processes to be run . for aida online , when the simulator is invoked , a process identification ( pid ) number is assigned . the files ( graphics and data ) are stored uniquely with the pid as part of the filename so even if multiple users from different parts of the world submit simulations at the same time , the simulations can all be processed separately . this explains how it is possible to run a diabetes simulation from , for example , auckland , new zealand , simultaneously with a simulation from basel , switzerland on a computer originally in durham , north carolina and now in london , uk all in a matter of seconds . the aida online simulations can be run from any machine ( pc , apple mac , linux , unix server , webtv , smartphone , etc . ) from anywhere in the world , provided the device has access to the internet and a graphical display . there is also a free registration / announcement list that aida online users can subscribe to at the website ( or by emailing subscribe@2aida.org ) so that they can be immediately notified by email about any enhancements or modifications to the system as they become available worldwide . furthermore , people can also follow developments with aida online on facebook at https://www.facebook.com/www.2aida.org and/or at http://www.facebook.com/aida.diabetes.simulator1 or on twitter at http://www.twitter.com/aida_diabetes . there are basically two periods of delay between data being submitted by the user and the full results being presented back to the user . firstly , the time for the simulator to be invoked and run depends on the amount of load on the server . secondly , there is a time delay for the transfer of the html page and graphics files back to the end user . the html ( text only ) file itself is only 9 kb ( kilobytes ) in size . by contrast , the standard simulation produces 17 kb , and the advanced ( fluxes ) simulation produces 25 kb of graphics data . while the total elapsed time for the user may initially have depended on the speed of the connection to the internet and geographical location , these file sizes are tiny compared with usual web transfers , so apparent simulator response times for users , wherever they are in the world , are generally very rapid ( typically 1 - 2 seconds ) . there is also a http://www.sitemeter.com/ embedded graphic on the main aida online simulation web page which helps to monitor visitors running simulations and provides some anonymised insight into ongoing aida online usage . loading this embedded graphic may delay the apparent simulation time but the simulations are still incredibly quick , even via a home broadband connection . designed to be accessible and usable by anyone who desires to learn more about diabetes , aida online version 1 was initially made available on a shodor education foundation web server in the eastern united states ( in north carolina ) . shodor ( http://www.shodor.org ) is a north - carolina - based research and education organisation dedicated to the advancement of science and maths education , specifically through the use of modelling and computers . however , since then , with the development and release of aida online version 2 ( aida online ) , the main aida online facility was relocated by one of the authors ( edl ) to london , england , and the web - based simulator has been updated further since , and merged in with the rest of the aida ( http://www.2aida.org ) website . aida online has seen glycosylated haemoglobin ( hba1c ) calculations as reported by lehmann [ 38 , 39 ] for aida v4based on work by nathan et al . incorporated into the web - based simulations , together with user - definable upper and lower normoglycaemic limits ( bounds ) which can be overlaid on the bg graph to assist users in identifying out - of - range high and low bg values . the main aida online website currently resides on a shared web server with an intel xeon single core processor running at 2 ghz with 4 gb of ram . the current operating system is suse linux enterprise server 10 ( suse inc , ca , usa ) . writing the program in perl and then utilising a fast linux server allows many users to access aida online simultaneously . the main educational utility seems to lie in aida online 's ability to compare results from different simulation runs . for instance , by modifying the inputted insulin injections , users can learn to exert more control over the bg level of the simulated virtual diabetic patient . the working hypothesis underlying use of aida online is that , while modifying the input parameters , users whether they be patients , carers , students , or hcps can also learn more about how to manage glucose concentrations in insulin - dependent diabetes mellitus ( iddm ) . the advantage of using multiple injection times and different insulin types becomes apparent as different regimens can be compared . large swings in meal carbohydrate intakes result in obvious swings in bg levels that require adjustments to be made to insulin injections to correct the perturbations in the simulated patient . as has been highlighted previously by blanchard et al . , the advantage of having multiple preprogrammed simulated patients allows the user to see the effect of these changes on different body types that have various insulin sensitivities . aida online is accessed directly at http://www.2aida.net or from the main aida website ( http://www.2aida.org ) via menu options on the left frame menu . selecting online simulation takes the user through a short selection procedure , in which a case scenario may be chosen if desired and a summary of the key features of the aida online web - based diabetes simulator at http://www.2aida.net is as follows . availability and functionality accessible via the aida website at http://www.2aida.org or directly at http://www.2aida.net.standard web browser software using mouse to navigate ( by contrast to the downloadable standalone pc version of aida , also available at the website , which is dos based and employs key and tab functions).simulated blood glucose ( bg ) and plasma insulin levels are derived and displayed in graphical format.carbohydrate intake and insulin injection data are also displayed graphically.simulated glycosylated haemoglobin ( hba1c ) value derived.forty different case scenarios are available . dedicated cases can also be created and simulated.data entry forms allow the user to change various values and rerun simulations . standard web browser software using mouse to navigate ( by contrast to the downloadable standalone pc version of aida , also available at the website , which is dos based and employs key and tab functions ) . simulated blood glucose ( bg ) and plasma insulin levels are derived and displayed in graphical format . carbohydrate intake and insulin injection data options bg units : choose either mmol / l or mg / dl.display : choose either standard or advanced.standard displays the simulated bg and plasma insulin curves.advanced displays the simulated bg and plasma insulin curves , together with graphs of the model 's glucose fluxes ( glucose absorption from the gut , glucose excretion from the kidneys , glucose utilisation in the periphery , and the net hepatic glucose balance : the production or utilisation of glucose by the liver).insulin types utilised ( when defining new case only ) : choose either standard or premixed.display upper and lower bounds / limits ( the user can define upper and lower normoglycaemic ranges to be indicated on bg graphs ) : choose either display bounds or do not display bounds . bg units : choose either mmol / l or mg / dl . advanced displays the simulated bg and plasma insulin curves , together with graphs of the model 's glucose fluxes ( glucose absorption from the gut , glucose excretion from the kidneys , glucose utilisation in the periphery , and the net hepatic glucose balance : the production or utilisation of glucose by the liver ) . insulin types utilised ( when defining new case only ) : choose either standard or premixed . display upper and lower bounds / limits ( the user can define upper and lower normoglycaemic ranges to be indicated on bg graphs ) : choose either display bounds or do not display bounds . data entry forms ( user defined ) six data entry points for the timing and quantity of carbohydrate intake over a 24 hour period.insulin(s)choose from a selection of short - acting and from a selection of intermediate- or long - acting preparations ; up to four injections per day of each insulin type can be entered , along with the time and dose in units for each injection.body weight is user defined ( in kg or lb).kidney function : renal glucose threshold can be normal and renal function can be reduced or normal.hepatic and/or peripheral insulin sensitivity may be normal or reduced . six data entry points for the timing and quantity of carbohydrate intake over a 24 hour period . insulin(s)choose from a selection of short - acting and from a selection of intermediate- or long - acting preparations ; up to four injections per day of each insulin type can be entered , along with the time and dose in units for each injection . there are currently 40 different case scenarios to choose from as a starting point ; each presents a virtual patient with iddm on a given insulin regimen , experiencing real - life problems associated with bg control . case may be defined by the user ( figures 3(a ) and 3(b ) ) . if the user wishes to have further details about the case scenarios , these can be accessed on the options.htm page via the click here for a better description of the cases link ( see at the bottom of figure 3(a ) ) , which loads the cases.htm page ( figure 3(c ) ) giving full details of each case scenario . the compartmental design of the aida model allows for a number of variables to provide a more tailored and realistic simulation . hepatic and peripheral insulin sensitivities are modifiable into three categories ( increased , normal , and reduced ) , as is the renal threshold of glucose ( the level of bg at which sugar spills into the urine ) . renal function is also modifiable into two levels , normal and reduced creatinine clearance ( table 1 ) , which is then used for an estimate of the glomerular filtration rate ( gfr ) . the final two variables are probably of most interest to casual users , and these are the carbohydrate intake and insulin injections . the insulin input allows for a time of injection , dosage , and type of insulin ( short acting , intermediate acting , and/or long acting , with a variety of brand names to choose from ) . additional input variables include the patient 's name , weight , and definable limits of the recommended , allowed , normoglycaemic range . having selected a case scenario via the pull - down menu on the options.htm page ( figure 4(a ) ) or via the cases.htm page ( figure 3(c ) ) and/or indicated preferred options , the ( continue ) button is clicked and a perl script ( intro.cgi ) stored in the cgi - bin area is invoked . a new dynamically generated page is loaded , giving a description of the selected case ( see figure 4(b ) ) . data entry forms are displayed , with prestored entries shown for carbohydrate intake ( amount and time ) and insulin dosage ( type , amount , and time ) , according to the selected case scenario . if the option has been selected , then this form is presented blank ( figure 3(b ) ) . there are up to six data entry times for carbohydrate intake ( for breakfast , midmorning snack , lunch , afternoon snack , supper , and bedtime snack ) and up to four data entry times for each of two insulin types , to cover a 24-hour period , which enables a wide range of insulin regimens to be accommodated . preselected data pertaining to the chosen case scenario can be modified at this stage , if desired , prior to running the simulation . a number of other parameters can also be changed , as outlined previously and intable 1 , and these help in further defining the virtual patient 's clinical situation . when all of the data entries have been made and the ( run simulation ) button is clicked ( figure 4(b ) ) , then the simulation is performed in real time . if the online simulator is accessed directly from the popup window that is automatically generated when the aida website is loaded or if quick simulation is selected from the left frame menu , then the user is taken straight into a preselected case example ( case number 0001 , joy wilson in the aida online case scenario database ) , in which the baseline simulation has already been performed ( see http://www.2aida.org/aida/example.htm ) . once a simulation is run , a new page is loaded , which gives the simulated bg and plasma insulin levels for the 24-hour period ( figure 4(c ) ) . the first panel shows bg fluctuations plotted against time , with a dotted line indicating preferred limits ( normoglycaemic range ) , if this option was selected . directly below , on the same chart , carbohydrate intake is quantitatively indicated across the 24-hour period . the bars at the bottom of the graph , showing the carbohydrate intake , help the user to visually line up the changes in the bg graph based on carbohydrate intake . the second panel shows simulated plasma insulin levels over the same 24-hour period ; injected insulin doses are indicated below , on the same chart . once again , the bars below the plasma insulin levels are similar to the carbohydrate graph , except showing insulin injections . this graphical presentation of both user - defined and simulated data clearly illustrates the relationship between meal times , carbohydrate consumption , and associated bg excursions , insulin injections , and plasma insulin levels . by the same token , the graphs provide an uncomplicated presentation of the characteristics of various insulin types , particularly in terms of their action profiles . a key feature of aida online is the facility that enables one simulation to be compared with the previous . thus , the html data forms are reproduced on the same page below the simulation panels , allowing the user to make any number of changes to the input data ( figure 4(d ) ) . re - running the simulation derives similar graphs ; however , the bg and plasma insulin levels from the previous simulation are also shown . taken together , the two panels illustrate how various features of the insulin - diet regimen interact , and the user can observe and identify specific effects arising from any changes made prior to rerunning the simulation . in addition , the computed glycosylated haemoglobin ( hba1c ) value gives a predicted indication of overall bg control , and this helps to guide the learning process , especially when simulations are rerun repeatedly ( figures 5(a ) , 5(b ) , and 5(c ) ) . the user has the option to view several additional graphs that help explain how the bg simulations are derived . these additional graphs include glucose absorption rate , renal excretion of glucose into the urine , peripheral glucose utilization , and net hepatic glucose balance ( figure 6(a ) ) , which are fluxes in the original standalone pc aida v4 program . throughout the simulation web pages are html links to an online glossary ( figure 6(b ) ) , which gives clear but concise explanations of basic terms for the newcomer such as bg level , plasma insulin level , and hba1c . so , clicking on underlined links shown , for instance , in figures 4(a)4(d ) , 5(a)5(c ) , and 6(a ) all link to the online glossary for further explanations . more advanced scientific concepts associated with the model used in the simulations are also described . thus , for those users already familiar with the relevant basic concepts , glucose flux options are described , and html links provide access to graphical representations of the model of glucose utilisation that is used ( also available by selecting model graphics from the left frame menu ) . furthermore , there are links to graphs showing insulin absorption and elimination for various types of insulins covered by the simulator . additional detailed technical information about the aida model is also freely available to users at the aida website , accessible directly at http://www.2aida.org/technical . a summary of how aida online works is also explained via a link at the bottom of the online simulation web page . employing standard web - browser technology , aida online is easy to use , and simulator response times are generally very rapid . it is emphasised that the model upon which aida online is based is not sufficiently accurate for individual patient bg prediction or therapy planning [ 15 , 42 , 43 ] . the user is therefore reminded that the simulation approach is suitable only as an educational tool and not for generating individual therapeutic advice or treatment planning . the general caveats displayed at the aida website can be seen at the top of figures 3(a ) and 3(c ) , and the aida online specific caveats can be seen top right in figure 3(b ) . current applications of aida online are numerous and wideranging , as exemplified through the variety of user - generated feedback received which has on the whole been very positive . independent user reviews of aida / aida online have been published previously elsewhere [ 44 , 45 ] , and user feedback can also be found at the aida website at http://www.2aida.org/aida/online-reviews.htm and is referred to in the results section of this paper . to make the most effective use of aida online , it may help for the web - based simulator to be incorporated into structured learning materials . one way in which the simulation approach may be tied in with static textual educational material to effectively enhance the learning experience however , use of aida online is not limited to personal or individual distance learning via the internet ; it also has the potential to be used successfully in a variety of settings , including group education sessions . as has been highlighted by dewolf , computer - assisted education has the potential to accommodate different speeds of learning . some diabetes education sessions have often been provided to groups of patients / students and have necessarily been administered at the average learning speed of the group . however , computers can also offer education directly to individuals , allowing individuals to control the pace of their own learning and at the same time receive immediate feedback . aida online can service both group and individual teaching sessions . there have been some technical issues with aida online , particularly in the early years . this converted .ppm format files generated by gnuplot , which could not be viewed by browsers , into .gif format files , which are very well supported by web browsers . while .gif files have been very widely used on the internet , there were unfortunately some issues about programs which produce .gif files online needing to be licenced prior to use . the licence fee , apparently , cost us$5,000 , and therefore , the ppmtogif program could no longer be provided with a gnu public licence . given this , when aida online was moved from north carolina to london , uk .gif files could no longer be generated in realtime by the web - based simulator . as a result , the initial release of aida online version 2 ( aida online ) was modified to make use of .jpg ( joint photographic experts group ) and .png ( portable network graphics ) image files , avoiding any problems with dynamically generating .gif files . subsequent versions of aida online have been modified further to just concentrate on .png format graphics , which actually reduce the file sizes , contributing further to the apparent speed of aida online ( table 2 ) . the conversion of the aida v4 pascal program from the dos - based standalone , downloadable version to perl has been successful , and aida online has been accessible via http://www.2aida.net for a number of years . the purpose of developing a world wide web accessible glucose - insulin simulator has been to provide an educational opportunity for as many people as possible ( patients with diabetes , their relatives , students , and hcps ) . in this respect , aida online has exceeded all expectations . a range of user comments / reviews can be found in figures 7(a ) , 7(b ) , 7(c ) , and 7(d ) . aida online first went live on the internet for beta testing in december 1997 . aida online version 1 was formally launched in august 1998 , following 8 months of extensive beta testing by the authors and others . aida online has been thoroughly tested with a range of web browsers and works well with microsoft internet explorer , mozilla firefox , google chrome , and apple safari , as well as with netscape navigator ( in earlier years ) . since records began , over 637,500 diabetes simulations have been run at aida online ( figure 8) . since aida online was relocated to a server in london , uk , visitors to the website have been logged from over 115 countries , including ( in alphabetical order ) : andorra , argentina , armenia , aruba , australia , austria , bahrain , belarus , belgium , bermuda , bhutan , bolivia , bosnia - herzegovina , brazil , brunei darussalam , bulgaria , canada , chile , china , christmas island , cocos ( keeling ) islands , colombia , comoros , costa rica , croatia ( hrvatska ) , cuba , cyprus , czech republic , denmark , dominican republic , ecuador , egypt , estonia , ethiopia , faroe islands , fiji , finland , france , french polynesia , georgia , germany , gibraltar , greece , guam , guatemala , hong kong , hungary , iceland , india , indonesia , iran , ireland , israel , italy , jamaica , japan , jordan , kazakhstan , kuwait , latvia , lebanon , lesotho , lithuania , luxembourg , macau , macedonia ( former yugoslav republic ) , malaysia , malta , mauritius , mexico , moldova , morocco , nepal , netherlands , new caledonia , new zealand ( aotearoa ) , nicaragua , nigeria , norway , oman , pakistan , panama , papua new guinea , paraguay , peru , philippines , poland , portugal , qatar , romania , russia / russian federation , samoa , san marino , saudi arabia , singapore , slovakia / slovak republic , slovenia , south africa , south korea , spain , sri lanka , sweden , switzerland , taiwan , thailand , tonga , trinidad and tobago , turkey , ussr ( former ) , ukraine , united arab emirates , united kingdom , united states of america , uruguay , venezuela , yemen , and yugoslavia ( former ) , as well as from nonprofit organizations , unknown ip addresses , us commercial , educational , government and military ip addresses , and dot.net and dot.arpa ( old arpanet ) addresses . independent comments about the simulations have been very encouraging [ 44 , 4951 ] . on average , more than 3,200 simulations have been run each month at aida online ( in 20112013 ) . figure 9 shows a series of simulations using case scenario 0026 in the aida online database . the case details for this scenario record that this young woman is on a twice daily insulin regimen , injecting a biphasic preparation which has a premixed 30% to 70% ratio of short versus intermediate acting insulin . while this does not permit quite as much flexibility in selecting a dose it does save on having to mix insulin in the syringe . use the simulator to see what would happen if you switched this woman onto other biphasic preparations with , say , premixed 10/90 , 20/80 , 40/60 , or 50/50 percent constituents the simulations given in figures 9(a ) , 9(b ) , and 9(c ) demonstrate the usage of aida online with premixed ( biphasic ) insulin injections containing different proportions of short - acting and intermediate - acting insulin preparations . the work overviewed in this paper shows how it has been possible to make use of the internet not just as a static repository of information but also as an interactive medium to perform quite complex dynamic simulations in a clinically useful manner . translating the original pc aida v4 pascal source code into perl and then utilising a fast web server the authors are not aware of any diabetes simulation facility as sophisticated as aida online that is available anywhere else on the internet . the main educational utility of aida online lies in the ability to rapidly compare results from different diabetes simulations . by modifying the simulated insulin injections , users can learn to exert more control over the bg level of the simulated patient . for instance , possible advantages of using multiple injections and different insulin types can become apparent as different therapeutic combinations are compared . modifying carbohydrate inputs can also demonstrate the important contribution of diet to bg control . in this respect , large swings in meal carbohydrate intake result in obvious swings in the bg level , which requires insulin injections to be adjusted for the simulated virtual diabetic patient . patients allows users to see the effects of these changes on different patients with different sensitivities to insulin . the hope is that patients , their relatives , and students can learn how to balance insulin and diet in diabetes by modifying the simulations . the concept underlying this diabetes simulation approach is that patients with diabetes may also improve their ability to actually manage their own diabetes by experimenting in this way . clearly , this hypothesis remains to be tested in a clinical randomised controlled trial ( rct ) setting [ 52 , 53 ] . however , it is hoped that the relatively widespread use and widespread availability of the aida pc and aida online diabetes simulation approaches will encourage the undertaking of such clinical trials . there are potential advantages and limitations to both the downloadable and web - based versions of aida . one benefit of aida online has been to allow visitors to the aida websites to try out the simulator on the web before downloading a copy to use locally on their pc / mac . although not specifically studied , the working hypothesis for this complementary approach is that if people like what they see using aida online , they may be more likely to download the standalone ( pc ) version for further use locally on their home / office computer . aida online extends the concept of the downloadable aida pc software by making the simulator accessible via the world wide web from any device ( e.g. , unix based , apple mac , or network server computer ) , smartphone , or webtv anywhere in the world provided it has internet access and a graphical display . there is no longer a need to have a pc nor to download and install software on a local machine . furthermore , upgrades to aida online require only one version of the program to be changed , and these changes then automatically become accessible to anyone from anywhere in the world . in addition , many users have asked for a windows - based , mouse - controlled point and click version of aida . aida online provides just such an easy to use simulator with a standard user interface accessible in a familiar web browser format . physical activity is currently not addressed and assumed to be constant over the course of the steady - state simulation period . it is recognised that different carbohydrate types are absorbed at different rates by the body , but for the purpose of the model , all carbohydrates are considered equal . in addition , although there are modifiable variables for medical conditions , such as renal disease , they are not able to be specifically tailored to exactly recreate a specific patient 's condition . some of this is done intentionally , to keep patients from feeling that they can model their own particular treatment regimen and expect the results obtained to exactly mimic their own bg values . however , the more variables ( parameters ) included in a model , the more complex it becomes . one of the not - so - obvious limitations of the model results not from the model itself , but from the patient 's own day to day variances , be it diet , insulin dosing , bg data collection , or otherwise , which can affect their own bg readings . many of these variables ( such as exercise , stress levels , or food intake ) can change minimally and not be noted by the patient . this was especially notable during the validation process for the original aida model . these and other limitations may help to explain why there has been difficulty in applying these models to individual patient therapy planning in clinical practice . however , this does not negate the educational benefit of allowing users to see how carbohydrate intake and insulin usage will affect bg levels on an ongoing basis throughout the day and allow users the opportunity to see how plasma insulin and bg levels interact over a 24-hour time period . other limitations of the aida online approach relate to the fact that a user clearly needs continuous internet access to use the web - based simulator . in earlier times , this was fine for those in academic / work establishments with continuous web access and for those with unmetered access , for example , in first world countries . however , broadband / dial - up usage does remain an issue for those who still pay by the minute for internet access , for example , via dial - up in certain parts of the world , or who only have time - limited satellite - based internet access , for instance , in some parts of africa . as there are still many parts of the world with intermittent / no web access , it is recognised that a web - based application like aida online will be only of limited benefit in those places , and perhaps the downloadable standalone pc version of the simulator will actually remain of more use in those areas . interestingly , the centralised versus distributed computing idea of aida online versus aida v4 revisits a number of early discussions about pcs versus central servers in years gone past . at present with its current level of usage aida online does not require a dedicated server . however , potentially one web - based server could have difficulty supporting the extent of usage which has been reported with the downloadable aida pc software . for instance , a detailed survey of aida v4 users has revealed that 16,790 simulations were run by 200 users . over a similar period around the same time over 16,700 downloads of the aida pc software were logged at the main aida website . if the survey usage was typical for most aida v4 pc downloads , this would suggest that in the region of 16,700 ( 16,790/200 ) = 1,400,000 simulations might have been run on these 16,700 downloaded copies of the pc software . from past experience , 2,600 aida online simulations run per month account for approximately 610 mb of website data traffic each month ( dr . e. d. lehmann , personal communication ) . over a comparable two - year period , based on the aida v4 pc survey data , aida online would have needed to support roughly 23 times as many simulations ( approximately 58,300 simulations / month ) to run as many simulations as are estimated to have been executed by the aida pc software . this would equate to approximately 14 gb of data traffic per month ( 23 610 mb ) . while these are only estimates , 14 gb of data transfer per month just for aida online would be perfectly feasible to support in internet / web / computing terms . however , together with static html traffic from the diabetes / insulin tutorial and aida online glossary and pc aida v4 software downloads which independently account for 4 - 5 gb of data traffic each month ( dr . e. d. lehmann , personal communication ) , careful consideration may need to be given to the web hosting company used to permit such an ongoing level of sustained activity . furthermore , as an unfunded , not - for - profit , and noncommercial venture , the cost of a dedicated server may be hard to justify just to run aida online . therefore , the web - based simulator is currently hosted on a shared server , which substantially reduces the hosting cost . however , in order to render the graphical simulations , aida online relies on local access on the same server to the gnuplot program . access to gnuplot is not always included as standard on shared linux servers ; gnuplot needs to be compiled on the server for installation , which requires root access . this clearly is possible with a dedicated server , where root access and installation are feasible , but on shared servers , this necessitates the support and cooperation of the web host . fortunately , gnuplot is accessible as part of the ruby on rails open - source web framework ( http://www.rubyonrails.org ) , which appears to be supported on more shared linux servers , and in this way , it seems to be possible to find web - hosting companies that will support longer - term access to gnuplot even on shared linux server platforms . clearly , at times , the number of simulations run at aida online will be high , for example , when people are running diabetes education classes making use of the web - based simulator . at other times , the load on the server will be much lower . it is difficult to predict these surges in usage , but to maintain its position as a well - established , much used web - resource , the aida online simulation facility needs to be able to cater for peak demand . with only one web server running the online simulator one solution would be to make use of mirror sites , but in the early days of aida online , cost may have been an issue . however , web hosting costs have fallen dramatically since , so hosting multiple aida online simulators on different shared web servers now becomes more practical . this consideration is of importance particularly if the intention is to encourage third party websites to link to aida online and the diabetes / insulin tutorial . for such links , it becomes imperative to offer a good service , because if aida online becomes unavailable , then the service at the other websites will also be restricted . clearly , other website owners will not wish to make a commitment to using interactive educational simulations , however good , unless they can be assured that the service will be reliable . therefore , in the longer term , if aida online is to be used even more extensively , a distributed network of mirrored low - cost shared web server accounts might be an appropriate way to proceed . this could be less costly and more flexible than one large dedicated ( expensive ) server and could help to ensure that the web - based simulator would remain available irrespective of local geographic server issues . computing cluster , so that usage of the simulator could be scalable and increased , as required by user demand . obviously , keeping aida online working on multiple servers would raise issues about longer - term maintenance , aside from cost . however , it seems preferable to keep aida online operating on a number of separate , distributed web - servers in different geographical locations to ensure the web - based simulations can continue to operate irrespective of local issues with any one single web server . for this reason , the process of mirroring aida online has started , and one of the authors ( edl ) has set up a mirror server for aida online in california , usa . the authors believe that the full potential of such web - based interactive educational diabetes simulators is yet to be fully realised . as a result , aida online and the aida website form part of an ongoing development . a number of simple refinements are already planned , but with collaboration and further resources , the novel web - based simulator could be incorporated into a wider range of diabetes educational resources in a number of innovative ways and make an even more significant contribution to the rapidly evolving field of web - based learning . recent years have seen the successful introduction of a number of insulin analogues with markedly different action profiles compared with earlier insulin preparations . as these types of insulin are becoming ever more popular with patients and hcps alike , it will be pertinent to adapt aida online to accommodate use of these newer insulins . v4 [ 5558 ] , and now the concept needs to be ported to an updated release of aida online ( version 3 ) . insulin pumps are increasingly being recognised as an effective way to provide physiological insulin replacement in people with iddm . as it stands , the aida model does not explicitly cater for continuous subcutaneous insulin delivery ; however , user feedback has indicated that useful insight can still be gained from the software by using basal levels of insulin and adding in insulin boluses , ways in which aida and aida online can be applied for insulin pump simulations , derived from : http://www.2aida.org/pump , are as follows . to set up a near basal level of insulin administration , it is possible to make two entries within aida online using a long - acting insulin preparation ( e.g. ultratard , humulin - u , or ultralente ) . these two injections need to be set 12 hours apart ( say in the morning and evening ) . as aida and aida online are currently limited to 4 insulin administrations ( injections ) per day , if a user can make one of these long - acting entry times correspond to a meal ( and therefore a bolus ) , that will permit 3 boluses to be given to aida / aida online , at other times . for example , if a user would like to simulate a basal regimen with 10 units / day , with aida or aida online the user could enter 4 units of ultratard in the morning ( at 7 am ) and 6 units in the evening ( at 7 pm ) . this would give a background insulin profile that is slightly elevated in the morning , which might be fairly typical for a patient who does not have too much of a blood glucose ( bg ) rise in the morning . if a user would like to simulate a bigger morning bg rise ( e.g. , as in the dawn phenomenon ) , then with aida / aida online , it is possible to use a slightly more lopsided ratio . for instance , for the 10 units / day ultratard example given above , a user could split the dose to 3 units in the morning and 7 units in the evening , for the basal background . however the user would need to be careful to not split the basal dose , say 1 unit am and 9 units pm , since the simulator will need some morning long - acting insulin to help flatten out the basal curve in the afternoon . for the bolus doses , aida / aida online will allow users to give the simulator up to 4 boluses ( injections ) of short - acting ( regular ) insulin , although one limitation is that 2 of these will need to be at the same times as the basal doses . nevertheless , by giving the simulator a basal and bolus dose together ( for instance in the morning ) , this will still leave users with at least 2 more ( and possibly 3 ) boluses to play with it is hoped that in this way by experimenting with different basal and bolus regimens users might be able to learn a bit more about balancing insulin and diet in diabetes , even with an insulin pump . a version of the simulator software which would enable pump regimens to be more completely simulated could be planned . ultimately , it is hoped that aida online may be usefully incorporated into insulin pump training programmes , as a means of illustrating some of the principles of physiological insulin therapy . the diabetes control and complications trial ( dcct ) demonstrated the long - term benefits of intensive insulin treatment ; however , the increased risk of hypoglycaemia and increased staff resources used in the dcct have perhaps limited the adoption of this approach even more widely . however , more recent evidence suggests that appropriate training in insulin adjustment can improve hba1c without significantly increasing the risk of severe hypoglycaemia [ 60 , 61 ] . diabetes self - management skills training enables patients to adapt insulin doses on a daily basis , in order to accommodate increased dietary freedom . the dose adjustment for normal eating ( dafne ) trial in the uk has led to the approach receiving increased support , and this educational material is even being adapted for school - age children . modular outpatient education for flexible insulin treatment adapted for pregnancy has also been shown to improve pregnancy outcome . web - based diabetes simulators , such as aida online , could conceivably be used in such skills - based training programmes to aid understanding of the relationships between insulin dose / timing and meal content / timing . further refinement of the software to allow for the effects of exercise and/or illness or stress would obviously confer even greater potential . additionally , the use of web - based resources in training programmes such as those used in the dafne approach may help to address the perceived problems associated with staffing levels , time , and resources . the interactive educational diabetes / insulin tutorial at the aida website demonstrates how aida online can be integrated with it is envisaged that this concept could be expanded further . in particular , aida online could potentially be integrated with teaching materials for various different specific audiences , with the text and format of the informational material being tailored appropriately to the target audience . for example , aida online could be used in structured training programmes for diabetes hcps , educators , and students . specific educational materials could be directed towards various patient populations : teenagers , young women considering pregnancy , patients with low health literacy , patients with noninsulin dependent diabetes mellitus ( niddm ) beginning insulin treatment , and so on . an interactive learning game for school - age children may also be of tremendous value . while it is noted that the aida simulator model is based on data for adults with an absolute insulin deficiency , it can still be used effectively to demonstrate the principles of insulin adjustment even if some endogenous insulin secretion is still occurring . aida online can also be run across the internet from third - party websites , in a separate pop - up or new web browser window . this increases the possibilities for further collaborative intelligent use of the simulator in web - based diabetes education resources . in particular , there are large numbers of static graphical / fixed textual diabetes educational resources on the internet but not so many online diabetes simulators . in reed and lehmann , the concept is developed of one simulation engine being able to service a number of educational websites . so , for instance , not all diabetes educational websites need to have the complexity of running aida online locally , with ( common gateway interface ) cgi - bin programs / scripts and gnuplot available . with the interconnected web , each educational diabetes resource does not need to run its own copy of the simulator locally . therefore , a diabetes / insulin tutorial has been developed at http://www.2aida.info and integrated with aida online , which can also now be accessed at other websites for example http://medweb.bham.ac.uk/easdec/aidadevelopment/tutorial.htmand the tutorial has even been translated into other languages for example , spanish ( tutorial sobre insulina y diabetes ) at http://www.um.es/grupo-cirrosisinsulintutorial/tutorial87.htm further collaborative website developments like this are expected . refinement of the simulator to incorporate the effects of exercise , illness , and stress would be useful . additional benefits may also be derived from adding in functions to allow for some variation in the glycaemic index of carbohydrates consumed . the aida model thus far has been centred on people with iddm and focuses on the diet and insulin interactions instituted by the patient / student . of necessity this is the nature of modelling to present as simple and as accurate a version of an event as possible . the validation work performed on the original downloadable aida software precluded using this method as a treatment planner but was found to be sufficiently accurate to warrant use as an educational tool . the same issues apply to aida online , and this fact is extensively noted at the aida website to try and prevent patients from directly applying data from aida online simulations to their personal treatment plans ( e.g. see caveats in figures 3(a ) and 3(b ) ) . by focusing predominantly on insulin therapy and carbohydrate intake , while unobtrusively accounting for some of the significant medical modifiers , aida online allows users to experiment with various regimens and see how they may affect the overall glucose - insulin balance . this is done by providing a relatively simple interface for the user to input / modify times and amount of carbohydrate intake , as well as insulin dosages , and providing a wide variety of insulin types to choose from . the simulation can then be run in either a basic ( standard ) or advanced mode , with the difference being in the number and type of graphs that are displayed . as part of future work , it is intended to increase the number of aida online case scenarios available on the web . already , three extra cases have been generated , which are accessible directly at http://www.2aida.org/aida/fast-track8.htm , http://www.2aida.org/aida/fast-track1.htm , and http://www.2aida.org/aida/fast-track14.htm these just now remain to be included in the main aida online case scenario database . additional case scenarios will be added , particularly when new cases are generated for the lispro / glargine release of aida v4 ( v4.5c ) and once the new insulin analogues ( lispro and glargine [ 5558 ] ) have been incorporated into aida online version 3 ( aida online ) . eventually , if arrangements could be made to track users ( teachers ) at aida online , it would be possible to have a tutorial or series of lessons that teachers could go through to be credentialed to teach with the simulator . the idea could be developed to get teachers up to speed with using the diabetes simulations and what aida is about . technically , it is likely to be less challenging to track users of an online simulator than a downloadable / offline simulator like aida v4 . one could even envisage a logon perl script to monitor usage of aida online by patients as part of a rct with hba1c measured before and after lessons with the simulator , possibly using home hba1c monitoring kits . something similar ( without hba1c monitoring ) has been tried for a small number of medical students by dewolf , but the approach might be more powerful with hundreds , or thousands , of people with diabetes accessing aida online and the accompanying diabetes / insulin tutorial . there have also been suggestions to permit some user selectable different colours to be chosen for the graphs in aida online . allowing grey scale or other colour schemes to be selected may assist patients with visual impairments / colour blindness to maximize their access to and the usability of the web - based simulations , and this is planned to be investigated as part of future development work with aida online . in terms of its functionality , the current version of aida online is similar in many ways to the pc version of aida ( v4.3d ) . however , with its improved mouse - based user interface and the fact that only one version of the program needs to be changed to distribute updates it is envisaged that aida online may become more amenable to further upgrades and improvements in functionality , in due course . it is recognised that making use of a simulator in a standard web - browser window , which most people know how to use , could also potentially help decrease the learning curve for generating simulations , compared with a downloadable version of the program . nevertheless , the designated purpose of the standalone pc software and aida online remains the same . in this respect , aida online should be solely regarded as an educational tool which some patients and hcps have found fun and interesting to use . it is provided free of charge from a not - for - profit website at : http://www.2aida.netas a noncommercial contribution to continuing diabetes education , in the hope that more people may find it of some use . a number of verification , validation , and clinical evaluation studies have been performed with the downloadable versions of aida v4 ( e.g. , see ) . thus far , relatively little evaluation work has been done with aida online , aside from a small study by dewolf investigating the value of the web - based simulator as an educational tool for medical students and an assessment of aida online for teaching high school students . part of the reason for the relatively limited assessment of the aida online version of the simulator , to date , may simply be its self - evident use . however , in view of the fact that the mathematical model underlying aida online is the same as that in the downloadable software , going forward evaluation work for aida online may benefit from focusing particularly on human factor assessments , as well as identifying optimal ways to actually apply aida online in clinical / teaching settings . one major advantage to utilising a server - based application ( as opposed to a downloadable program ) is the flexibility that is offered . new modules can be added to the simulator which become available instantaneously around the world . this has facilitated the development of aida online version 2 ( aida online ) , as well as various more recent refinements to the web - based simulator . the most up - to - date version of aida online ( version 2 ) is available without charge at the http://www.2aida.net website . following completion of further programming and bench testing work , it is expected that new , improved versions of aida online will become available in due course . people who wish to be automatically informed by email about updates to aida online are welcome to join the very low volume aida registration / announcement list by sending a blank email note to subscribe@2aida.org or by following aida on facebook at http://www.facebook.com/www.2aida.org and/or at http://www.facebook.com/aida.diabetes.simulator1 or on twitter at http://www.twitter.com/aida_diabetes .

background . aida is a widely available downloadable educational simulator of glucose - insulin interaction in diabetes . methods . a web - based version of aida was developed that utilises a server - based architecture with html form commands to submit numerical data from a web - browser client to a remote web server . aida online , located on a remote server , passes the received data through perl scripts which interactively produce 24 hr insulin and glucose simulations . results . aida online allows users to modify the insulin regimen and diet of 40 different prestored virtual diabetic patients on the internet or create new patients with user - generated regimens . multiple simulations can be run , with graphical results viewed via a standard web - browser window . to date , over 637,500 diabetes simulations have been run at aida online , from all over the world . conclusions . aida online 's functionality is similar to the downloadable aida program , but the mode of implementation and usage is different . an advantage to utilising a server - based application is the flexibility that can be offered . new modules can be added quickly to the online simulator . this has facilitated the development of refinements to aida online , which have instantaneously become available around the world , with no further local downloads or installations being required .

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

the software incorporates a compartmental / physiological model describing glucose - insulin interaction in insulin - dependent diabetic patients ( lacking endogenous insulin secretion ) . although a range of other interactive simulation programs of glucose - insulin interaction in diabetes have been described in the literature [ 412 ] , to date , most of these do not seem to have been distributed so widely via the internet or been made particularly widely available . by contrast , with aida , multiple updates and versions of the software have been freely available on the web since 1996 , and before that made available to researchers on diskette [ 1315 ] . with the recent move towards cloud computing , the interest has focused less on a single central server and more on distributed services that can be hosted on a range of servers , with more resources ( bandwidth , central processing units [ cpus ] , memory , and disk space ) commissioned as usage requires . the purpose of the aida online diabetes simulator has not been to provide individual patient bg prediction or medical advice or therapy planning but instead to provide an interactive educational web - based tool to help people with diabetes and their relatives / carers , as well as hcps and students , to better understand how meal ( carbohydrate ) and insulin interactions can affect bg levels ; that is the intended role for self - learning / teaching / demonstration / education purposes . while the downloadable aida software has been widely applied , there may be a number of practical and theoretical limitations to the standalone aida pc software approach . to address these issues , the authors set out to collaborate on the development of a web - based version of the aida diabetes simulator , called aida online , which would be totally internet based and which therefore would be able to operate from any computer , anywhere in the world , provided it had a connection to the internet and a graphical display . the intention was that via such a web - based simulator enhancements and upgrades could be made available instantaneously , worldwide . in fact , it would not even be necessary to have a computer to use aida online , with usage being possible through any internet - enabled device , for example , webtv or a smartphone . server - side processing , the user types data into a form , and this is sent to a web server , which calculates the results and sends them back as html ( hypertext markup language ) data . clearly , such a centralised approach might lead to issues with load on the server , although this has not been a problem to date with the web - based version of aida . have suggested that in contrast to conventional computer - assisted instruction ( cai ) programs , web - based training ( wbt ) programs can be considered as four main types : client - based ; remote data and knowledge ; distributed teaching ; and server - based . using this classification , the web - based version of aida based on this , a decision was taken to make use of server - side processing and translate the original aida v4 program source code from pascal into perl ( practical extraction and report language ) and avoid java and javascript . perl is the language used in cgi ( common gateway interface ) scripts , which are used by web servers to allow for communication with the outside world , and therefore must be recognised by web browsers . perl is distributed under the gnu general public license and is maintained by a worldwide network of volunteers , a factor which also contributed to its choice for the implementation of aida online , which is itself a free web resource implementation of a freeware pc program . using forms is supported by the use of perl , that can be used to create an output html page on the fly , which will incorporate both text and graphs to provide the required information for the user . aida online has been designed to allow the user to run multiple simulations in order to compare the results of changes made to a patient 's diet and/or insulin regimen . under edl 's direction , the existing aida v4 pascal source code was translated , and an online version of aida was developed in order to produce a more accessible version of the simulator for a wider ( different ) audience . the aida online website utilises two divisions of a web server , the html public domain area and the cgi - bin script / program area ( figure 1 ) . the perl scripts have been set up to accept input from the web server ( html form submission ) as well as from static text files stored at the website . for example , users can select their preferred units for bg display ( mmol / l versus mg / dl ) , select the insulin types to be used , and decide whether or not they would like to see the advanced fluxes plots underlying the simulations ( net hepatic glucose balance , peripheral glucose utilization , renal excretion of glucose into the urine , and glucose absorption from the gut ) as part of their simulation runs . if an existing case is chosen , the script populates an html template form with data extracted from static text files obtained from aida v4 , which are stored on the aida online web server for that individual case scenario ( figure 2(a ) ) . using this form , when the user opts to run the simulation , this html form data and the user 's preferences are passed to the simulation engine , implemented in perl , and which makes use of the same model as that adopted for the downloadable aida v4 simulator to generate the bg and plasma insulin profiles . an early prototype version of aida online incorporated observed bg data points for example , glucostix measurements as in the downloadable version of aida , but these were not retained when aida online went live on the web , in order to try and reduce the amount of user entered data required to run a simulation and thereby simplify user interaction with aida online . once these files have been closed , the simulator makes system calls that pass these files to the gnuplot graphing program , which takes in the x , y coordinates from the ascii text data files and produces a graphic file . the aida online website is designed so that multiple simulations can be run in succession . the apache web server used on the linux platform utilises a multithreading algorithm that allows multiple simultaneous processes to be run . the aida online simulations can be run from any machine ( pc , apple mac , linux , unix server , webtv , smartphone , etc . ) from anywhere in the world , provided the device has access to the internet and a graphical display . there is also a free registration / announcement list that aida online users can subscribe to at the website ( or by emailing subscribe@2aida.org ) so that they can be immediately notified by email about any enhancements or modifications to the system as they become available worldwide . while the total elapsed time for the user may initially have depended on the speed of the connection to the internet and geographical location , these file sizes are tiny compared with usual web transfers , so apparent simulator response times for users , wherever they are in the world , are generally very rapid ( typically 1 - 2 seconds ) . however , since then , with the development and release of aida online version 2 ( aida online ) , the main aida online facility was relocated by one of the authors ( edl ) to london , england , and the web - based simulator has been updated further since , and merged in with the rest of the aida ( http://www.2aida.org ) website . incorporated into the web - based simulations , together with user - definable upper and lower normoglycaemic limits ( bounds ) which can be overlaid on the bg graph to assist users in identifying out - of - range high and low bg values . selecting online simulation takes the user through a short selection procedure , in which a case scenario may be chosen if desired and a summary of the key features of the aida online web - based diabetes simulator at http://www.2aida.net is as follows . availability and functionality accessible via the aida website at http://www.2aida.org or directly at http://www.2aida.net.standard web browser software using mouse to navigate ( by contrast to the downloadable standalone pc version of aida , also available at the website , which is dos based and employs key and tab functions).simulated blood glucose ( bg ) and plasma insulin levels are derived and displayed in graphical format.carbohydrate intake and insulin injection data are also displayed graphically.simulated glycosylated haemoglobin ( hba1c ) value derived.forty different case scenarios are available . standard web browser software using mouse to navigate ( by contrast to the downloadable standalone pc version of aida , also available at the website , which is dos based and employs key and tab functions ) . employing standard web - browser technology , aida online is easy to use , and simulator response times are generally very rapid . current applications of aida online are numerous and wideranging , as exemplified through the variety of user - generated feedback received which has on the whole been very positive . to make the most effective use of aida online , it may help for the web - based simulator to be incorporated into structured learning materials . there have been some technical issues with aida online , particularly in the early years . given this , when aida online was moved from north carolina to london , uk .gif files could no longer be generated in realtime by the web - based simulator . subsequent versions of aida online have been modified further to just concentrate on .png format graphics , which actually reduce the file sizes , contributing further to the apparent speed of aida online ( table 2 ) . aida online first went live on the internet for beta testing in december 1997 . since records began , over 637,500 diabetes simulations have been run at aida online ( figure 8) . since aida online was relocated to a server in london , uk , visitors to the website have been logged from over 115 countries , including ( in alphabetical order ) : andorra , argentina , armenia , aruba , australia , austria , bahrain , belarus , belgium , bermuda , bhutan , bolivia , bosnia - herzegovina , brazil , brunei darussalam , bulgaria , canada , chile , china , christmas island , cocos ( keeling ) islands , colombia , comoros , costa rica , croatia ( hrvatska ) , cuba , cyprus , czech republic , denmark , dominican republic , ecuador , egypt , estonia , ethiopia , faroe islands , fiji , finland , france , french polynesia , georgia , germany , gibraltar , greece , guam , guatemala , hong kong , hungary , iceland , india , indonesia , iran , ireland , israel , italy , jamaica , japan , jordan , kazakhstan , kuwait , latvia , lebanon , lesotho , lithuania , luxembourg , macau , macedonia ( former yugoslav republic ) , malaysia , malta , mauritius , mexico , moldova , morocco , nepal , netherlands , new caledonia , new zealand ( aotearoa ) , nicaragua , nigeria , norway , oman , pakistan , panama , papua new guinea , paraguay , peru , philippines , poland , portugal , qatar , romania , russia / russian federation , samoa , san marino , saudi arabia , singapore , slovakia / slovak republic , slovenia , south africa , south korea , spain , sri lanka , sweden , switzerland , taiwan , thailand , tonga , trinidad and tobago , turkey , ussr ( former ) , ukraine , united arab emirates , united kingdom , united states of america , uruguay , venezuela , yemen , and yugoslavia ( former ) , as well as from nonprofit organizations , unknown ip addresses , us commercial , educational , government and military ip addresses , and dot.net and dot.arpa ( old arpanet ) addresses . on average , more than 3,200 simulations have been run each month at aida online ( in 20112013 ) . translating the original pc aida v4 pascal source code into perl and then utilising a fast web server the authors are not aware of any diabetes simulation facility as sophisticated as aida online that is available anywhere else on the internet . the hope is that patients , their relatives , and students can learn how to balance insulin and diet in diabetes by modifying the simulations . there are potential advantages and limitations to both the downloadable and web - based versions of aida . one benefit of aida online has been to allow visitors to the aida websites to try out the simulator on the web before downloading a copy to use locally on their pc / mac . aida online extends the concept of the downloadable aida pc software by making the simulator accessible via the world wide web from any device ( e.g. furthermore , upgrades to aida online require only one version of the program to be changed , and these changes then automatically become accessible to anyone from anywhere in the world . other limitations of the aida online approach relate to the fact that a user clearly needs continuous internet access to use the web - based simulator . as there are still many parts of the world with intermittent / no web access , it is recognised that a web - based application like aida online will be only of limited benefit in those places , and perhaps the downloadable standalone pc version of the simulator will actually remain of more use in those areas . however , potentially one web - based server could have difficulty supporting the extent of usage which has been reported with the downloadable aida pc software . therefore , the web - based simulator is currently hosted on a shared server , which substantially reduces the hosting cost . clearly , at times , the number of simulations run at aida online will be high , for example , when people are running diabetes education classes making use of the web - based simulator . with only one web server running the online simulator one solution would be to make use of mirror sites , but in the early days of aida online , cost may have been an issue . however , it seems preferable to keep aida online operating on a number of separate , distributed web - servers in different geographical locations to ensure the web - based simulations can continue to operate irrespective of local issues with any one single web server . a number of simple refinements are already planned , but with collaboration and further resources , the novel web - based simulator could be incorporated into a wider range of diabetes educational resources in a number of innovative ways and make an even more significant contribution to the rapidly evolving field of web - based learning . as it stands , the aida model does not explicitly cater for continuous subcutaneous insulin delivery ; however , user feedback has indicated that useful insight can still be gained from the software by using basal levels of insulin and adding in insulin boluses , ways in which aida and aida online can be applied for insulin pump simulations , derived from : http://www.2aida.org/pump , are as follows . as aida and aida online are currently limited to 4 insulin administrations ( injections ) per day , if a user can make one of these long - acting entry times correspond to a meal ( and therefore a bolus ) , that will permit 3 boluses to be given to aida / aida online , at other times . web - based diabetes simulators , such as aida online , could conceivably be used in such skills - based training programmes to aid understanding of the relationships between insulin dose / timing and meal content / timing . aida online can also be run across the internet from third - party websites , in a separate pop - up or new web browser window . therefore , a diabetes / insulin tutorial has been developed at http://www.2aida.info and integrated with aida online , which can also now be accessed at other websites for example http://medweb.bham.ac.uk/easdec/aidadevelopment/tutorial.htmand the tutorial has even been translated into other languages for example , spanish ( tutorial sobre insulina y diabetes ) at http://www.um.es/grupo-cirrosisinsulintutorial/tutorial87.htm further collaborative website developments like this are expected . the same issues apply to aida online , and this fact is extensively noted at the aida website to try and prevent patients from directly applying data from aida online simulations to their personal treatment plans ( e.g. by focusing predominantly on insulin therapy and carbohydrate intake , while unobtrusively accounting for some of the significant medical modifiers , aida online allows users to experiment with various regimens and see how they may affect the overall glucose - insulin balance . already , three extra cases have been generated , which are accessible directly at http://www.2aida.org/aida/fast-track8.htm , http://www.2aida.org/aida/fast-track1.htm , and http://www.2aida.org/aida/fast-track14.htm these just now remain to be included in the main aida online case scenario database . additional case scenarios will be added , particularly when new cases are generated for the lispro / glargine release of aida v4 ( v4.5c ) and once the new insulin analogues ( lispro and glargine [ 5558 ] ) have been incorporated into aida online version 3 ( aida online ) . allowing grey scale or other colour schemes to be selected may assist patients with visual impairments / colour blindness to maximize their access to and the usability of the web - based simulations , and this is planned to be investigated as part of future development work with aida online . in terms of its functionality , the current version of aida online is similar in many ways to the pc version of aida ( v4.3d ) . however , with its improved mouse - based user interface and the fact that only one version of the program needs to be changed to distribute updates it is envisaged that aida online may become more amenable to further upgrades and improvements in functionality , in due course . it is recognised that making use of a simulator in a standard web - browser window , which most people know how to use , could also potentially help decrease the learning curve for generating simulations , compared with a downloadable version of the program . a number of verification , validation , and clinical evaluation studies have been performed with the downloadable versions of aida v4 ( e.g. thus far , relatively little evaluation work has been done with aida online , aside from a small study by dewolf investigating the value of the web - based simulator as an educational tool for medical students and an assessment of aida online for teaching high school students . part of the reason for the relatively limited assessment of the aida online version of the simulator , to date , may simply be its self - evident use . one major advantage to utilising a server - based application ( as opposed to a downloadable program ) is the flexibility that is offered . new modules can be added to the simulator which become available instantaneously around the world . this has facilitated the development of aida online version 2 ( aida online ) , as well as various more recent refinements to the web - based simulator .

alternative splicing is the process by which splice sites in precursor messenger rnas ( pre - mrnas ) are differentially selected and paired to produce multiple mature mrnas and protein isoforms with distinct structural and functional properties . the first example of alternative splicing was discovered almost 30 years ago , when membrane - bound and secreted antibodies were demonstrated to be encoded by the same gene [ 1 , 2 ] . now , we know that alternative splicing is a very accurate , efficient , and extraordinarily flexible process that regulates all major aspects of eukaryotic cell biology . affecting approximately 94% of human genes [ 3 , 4 ] , it represents the major source of the human proteomic diversity . regulation of alternative splicing decisions involves the recognition of target sequences in the pre - mrna by a number of splicing regulatory factors with antagonistic functions such as sr ( serine - arginine - rich ) and hnrnp ( heterogeneous nuclear ribonucleoprotein ) protein families . generally , sr proteins promote exon recognition by binding to exonic or intronic splicing enhancer sequences ( eses and ises , resp . ) , while hnrnp factors typically interact with exonic or intronic splicing silencers ( esss and ises ) inhibiting splice sites recognition . the regulation of alternative splicing has been discussed in several excellent reviews [ 68 ] . changes in alternative splicing patterns have an essential role in normal development , differentiation , and in response to physiological stimuli , but aberrant splicing generates variants that contribute to multiple aspects of tumor establishment and progression and in the resistance to therapeutic treatments [ 5 , 9 , 10 ] . many cancer - associated splicing isoforms are expressed during embryonic development , but not in normal adult tissues , whereas others are entirely novel transcripts . central to the splicing oncogenic switch are changes in the expression , activity , or post - translational modification of splicing regulatory factors , such as sr and hnrnp proteins [ 5 , 9 ] . thus , modification of alternative splicing profiles contemporaneously affects multiple key aspects of cancer cell biology , including control of cell proliferation , cancer metabolism , angiogenesis , evasion from apoptosis , invasiveness , and metastasis [ 5 , 9 , 10 ] . here , we discuss aberrant alternative splicing networks that contribute to the oncogenic phenotype and have a prominent role in important aspects of tumorigenesis process , including response to hypoxia and cancer cell invasion and metastasis . in addition , we also discuss important questions connected to the role of alternative splicing in cancer : what are the relevant splicing switches that are critical to malignant transformation ? what are the main functions of cancer - associated alternatively spliced variants ? by illustrating specific examples , it will be clear how the production of cancer - related isoforms offers the potential to develop novel diagnostic , prognostic , and more specific anticancer therapies . through the activation of oncogenes and inactivation of tumor suppressor genes , cancer cells become able to proliferate , survive , and resist to apoptosis . nevertheless , also microenvironmental signaling plays a crucial role in controlling cancer cell homeostasis , metabolism , growth , and differentiation . the microenvironment in solid tumors is very distinct from that in normal tissues and the cross - talk between cancer and stromal cells contributes to the formation of a clinically relevant tumor and to response to antitumor therapy [ 13 , 14 ] . modifications of the microenvironment ( most of these start early during tumor progression ) result from metabolic alterations in cancer cells and from recruitment or activating of nontumoral cells , including blood and lymphatic endothelial cells , pericytes , carcinoma - associated fibroblasts , bone marrow - derived cells , and immune and inflammatory cells [ 15 , 16 ] . in this altered microenvironment cancer cells in addition , transformed cells often hijack the signaling circuits acting on normal cells in order to become independent from external stimulation to grow and proliferate [ 12 , 13 ] . due to deregulated cancer cell metabolism ( the consequence of uncontrolled and rapid proliferation ) and to an altered structure and functionality of tumor blood vessels , the tumor microenvironment is characterized by hypoxia and acidosis [ 15 , 17 , 18 ] . hypoxic tumor microenvironments are now recognized as a selective pressures that promote tumor aggressiveness , inducing cancer cells to metastasize and making them refractory to radiotherapy and chemotherapy . cells cope with hypoxia by activating transcription factor hif-1 ( hypoxia - inducible factor-1 ) , a heterodimer formed by the constitutively expressed subunit ( hif-1 ) and the inducible subunit ( hif-1 ) . the regulation of hif-1 activity is mainly at the protein level . under non - hypoxic conditions , the hif-1 subunit is rapidly ubiquitinylated and degraded by the pvhl ( von hippel - lindau tumor suppressor protein ) and the proteasome , thus preventing the dimerization with hif-1 . under hypoxia , hif-1 degradation is suppressed and its level increases rapidly . the hif-1/hif-1 heterodimer translocates to the nucleus where it binds to hypoxia response elements ( hre ) in the promoters of target genes . through the activation of more than 150 genes , hif-1 affects important biological processes such as angiogenesis , glucose metabolism , cell proliferation , survival , apoptosis , and invasion / metastasis . target genes include several enzymes involved in glycolysis ( glut-1 and pdk-1 ) , angiogenesis , iron metabolism ( transferrin ) , cell adhesion molecules ( mic2 ) , fibronectin and matrix metalloproteinase ( mmp-2 ) , extracellular matrix ( ecm ) modifying enzymes such as lysyl oxidase , and pluripotency factors including oct4 , nanog , and sox2 [ 1921 ] . one of the primary targets of hif-1 is vegfa ( vascular endothelial growth factor a ) , a cytokine that promotes blood vessel growth and stimulates angiogenesis . vegf , secreted by hypoxic cancer cells , diffuses through the tumor mass to neighbouring host normal vessels , where , upon binding to its receptor vegfr2 , it stimulates endothelial cells to proliferate , migrate toward the tumor , and form new capillaries . even though the angiogenesis process does not initiate malignancy , it promotes tumor growth by allowing oxygen and nutrients to reach proliferating cancer cells . alternative splicing occurs extensively within vegf pre - mrna , generating various isoforms with different c - terminal domains and distinct affinity for its receptors and with a non - redundant role in angiogenesis [ 24 , 25 ] . vegf isoforms are classified in two families , called vegfxxx ( pro - angiogenic ) and vegfxxxb ( anti - angiogenic ) , where xxx denotes the position of the amino acid residue in the mature protein . pro- or anti - angiogenic isoforms are generated through alternative splicing of the mutually exclusive terminal exons 8a and 8b . selection of the proximal 5 splice site ( pss ) in exon 8a generates vegfxxx whereas the distal 5 splice site ( dss ) in exon 8b results in vegfxxxb isoforms . the two types of isoforms bind vegfr2 ( vascular endothelial growth factor receptor 2 ) with equal affinity . however , vegfxxx activates the downstream signaling pathways and induces angiogenesis , while vegfxxxb blocks this process . given these characteristics , it is not surprising that vegfxxxb is preferentially expressed in normal tissues and it is downregulated in cancer . the choice between pss or dss depends upon the activity of the splicing factors of the sr family srsf1 and srsf6 : srsf6 preferentially selects the dss and promotes the production of anti - angiogenic vegf isoforms , whereas srsf1 mainly activates the proximal pss . thus , the altered expression of srsf1 and srsf6 in many types of tumors could impact malignant transformation by ensuring the proper balance of pro- and anti - angiogenic isoforms in cancer cells [ 28 , 29 ] . for example , treatment with tgf-1 increases the anti - angiogenic vegfxxxb isoforms via p38 mapk signaling . conversely , insulin - like growth factor 1 ( igf-1 ) decreases the expression of vegfxxxb isoforms in retinal pigmented epithelial cells through pkc and srpk1 . srpk1 is a protein kinase that specifically phosphorylates proteins containing sr domains and is upregulated in various types of cancer , like pancreatic , breast , and colon carcinoma . recently , a genome - wide analysis of hypoxia - induced alternative splicing changes in endothelial cells has identified target genes implicated in angiogenesis - mediated cytoskeleton remodeling ( cask , itsn1 , larp6 , sptan1 , tpm1 , and robo1 ) , in the synthesis of membrane anchors ( pign ) and in the regulation of gene expression ( cugbp1 and max ) . the impact of hypoxia on post - transcriptional events is proven also by lama3-a ( laminin alpha 3 ) , a splicing variant of the lama3 gene induced by reduction of oxygen supply which promotes cell invasion and is associated with a poor prognosis in head and neck cancer [ 33 , 34 ] . as mentioned before , hif is a heterodimer that acts as a dominant regulator of adaptive cellular responses to hypoxia . there are three principal isoforms of the hif- subunit encoded by distinct genes : hif-1 , hif-2 ( epas1 ) , and hif-3. hif-3 gene consists of 19 exons and it is subjected to extensive alternative splicing leading to the production of at least six isoforms . hif-34 is one of the hif-3 splicing variants whose functions have recently been linked the development of hypervascular malignant meningiomas . importantly , hif-34 is able to inhibit proliferation and invasion , to reduce neovascularization and glucose metabolism in hypervascular meningiomas . another gene induced by hypoxia is cyr61 ( cysteine rich 61 ) encoding for a secrete protein that functions as a proangiogenic factor promoting adhesion , migration , and survival of vascular endothelial cells . cyr61 is supposed to be a promoter of tumor progression since its high expression levels were detected in various cancer types [ 38 , 39 ] . notably , in addition to transcriptional level , the expression of cyr61 can be also regulated through an alternative splicing event that stimulates the retention of intron 3 in cyr61 mrna leading to the production of a nonsense - mediated decay ( nmd ) sensible transcript . importantly , this alternative splicing event coupled with nmd pathway ( called as - nmd ) was reported to be altered in breast cancer and associated with a shift from an intron 3 retained transcript ( ir ) toward an intron 3 spliced mrna ( is ) encoding for the biological active cyr61 protein . moreover , in several breast cancer cell lines under hypoxic conditions as - nmd of cyr61 was reported to enhance the expression of the is transcript , suggesting that hypoxia - mediated alternative splicing changes could be a central mechanism regulating the cyr61 expression and its tumor - promoting activity . cd44 is a transmembrane molecule able to bind several ligands , including important components of ecm , such as hyaluronic acid , collagen , fibronectin , laminin , and matrix metalloproteinases ( mmps ) , and involved in cell - cell and cell - matrix interactions , migration , and invasion . cd44 pre - mrna consists of 20 exons , 10 of which ( v1v10 ) undergo alternative splicing events , thus generating multiple cd44 isoforms with different molecular sizes and with diverse extracellular domains [ 41 , 42 ] . the predominant cd44 isoform , the low - molecular - weight cd44s ( 90 kda standard form ) , is expressed by several tissues , including hematopoietic , fibroblast , and glial cells , whereas high - molecular - weight cd44v variants ( 140230 kda ) are restricted to epithelial cells and abundant in epithelial - type carcinomas [ 41 , 42 ] . however , it has been observed that some variants are over - expressed in various tumors and implicated in cancer cell invasion and metastasis [ 41 , 43 , 44 ] . in particular , cd44v6 and cd44v8 variants are associated with tumor progression and poor diagnosis in several types of carcinoma including breast and colorectal cancers [ 4547 ] . interestingly , hif-1 is able to increase the expression of the cd44 mrna and to upregulate cd44 variants containing exons v6 and v7/8 . in line with this , krishnamachary and collaborators have reported that hypoxic regions of breast cancer specimens contain cells with elevated expression of cd44 . additional studies are necessary to identify the molecular mechanisms and signaling pathways regulating cd44 alternative splicing in response to hypoxia . hypoxia - induced alternative splicing changes were also recently investigated in the study of hirschfeld and colleagues . the yt521 ( yth domain containing 1 ) is a ubiquitously expressed nuclear splicing factor containing a novel rna - binding domain ( yth domain ) necessary for yt521 to directly influence splice site selection . importantly , low yt521 expression was associated with clinical outcome in patients with type i endometrial cancer , suggesting its potential role as a tumor suppressor . furthermore , yt521 alternative splicing targets are well - known cancer - associated genes such as brca2 , esr1 , mdm2 , vegf , and cd44 [ 49 , 5155 ] . similar to other proteins involved in mrna processing , the expression of yt521 is regulated by alternative splicing . interestingly , it was reported that under hypoxic conditions the alternative splicing of yt521 pre - mrna containing exons 8 and 9 generates two transcripts that are subjected to degradation through nmd , suggesting that these as - nmd events of yt521 could simultaneously affect the processing of several cancer - associated genes . further functional investigations on hypoxia and its impact in regard to alternative splicing of target genes may contribute to better understanding of a key regulatory epiphenomenon in tumor growth involved in the development of an aggressive cancer phenotype . the term metastasization is currently used to indicate the ability of tumor cells to invade adjacent tissues and disseminate toward distant organs . this is a multistep process that involves ( i ) local invasion of tumor cells through the basement membrane and endothelial walls into the host stroma , ( ii ) intravasation within the blood and the lymphatic circulatory systems ; ( iii ) extravasation into distant tissues , and ( iv ) proliferation of tumor cells allowing growth and efficient metastatic colonization . as observed for other hallmarks of cancer , gene expression programs implicated in the metastatic process are the same that participate in embryonic development , morphogenesis , and wound healing [ 57 , 58 ] . one of these programs that physiologically pertain to embryogenesis is the epithelial - to - mesenchymal transition ( emt ) . the emt process involves dedifferentiation steps in which cells lose their epithelial phenotype to acquire mesenchymal traits [ 58 , 59 ] . the epithelial cell layer consists of polarized cells with cohesive cell - cell junctions . through emt cells undergo an extensive reorganization of the cytoskeletal architecture with the loss of intercellular junctions and cell polarity and the acquisition of an elongated , fibroblast - like shape . during tumor progression , emt is one of the major routes through which cancer cells acquire invasive capabilities and spread throughout the body as single cells [ 58 , 59 ] . importantly , tumor emt is a transient process that occurs in a subset of cells at the invasive front of the metastasizing primary carcinoma and is reversed at the final metastatic sites , where cells undergo the reverse process , namely , the mesenchymal - to - epithelial transition ( met ) [ 58 , 59 ] . this plasticity and the redifferentiation of metastatic cells to an epithelial identity help in organ colonization , ensuring metastasis formation . moreover , it clearly indicates that emt is not driven by stable genetic mutations but by activation of gene expression programs in response to external cues in the tumor microenvironment . several relevant players involved in emt / met cycles have been identified including transcription factors , growth factors , cytokines and chemokines , pro - angiogenic factors , cell adhesion molecules , modifiers of cytoskeletal organization , and extracellular matrix - remodeling enzymes . several pathways activate a network of transcription factors that promote the expression of mesenchymal markers , such as n - cadherin and vimentin , while inhibiting e - cadherin production , not only a key component of adherent junctions but also a tumor suppressor frequently repressed , mutated or degraded during tumor transformation [ 58 , 59 ] . however , in the last few years , several studies have highlighted additional layers of emt control , including epigenetic reprogramming , small noncoding rnas , translational and post - translational regulations , and alternative splicing changes [ 10 , 57 , 61 , 62 ] . in particular , an increasing body of evidence indicates that splicing regulation alone can drive critical aspects of emt - associated phenotypic changes . below , we discuss some interesting examples of specific alternative splicing events that are implicated not just in the emt / met program but also in the different stages of the metastatic cascade . alternative splicing of the ron proto - oncogene ( also known as mst1r ) has provided the first example of an alternative splicing variant linked to the activation of tumor emt . ron encodes for a tyrosine kinase receptor involved in control of cell dissociation , migration , and matrix invasion . in particular , a constitutively active isoform ( called ron ) , which confers increased motility to expressing cells and accumulates during tumor progression of epithelial cancers [ 63 , 64 ] , is generated through skipping of exon 11 . the oncoprotein srsf1 deeply impacts cell physiology since its upregulation stimulates skipping of exon 11 , thus promoting the production of ron isoform that in turn triggers activation of the emt program increasing the invasive properties of the cells . interestingly , srsf1 expression levels are dynamically controlled in epithelial and mesenchymal cells through an as - nmd event . as - nmd of srsf1 , which involves an intron in the 3utr region of the gene , decreases mrna stability and srsf1 protein levels and , sam68 , the 68 kd src - associated protein in mitosis , is a member of the star ( signal transduction and activation of rna ) family of rna - binding proteins . it contains a single kh - type rna - binding domain and several protein - protein interaction motifs including potential binding sites for sh2 , sh3 , and ww domains , which are characteristic of signaling transducers . sam68 has been recognized as a substrate of several kinases , such as members of the src family , erk1/2 and brk ( breast tumor kinase ) [ 65 , 6870 ] . as such , sam68 is the first identified hub factor able to communicate extracellular stimuli to specific rna splicing decisions . in particular , directed by erk1/2 signaling , sam68 controls as - nmd of srsf1 transcript , thus modulating its protein level . notably , epithelial cell - derived soluble factors are able to inhibit erk1/2 signaling thereby repressing sam68 phosphorylation , which increases the production of the nmd - sensitive transcript of srsf1 . the matrix metalloproteinases ( mmps ) are the most important family of proteinases of the tumor microenvironment that degrade structural components of the extracellular matrix ( ecm ) , thus regulating proliferation , cell - cell adhesion , angiogenesis , invasion , and metastases . in line with this , mmps are upregulated in almost all types of human cancer and associated with poor survival . notably , over - expression of mmp-3 in mammary and lung epithelial cells triggers a cascade of events that determine activation of emt process [ 72 , 73 ] . notably , these events , which induce tumorigenesis process in transgenic mice , are dependent on the expression of a constitutively active alternatively spliced isoform of the rac1 gene , encoding for a small gtpase of the mammalian rho family involved in actin cytoskeleton organization , cell growth , cell - cell adhesion , and migration . this splice variant ( known as rac1b ) is produced by inclusion of the exon 3b that contains the encoding region for a 19-amino acid domain involved in the interaction with regulator and effector molecules . since rac1b shows increased expression during progression of several cancer types [ 72 , 75 , 76 ] , it is tempting to speculate that the exon 3b ( or the 19-amino acid insertion ) could offer the opportunity for a selective targeting to develop anticancer therapies that block emt - associated progression towards advanced tumor stages . the complexity of networks regulating the emt program makes it very difficult to identify early inducers and a unifying molecular basis for this transition . many transcription factors have been extensively studied for their involvement in activation of emt . among these are slug ( referred also to as snai2 ) and twist , two repressors of e - cadherin promoter activity . recent studies have revealed important roles for specific alternatively spliced variants of upstream regulators of slug and twist activity . this is the case of oncogenic splice variant 1 of klf6 ( klf6-sv1 ) , a tumor suppressor gene belonging to the krppel - like family of transcription factors , able to act as functional driver of the entire metastatic cascade through twist induction [ 77 , 78 ] . notably , klf6-sv1 antagonizes the tumor suppressive activity of the full - length klf6 protein and sustains tumor growth and dissemination in ovarian and prostate cancer models [ 7981 ] . interestingly , increased expression of klf6-sv1 occurs in many tumors and is associated with poor prognosis in prostate , lung , and ovarian cancers . klf6-sv1 has been recently shown to induce emt and to drive aggressive multiorgan metastasis formation in both subcutaneous and orthotopic mouse models of breast cancer . in this process , it acts through a twist - dependent mechanism and twist downregulation reverts the phenotype of klf6-sv1 over - expressing cells , restoring the expression of epithelial markers . finally , high levels of klf6-sv1 were found associated with increase in emt markers in a large cohort of primary breast cancer patients . fam3b is a member of the novel fam3 family of cytokine - like genes , predicted to produce at least 7 alternatively spliced isoforms [ 83 , 84 ] . the most studied variant is the secreted form pander , so called for its robust expression and activity in the pancreatic cells [ 85 , 86 ] . in addition , lower pander levels have been observed in human gastric cancers in respect to the corresponding normal tissues . recently , li and colleagues have identified fam3b-258 , a 258-amino acids non - secretory protein , as a novel splicing variant of fam3b up - regulated in colon adenocarcinoma cell lines as well as in human colorectal tumors . fam3b-258 is able to induce changes typical of an activated emt program , stimulating cell migration and invasion in vitro and promoting metastases formation in nude mice . the oncogenic abilities of fam3b-258 require a slug - mediated transcriptional repression of e - cadherin and jam ( junctional adhesion molecule ) . knock - down of slug in fam3b-258 over - expressing cells restores higher levels of e - cadherin and jam and prevents the fam3b-258-dependent cell invasion . whether slug is a direct target of fam3b-258 or whether other signaling effectors are involved remains to be investigated . to become migratory and invasive , cells must extend plasma membrane protrusions ( such as lamellipodia and filopodia ) forward and overcome the epithelial basement layer as a first barrier [ 56 , 88 ] . the formation of invadopodia has been recently characterized as another important step of the emt program [ 8991 ] . invadopodia are enriched with a variety of proteins , including actin and actin regulatory proteins [ 92 , 93 ] . the filamentous actin ( f - actin ) binding protein cortactin is one of the regulators of actin polymerization / branching involved in invadopodia assembly and maturation [ 92 , 94 ] . unlike the full - length protein ( fl ) , the sv1 and sv2 variants lack , respectively , one and two of the six cortactin repeats that mediate the interaction with f - actin . as a consequence of their reduced capabilities in f - acting binding and polymerization , over - expression of sv1- or sv2-cortactin shows reduced cell migration when compared with fl - cortactin over - expressing cells . initially characterized as a component of the spliceosome , spf45 has been also implicated in the regulation of fas and fibronectin alternative splicing [ 97 , 98 ] . in their interesting article , liu and coworkers have demonstrated that spf45 mediates cell migration and invasion in ovarian cancer cells by promoting the formation of the fl - cortactin isoform . finally , spf45 over - expression correlates also with increased cortactin phosphorylation by erk , which enhances cortactin - mediated actin polymerization [ 96 , 99 ] . as for invadopodia , filopodia nucleation and extension require actin cytoskeleton remodeling and involve several regulators of actin dynamics , such as the ena / vasp protein family that in mammals includes three members : mena ( also called enah ) , vasp , and evl . up - regulation of mena has been detected in several human cancers , including breast cancer and melanoma and correlates with invasiveness of breast tumors [ 100 , 101 ] . mena pre - mrna is alternatively spliced to generate different isoforms , expressed in a tissue - specific manner . importantly , the alternative splicing profile of mena in invasive tumor cells is different from non - migratory resident cancer cells . non - invasive tumor cells as well as poorly invasive breast cancer cells with epithelial morphology express mena , the epithelial - associated variant generated from inclusion of exon 11a [ 102 , 103 ] . this exon is inserted within the evh2 domain , very close to the f - actin binding motif and the tetramerization site . this insertion has been predicted to affect the ability of mena tetramers to interact with f - actin and thus to drive filopodia and lamellipodia maturation . in agreement with this , the increment of mena reduces both the number and the length of filopodia in a 3d culture assay . on the contrary , invasive cancer cells lack mena and express mena , an isoform containing an additional exon , also referred to as exon + + + [ 103 , 105 ] . the relative abundance of mena and mena seems to be important to regulate key stages of the metastatic cascade in breast cancer cells . thus , high levels of mena enhance coordinated motility , transendothelial migration , and intravasation of tumor cells , promoting spontaneous lung metastases in a murine model of breast cancer [ 105 , 106 ] . in contrast , increased expression of mena correlates with decreased invasion , intravasation , and dissemination of cancer cells . recently , di modugno and coworkers have identified another splice variant of human mena lacking exon 6 and called hmenav6 . contrary to mena , hmenav6 is expressed selectively in invasive cancer cells with mesenchymal phenotype and is able to enhance invasiveness of breast cancer cells but only in the absence of mena . this evidence suggests that mena behaves as dominant anti - invasive player , making alternative splicing of this gene a potential target for anticancer therapies . furthermore , mena splicing occurs also in primary breast tumors and in particular mena - negative tumors display lower level of e - cadherin when compared to mena - positive samples , supporting the anti - migratory functions of this splicing isoform . splicing of mena exon 11a is regulated by the expression levels of epithelial - specific alternative splicing factors esrp1 and 2 ( epithelial splicing regulatory proteins 1 and 2 ) , two important regulators of the mesenchymal and epithelial splicing signatures . in particular , esrp proteins , by promoting inclusion of exon 11a and the production of mena isoform , cause a drastic reorganization of actin cytoskeleton as well as cell morphology and a reduction of invasive properties . in addition to mena , esrps control the alternative splicing of several genes involved in different aspects of the metastatic cascade , such as cell - cell and cell - matrix adhesion , actin cytoskeleton organization , cell polarity , and migration [ 108 , 109 ] . in line with this , modification of esrps expression levels results in alternative splicing changes of cd44 and p120-catenin , a protein found at cell - cell junctions and also involved in signal transduction [ 108 , 109 ] . the penetration of migrating cancer cells into tissue barriers , including the basement membrane , is supported by the proteolytic degradation of the extracellular matrix ( ecm ) through the activity of secreted enzymes , such as matrix metalloproteinases mmp-2 and mmp-9 . the expression and activity of mmps are regulated through several signals , mainly induced by growth factors and chemokines , as well as through integrin and extracellular matrix - mediated signals . recently , the alternative splicing of the cell adhesion molecule l1 ( l1cam ) has been found to control the invasive capabilities of tumor cells by regulating mmp-2 and mmp-9 expression and activity . more specifically , although initially the splicing variant considered as cancer - associated was sv - l1cam ( lacking of exons 2 and 27 ) , only the full - length fl - l1cam has been found up - regulated upon exposure of tumor cells to the pro - metastatic factors tgf- and hgf . importantly , the over - expression of fl - l1cam but not of the sv isoform is able to induce metastasis formation in mice . invasion and colonization of a secondary organ by disseminating cancer cells are influenced by the microenvironment and the cross - talk between cancer populations and cells in the niche of the receiving tissue [ 56 , 112 ] . the cell adhesion molecule svep1 has been recently involved in the interactive network that affects breast cancer cells homing to bone niches . svep1 expression is stimulated by tnf , a pro - inflammatory cytokine able to affect adhesion and migration , and to induce emt [ 114 , 115 ] . recently , glait - santar and colleagues have investigated the alternative splicing of svep1 transcripts in a co - culture model of pre - osteoblastic mda-15 and mammary adenocarcinoma da3 cells , which mimic the molecular interactions in the bone niche after invasion of breast carcinoma cells . similar to what observed after tnf treatment , several splicing isoforms of svep1 , such as the full - length isoform a and isoform e , are up - regulated in both cell lines upon co - culture conditions . in parallel embryonic variants g and f are silenced in adenocarcinoma da3 cells , whereas no effect is observed in pre - osteoblastoma cells . the same authors also observed that the ratio between splicing isoforms of svep1 is perturbed after treatment with epigenetic drugs such as dna demethylating or histone deacetylase inhibitors , supporting a link between the epigenetic organization and splicing of svep1 pre - mrna . however , further studies are needed to establish the pathological role of the different svep1 isoforms in the metastatic process . several microenvironmental stressors , including nutrient deficiency , low ph , mediators of inflammatory responses , and reactive oxygen species ( ros ) , can affect successful colonization by disseminating cells at the final metastatic tissues . disseminating cancer cells can take advantage of antioxidant systems to counteract the exposure to oxidative stress . the synthesis of reduced glutathione ( gsh ) , a reducing thiol peptide , protects cancer cells against ros - mediated damage and confers resistance to anticancer therapies [ 118 , 119 ] . a rate - limiting factor for gsh synthesis is the availability of cysteine and the cystine transporter system xc- ( composed by two subunits , xct and cd98hc ) is essential in the gsh antioxidant mechanism [ 119 , 120 ] . recently , a link between the gsh - dependent evasion from oxidative stress and alternative splicing of cd44 has been identified [ 121 , 122 ] . as mentioned before , cd44 has an important role in cell - cell and cell - matrix interactions , migration , and invasion . through alternative splicing , cd44 pre - mrna generates multiple cd44 high - molecular - weight isoforms with different extracellular domains [ 41 , 42 ] . intriguingly , cd44v8 - 10 has been demonstrated to interact with the cystine transporter xct , increasing the levels of gsh and , as a consequence , the ability of cancer cells to avoid ros damage . in line with this , cd44v - positive 4t1 mouse breast cancer cells display high levels of gsh and xct and enhanced ros defense compared to cd44v - negative 4t1 cells . thus , cd44v - positive 4t1 are able to establish lung metastatic lesions in mouse models with an incidence higher than cd44v - negative cells . interestingly , down - regulation of the splicing regulator esrp1 in metastatic 4t1 cells shifts the splicing pattern toward the production of cd44s and results in suppression of lung metastasis . on the contrary , forced expression of cd44v8 - 10 in esrp1-depleted cells is sufficient to restore high content of gsh and lung colonization potential . cancer chemotherapy relies on the expectation that anticancer drugs will preferentially kill rapidly dividing tumor cells , rather than normal cells . unfortunately , most pharmacological approaches for the treatment of solid tumors suffer from poor selectivity , which limits the overall dose of drug that can be administered because of unacceptable toxicities to normal tissues . as shown in previous sections , alternative splicing variants of many cancer - related genes can directly contribute to the oncogenic phenotype and to the acquisition of resistance to therapeutic treatments [ 5 , 9 , 10 ] . alternative splicing isoforms selectively expressed by tumors and not by normal tissues may represent suitable targets for new therapeutical approaches . in this section , we discuss some significant examples to illustrate how cancer - specific splicing events can be a powerful source of new diagnostic , prognostic , and therapeutic tools . several highly sensitive methods allowed the identification of cancer - specific splicing isoforms [ 124128 ] . for example , the splicing profile of abcc1 , mdm2 , and fibronectin transcripts has been used to distinguish normal ovary from epithelial ovarian cancer , whereas altered splicing of med24 , mfi2 , srrt , cd44 , and clk1 has been associated with metastatic phenotype in breast cancer and poor prognosis in patients . notably , splicing of hmena may improve the early diagnosis of breast cancer and clinical decision , whereas the balance between splicing variants of klf6 and caspase-9 genes could be useful to predict the susceptibility of cancer cells to chemotherapy [ 129 , 130 ] . along this line , a splicedisease database ( http://cmbi.bjmu.edu.cn/sdisease ) , which provides information for relationships among gene mutations , cancer - specific splice variants may not only serve as diagnostic and prognostic tumor biomarkers but also provide potential targets for the development of new therapeutic strategies . promising avenues towards the development of more selective anticancer drugs are ( i ) antibodies against tumor - associated markers , ( ii ) small molecules targeting the spliceosome or trans - acting splicing regulatory factors , and ( iii ) antisense oligonucleotides that prevent the production of specific aberrant alternative splicing variants . alternative splicing in cancer can generate unique epitopes in the extracellular domain of cell membrane proteins . indeed , many receptors involved in cell - cell and cell - matrix interactions undergo alternative splicing and specific splicing isoforms are associated with human malignancies . these novel , or embryo - restricted , epitopes seem to be ideally suited for tumor - targeting strategies consisting in the delivery of bioactive compounds , for example , monoclonal antibodies ( mabs ) . binding of mabs to tumor - associated biomarkers can determine down - regulation or inhibit the function of the target ( figure 1(a ) ) . moreover , when conjugated with radioemitters or chemotherapics , the mabs can efficiently ensure in situ delivery of the bioactive molecule to cancer cells , sparing normal tissues . a very well studied target for the mabs - mediated therapy is the epidermal growth factor receptor ( egfr ) , which is over - expressed in several tumors . two antibodies directed against the egfr , cetuximab ( c225 ) and panitumumab , are currently used in therapy . unfortunately , since egfr is expressed also in normal tissue , this therapy may have severe side effects . one of this is the egfrviii variant , and two mabs ( ch806 and ch12 ) , targeting the unique extramembrane epitope of egfrviii , are used in clinical trial [ 155 , 156 ] . another cancer - specific egfr isoform , de4 egfr , is also recognized by mab ch12 [ 157 , 158 ] . treatment with ch12 , but not with cetuximab , of mice over - expressing the de4 egfr variant significantly suppresses tumor proliferation and angiogenesis , leading to tumor apoptosis . an important correlation between aberrant in particular , cd44 isoforms containing the variant exon v6 and v8 ( cd44v6 or cd44v8 ) are commonly over - expressed in epithelial tumors . unfortunately , the expression of these isoforms is not confined to cancer cells , but it occurs also in normal tissues , as skin keratinocytes . anti - v6 mabs are effective in treatment of patients with head and neck squamous cell carcinoma but they show severe skin toxicity [ 161 , 162 ] . recently , masuko and collaborators have developed a mab ( gv5 ) against cd44r1 , a cd44 isoform containing exons v8 , v9 , and v10 . gv5 exhibited therapeutic effects in xenografts models , probably by inducing antibody - dependent cellular cytotoxicity , with undetectable reactivity with skin keratinocytes . however , despite these promising developments , solid tumors are frequently resistant to antibody - based therapies probably for the poor penetration of antibodies into the tumor tissue . neo - vascularization ( or angiogenesis ) is needed for growth of cancer cells and for the metastatic process . tumor endothelial cells have a central role in this process because they are readily accessible to drugs via the blood circulation . exploiting this feature , new cancer therapies have been developed to target the tumor vasculature with the aim to deprive the tumor of oxygen and nutrients and induce its regression . endothelial cells of tumor vessels express splicing isoforms of matrix proteins such as the fibronectin ( fn ) [ 165 , 166 ] . for example , the oncofetal isoform containing the extra domain edb of fn is exclusively expressed around newly developing tumor vasculature , whereas it is absent in adult tissue . notably , edb - specific radiolabeled antibodies are used in clinical trial for antiangiogenic cancer treatment . the first drugs used to target the spliceosome machinery , fr901464 and herboxidiene , are natural compounds extracted from bacteria [ 168 , 169 ] . subsequently , synthetic analogues , with less complex structure , therefore with minor synthesis costs , but with higher stability , solubility , and activity , have been obtained . all these molecules have been demonstrated to have selective toxicity and anti - cancer properties in human tumor xenografts . they bind to the splicing factor 3b ( sf3b ) , destabilizing the u2 snrnp - pre - mrna complex and altering the conformation of the branch site sequence . as a consequence , the correct selection of the 3 splice acceptor site fails to occur and alternatively spliced mrnas are generated . one more promising antitumor agent characterized for its anti - proliferative activity is the biflavonoid isoginkgetin , a natural product found in a variety of plants . interestingly , isoginkgetin inhibits the transition from pre - spliceosomal complex a to complex b , probably by preventing stable recruitment of the u4/u5/u6 nuclear ribonucleoproteins . sr proteins are targets of extensive phosphorylation that influences protein interactions and regulates their activity and sub - cellular localization ( figure 1(b ) ) . the benzothiazole compound tg003 has been described as a potent inhibitory of clk1/sty able to affect sfsr1-depending alternative splicing events . interestingly , the exposure of human colon carcinoma cells to tg003 has been described to determine accumulation of the tumor suppressor p53 , the most commonly mutated gene in human cancers . activation of p53 is promoted by down - regulation of mdmx and decreased stability of mdm2 , two key repressors of p53 . interestingly , tg003 seems to contribute to their degradation rather than to change their pre - mrna splicing . further studies are required to elucidate the role of p53 pathway as a sensor of alterations in the splicing machinery . as mentioned before , srsf1 and srsf6 control the choice between vegfxxx ( angiogenic ) and vegfxxxb ( anti - angiogenic ) isoforms . interestingly , phosphorylation of srsf1 by srpk1 promotes the use of the proximal splice site within exon 8 of vegf pre - mrna and thus the production of the angiogenic isoform , while phosphorylation of srsf6 activates the distal splice site in exon 8 . along this line , inhibitors of srpk1 and clk functions , as srpin340 or tg003 , have been shown to block srsf1 activation and to inhibit angiogenesis process both in vitro and in vivo [ 30 , 174 ] . indole derivatives represent a new class of strong splicing inhibitors able to interact with sr proteins and prevent their phosphorylation . some of them show anti - proliferative activity , with an acceptable toxicity . in our recent study , we have used indole derivatives to modulate the splicing event that generates the cancer - associated ron variant . binding of srsf1 to an ese sequence within exon 12 leads to skipping of exon 11 and to the production of the constitutively active ron isoform . , only idc92 is able to revert the invasive phenotype of cancer cells without affecting the splicing profile of other srsf1 targets , suggesting that this small molecule is suitable for further in vivo studies . because of their lack of specificity in modulating pre - mrna splicing , these compounds are expected to cause deleterious undesired events in normal cells as well . surprisingly , however , most of them have been found to possess selective tumor cytotoxicity . one hypothesis is that tumor cells respond differently from normal cells to changes in mrna splicing . alternatively , transformed cells may differ from normal counterparts for the expression of modified version of tumor suppressors originated by aberrant splicing and drug treatment may reverse this defect . however , molecular mechanisms underlying this specificity toward cancerous cells remain elusive and additional studies are necessary to characterize the effects of these drugs in both tumor and normal cells . several works have recently demonstrated that also inhibitors of oncogenic pathway components can indirectly target splicing reactions . for example , treatment of melanoma cells , harbouring b - raf ( v600e ) mutation with b - raf inhibitors , determines over - expression of srsf6 that in turn regulates alternative splicing of the bim gene , a member of the bcl-2 family , promoting the production of the proapoptotic short isoform bims [ 177 , 178 ] . antisense oligonucleotides ( asos ) are short oligonucleotides , usually 1525 bases in length , that are designed to anneal to a specific target region on a pre - mrna molecule , thus interfering with the splicing reaction ( figure 1(c ) ) . aso targeting an exon - intron junction may sterically block the access to the splicing machinery , redirecting splicing reaction to an adjacent splicing site . alternatively , aso can bind to splicing enhancer or silencer elements , masking the sequence to trans - acting regulatory factors and determining inclusion or skipping of specific exons . the latest generations of asos contain chemical modifications and appear more stable compared to conventional oligonucleotides . all asos share the following characteristics : ( i ) they bind tightly to rna through watson - crick base - pairs ; ( ii ) they are specific for rna molecule , and ( iii ) they do not alter the genomic sequence . indeed , the delivery technology , usually nanoparticle , is noninvasive , nontoxic , efficient , and very stable . clinical trials have been already started that exploit asos for treatments of human genetic disorders . even though the use of these molecules in anti - cancer therapy is still at early stages , several recent works report therapeutically relevant and encouraging results . below , we describe several studies , some of them performed on xenograft models of human tumors , while the more recent preliminary results on cancer cell lines are listed in table 2 . the first demonstration of in vivo anti - tumor efficacy of asos was reported by bauman and colleagues . the authors challenged a modified aso , targeting the downstream 5 alternative splice site of exon 2 in bcl - x pre - mrna ( bcl - x aso ) , in a mouse model of metastatic melanoma , an aggressive malignancy that shows poor prognosis when associated with increased expression bcl - xl splice variant . the oligo efficiently redirected splicing machinery to the upstream 5 splice site , decreasing the anti - apoptotic bcl - xl isoform , while increasing the pro - apoptotic bcl - xs variant . importantly , the administration of the oligo coupled to nanoparticles produced a significant reduction of tumor burden in rapidly growing and highly tumorigenic lung metastases . another example is the exploitation of asos to efficiently modify splicing of stat3 , another gene involved in apoptosis . the usage of an alternative acceptor site within exon 23 of stat3 pre - mrna leads to the production of the truncated stat3 isoform that promotes apoptosis and cell - cycle arrest . interestingly , by using a modified aso , targeting a splicing enhancer element that regulates alternative splicing of exon 23 , it was possible to promote a shift from stat3 to stat3 leading to tumor regression in a xenograft model of cancer . recently cartegni 's group showed that the antagonism / association between intronic polyadenylation and pre - mrna splicing can produce truncated soluble receptor tyrosine kinases ( rtks ) . these isoforms can act as dominant - negative regulators since they are deficient of the anchoring transmembrane and the intracellular kinase domains . notably , these secreted decoy receptors are able to inactivate the associated tumorigenic signaling pathways as a consequence of their ability to titrating the ligand or by blocking the wild - type receptors in non - functional heterodimers . interestingly , in the case of the vascular endothelial growth factor receptor 2 ( vegfr2/kdr ) , the key molecule involved in the control of the vegf signaling , morpholino antisense oligonucleotides ( asos extremely stable within biological systems because they are resistant to a wide range of nucleases ) were used to induce the expression of dominant - negative secreted vegfr2/kdr and more importantly to inhibit the angiogenesis process . a new generation of asos called toes ( targeted oligonucleotide enhancers of splicing ) have been developed to induce the inclusion of otherwise skipped exons . toes are complex modified antisense rna oligonucleotides formed by two functionally distinct regions : the 5 half of the oligo is complementary to a sequence within an exon of interest and is followed by a non - complementary rna tail , designed to mimic an ese sequence ( figure 1(c ) ) . in this manner the oligo recruits specific trans - acting regulatory factors ( such as sr proteins ) and provides a sort of enhancer in trans that promotes exon inclusion [ 180 , 186 ] . toes have been first tested for their ability to induce the inclusion of smn2 exon 7 in spinal muscular atrophy ( sma ) patient fibroblasts . the toes technology has been so far applied only once to correct splicing in cancer cells . as described before , srsf1 over - expression produces skipping of ron exon 11 and the production of the oncogenic ron isoform . in order to correct pathological ron splicing , we have designed a toes complementary to the first region of exon 11 and containing a tail of gga repeats , known to function as a strong enhancer . this treatment was able to revert ron splicing and to increase exon 11 inclusion , suggesting the exciting possibility to consider splicing of exon 11 as a possible target of new anti - metastatic therapeutic approaches . another well - established method to down - regulate a specific splicing isoform is through rna interference ( rnai ) . small interfering rnas ( sirna ) are a class of double - stranded rna molecules interfering with the expression of specific genes with complementary nucleotide sequence through endonucleases - mediated degradation mechanism . among the most recent studies on sirna technology performed in xenograft tumor models , it is worth mentioning the article of sangodkar and collaborators . klf6-sv1 , an oncogenic splice variant of the tumor suppressor klf6 gene , is significantly up - regulated in several human cancers [ 81 , 189 ] and its over - expression is associated with decreased survival in prostate and lung cancers [ 79 , 190 ] . sangodkar and colleagues demonstrated that knock - down of this variant via rnai restored chemotherapy sensitivity and induction of apoptosis in lung cancer cells both in vitro and in vivo . conversely , over - expression of klf6-sv1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model . another target for sirna - mediated anticancer therapy is hnrnp l. like to srsf1 , hnrnp l binds to a splicing regulatory element and regulates the splicing profile of caspase-9 gene , which is altered in a large percentage of human lung cancer . l is sufficient to increase the caspase-9a/9b ratio and , more importantly , to cause a complete loss of tumorigenic capacity in xenograft model . finally , some of asos prevent ribosomal assembly and hence mrna translation and seem to be well tolerated in patients . an example of this approach is provided by survivin , an inhibitor of apoptotic proteins . survivin is expressed in several human cancers and its over - expression is associated with a poor prognosis . these features make survivin an attractive target for anti - cancer therapy and several efforts , so far unsuccessful , have been made along this line . recently , down - regulation of survivin has been achieved by using ly2181308 , an aso targeting the translation initiation codon of survivin mrna and inducing its rnase h - mediated degradation . most importantly , in a xenograft tumor model , ly2181308 produced significant anti - tumor activity and sensitized tumor cells to chemotherapeutic - induced apoptosis . all these findings led ly2181308 to be evaluated for clinical trial ( phase ii ) in combination with docetaxel for the treatment of prostate cancer , acute myeloid leukemia , and non - small cell lung cancer [ 196 , 197 ] . the most important concept opened by the results reviewed here is that the rna - binding proteins are at the centre of the oncogenic alternative splicing switch that controls all the major aspects of cancer cell biology ( figure 1 ) . understanding the molecular basis and the effects of the splicing regulation on the transcriptome of cancer cells promises to identify key circuits that have a fundamental role in cell proliferation , apoptosis , and other aspects of tumor progression . moreover , despite the progress , significant challenges remain towards the rational design of more specific and selective approaches able to modulate alternative splicing events in order to control cancer growth . in the era of the personalized medicine , each therapy would have to fit the combination of markers specific for each patient . powerful and cost - effective methods are required to evaluate cancer markers , including those generated by alternative splicing , not only to provide a diagnosis and a prognosis but also to suggest the right personalized therapy .

alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment , progression and resistance to therapeutic treatments . notably , many cancer - associated genes are regulated through alternative splicing suggesting a significant role of this post - transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors . thus , the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis . understanding the molecular underpinnings of cancer - associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer , but will also offer unprecedented opportunities to improve the efficacy of anti - cancer treatments .

1. Introduction 2. Alternative Splicing Changes of Cancer Cells in Response to Hypoxia 3. Invasion and Metastases 4. Diagnostic, Prognostic, and Anticancer Therapy Potentials of Alternative Splicing 5. Conclusions

the first example of alternative splicing was discovered almost 30 years ago , when membrane - bound and secreted antibodies were demonstrated to be encoded by the same gene [ 1 , 2 ] . regulation of alternative splicing decisions involves the recognition of target sequences in the pre - mrna by a number of splicing regulatory factors with antagonistic functions such as sr ( serine - arginine - rich ) and hnrnp ( heterogeneous nuclear ribonucleoprotein ) protein families . the regulation of alternative splicing has been discussed in several excellent reviews [ 68 ] . changes in alternative splicing patterns have an essential role in normal development , differentiation , and in response to physiological stimuli , but aberrant splicing generates variants that contribute to multiple aspects of tumor establishment and progression and in the resistance to therapeutic treatments [ 5 , 9 , 10 ] . many cancer - associated splicing isoforms are expressed during embryonic development , but not in normal adult tissues , whereas others are entirely novel transcripts . central to the splicing oncogenic switch are changes in the expression , activity , or post - translational modification of splicing regulatory factors , such as sr and hnrnp proteins [ 5 , 9 ] . thus , modification of alternative splicing profiles contemporaneously affects multiple key aspects of cancer cell biology , including control of cell proliferation , cancer metabolism , angiogenesis , evasion from apoptosis , invasiveness , and metastasis [ 5 , 9 , 10 ] . here , we discuss aberrant alternative splicing networks that contribute to the oncogenic phenotype and have a prominent role in important aspects of tumorigenesis process , including response to hypoxia and cancer cell invasion and metastasis . in addition , we also discuss important questions connected to the role of alternative splicing in cancer : what are the relevant splicing switches that are critical to malignant transformation ? what are the main functions of cancer - associated alternatively spliced variants ? by illustrating specific examples , it will be clear how the production of cancer - related isoforms offers the potential to develop novel diagnostic , prognostic , and more specific anticancer therapies . through the activation of oncogenes and inactivation of tumor suppressor genes , cancer cells become able to proliferate , survive , and resist to apoptosis . modifications of the microenvironment ( most of these start early during tumor progression ) result from metabolic alterations in cancer cells and from recruitment or activating of nontumoral cells , including blood and lymphatic endothelial cells , pericytes , carcinoma - associated fibroblasts , bone marrow - derived cells , and immune and inflammatory cells [ 15 , 16 ] . due to deregulated cancer cell metabolism ( the consequence of uncontrolled and rapid proliferation ) and to an altered structure and functionality of tumor blood vessels , the tumor microenvironment is characterized by hypoxia and acidosis [ 15 , 17 , 18 ] . vegf isoforms are classified in two families , called vegfxxx ( pro - angiogenic ) and vegfxxxb ( anti - angiogenic ) , where xxx denotes the position of the amino acid residue in the mature protein . pro- or anti - angiogenic isoforms are generated through alternative splicing of the mutually exclusive terminal exons 8a and 8b . the choice between pss or dss depends upon the activity of the splicing factors of the sr family srsf1 and srsf6 : srsf6 preferentially selects the dss and promotes the production of anti - angiogenic vegf isoforms , whereas srsf1 mainly activates the proximal pss . thus , the altered expression of srsf1 and srsf6 in many types of tumors could impact malignant transformation by ensuring the proper balance of pro- and anti - angiogenic isoforms in cancer cells [ 28 , 29 ] . srpk1 is a protein kinase that specifically phosphorylates proteins containing sr domains and is upregulated in various types of cancer , like pancreatic , breast , and colon carcinoma . recently , a genome - wide analysis of hypoxia - induced alternative splicing changes in endothelial cells has identified target genes implicated in angiogenesis - mediated cytoskeleton remodeling ( cask , itsn1 , larp6 , sptan1 , tpm1 , and robo1 ) , in the synthesis of membrane anchors ( pign ) and in the regulation of gene expression ( cugbp1 and max ) . the impact of hypoxia on post - transcriptional events is proven also by lama3-a ( laminin alpha 3 ) , a splicing variant of the lama3 gene induced by reduction of oxygen supply which promotes cell invasion and is associated with a poor prognosis in head and neck cancer [ 33 , 34 ] . hif-3 gene consists of 19 exons and it is subjected to extensive alternative splicing leading to the production of at least six isoforms . notably , in addition to transcriptional level , the expression of cyr61 can be also regulated through an alternative splicing event that stimulates the retention of intron 3 in cyr61 mrna leading to the production of a nonsense - mediated decay ( nmd ) sensible transcript . moreover , in several breast cancer cell lines under hypoxic conditions as - nmd of cyr61 was reported to enhance the expression of the is transcript , suggesting that hypoxia - mediated alternative splicing changes could be a central mechanism regulating the cyr61 expression and its tumor - promoting activity . additional studies are necessary to identify the molecular mechanisms and signaling pathways regulating cd44 alternative splicing in response to hypoxia . hypoxia - induced alternative splicing changes were also recently investigated in the study of hirschfeld and colleagues . furthermore , yt521 alternative splicing targets are well - known cancer - associated genes such as brca2 , esr1 , mdm2 , vegf , and cd44 [ 49 , 5155 ] . similar to other proteins involved in mrna processing , the expression of yt521 is regulated by alternative splicing . interestingly , it was reported that under hypoxic conditions the alternative splicing of yt521 pre - mrna containing exons 8 and 9 generates two transcripts that are subjected to degradation through nmd , suggesting that these as - nmd events of yt521 could simultaneously affect the processing of several cancer - associated genes . further functional investigations on hypoxia and its impact in regard to alternative splicing of target genes may contribute to better understanding of a key regulatory epiphenomenon in tumor growth involved in the development of an aggressive cancer phenotype . as observed for other hallmarks of cancer , gene expression programs implicated in the metastatic process are the same that participate in embryonic development , morphogenesis , and wound healing [ 57 , 58 ] . however , in the last few years , several studies have highlighted additional layers of emt control , including epigenetic reprogramming , small noncoding rnas , translational and post - translational regulations , and alternative splicing changes [ 10 , 57 , 61 , 62 ] . in particular , an increasing body of evidence indicates that splicing regulation alone can drive critical aspects of emt - associated phenotypic changes . below , we discuss some interesting examples of specific alternative splicing events that are implicated not just in the emt / met program but also in the different stages of the metastatic cascade . alternative splicing of the ron proto - oncogene ( also known as mst1r ) has provided the first example of an alternative splicing variant linked to the activation of tumor emt . the oncoprotein srsf1 deeply impacts cell physiology since its upregulation stimulates skipping of exon 11 , thus promoting the production of ron isoform that in turn triggers activation of the emt program increasing the invasive properties of the cells . as - nmd of srsf1 , which involves an intron in the 3utr region of the gene , decreases mrna stability and srsf1 protein levels and , sam68 , the 68 kd src - associated protein in mitosis , is a member of the star ( signal transduction and activation of rna ) family of rna - binding proteins . notably , epithelial cell - derived soluble factors are able to inhibit erk1/2 signaling thereby repressing sam68 phosphorylation , which increases the production of the nmd - sensitive transcript of srsf1 . notably , these events , which induce tumorigenesis process in transgenic mice , are dependent on the expression of a constitutively active alternatively spliced isoform of the rac1 gene , encoding for a small gtpase of the mammalian rho family involved in actin cytoskeleton organization , cell growth , cell - cell adhesion , and migration . this splice variant ( known as rac1b ) is produced by inclusion of the exon 3b that contains the encoding region for a 19-amino acid domain involved in the interaction with regulator and effector molecules . notably , klf6-sv1 antagonizes the tumor suppressive activity of the full - length klf6 protein and sustains tumor growth and dissemination in ovarian and prostate cancer models [ 7981 ] . fam3b is a member of the novel fam3 family of cytokine - like genes , predicted to produce at least 7 alternatively spliced isoforms [ 83 , 84 ] . unlike the full - length protein ( fl ) , the sv1 and sv2 variants lack , respectively , one and two of the six cortactin repeats that mediate the interaction with f - actin . initially characterized as a component of the spliceosome , spf45 has been also implicated in the regulation of fas and fibronectin alternative splicing [ 97 , 98 ] . importantly , the alternative splicing profile of mena in invasive tumor cells is different from non - migratory resident cancer cells . non - invasive tumor cells as well as poorly invasive breast cancer cells with epithelial morphology express mena , the epithelial - associated variant generated from inclusion of exon 11a [ 102 , 103 ] . thus , high levels of mena enhance coordinated motility , transendothelial migration , and intravasation of tumor cells , promoting spontaneous lung metastases in a murine model of breast cancer [ 105 , 106 ] . this evidence suggests that mena behaves as dominant anti - invasive player , making alternative splicing of this gene a potential target for anticancer therapies . furthermore , mena splicing occurs also in primary breast tumors and in particular mena - negative tumors display lower level of e - cadherin when compared to mena - positive samples , supporting the anti - migratory functions of this splicing isoform . in particular , esrp proteins , by promoting inclusion of exon 11a and the production of mena isoform , cause a drastic reorganization of actin cytoskeleton as well as cell morphology and a reduction of invasive properties . in addition to mena , esrps control the alternative splicing of several genes involved in different aspects of the metastatic cascade , such as cell - cell and cell - matrix adhesion , actin cytoskeleton organization , cell polarity , and migration [ 108 , 109 ] . the expression and activity of mmps are regulated through several signals , mainly induced by growth factors and chemokines , as well as through integrin and extracellular matrix - mediated signals . recently , the alternative splicing of the cell adhesion molecule l1 ( l1cam ) has been found to control the invasive capabilities of tumor cells by regulating mmp-2 and mmp-9 expression and activity . more specifically , although initially the splicing variant considered as cancer - associated was sv - l1cam ( lacking of exons 2 and 27 ) , only the full - length fl - l1cam has been found up - regulated upon exposure of tumor cells to the pro - metastatic factors tgf- and hgf . importantly , the over - expression of fl - l1cam but not of the sv isoform is able to induce metastasis formation in mice . recently , glait - santar and colleagues have investigated the alternative splicing of svep1 transcripts in a co - culture model of pre - osteoblastic mda-15 and mammary adenocarcinoma da3 cells , which mimic the molecular interactions in the bone niche after invasion of breast carcinoma cells . however , further studies are needed to establish the pathological role of the different svep1 isoforms in the metastatic process . through alternative splicing , cd44 pre - mrna generates multiple cd44 high - molecular - weight isoforms with different extracellular domains [ 41 , 42 ] . intriguingly , cd44v8 - 10 has been demonstrated to interact with the cystine transporter xct , increasing the levels of gsh and , as a consequence , the ability of cancer cells to avoid ros damage . interestingly , down - regulation of the splicing regulator esrp1 in metastatic 4t1 cells shifts the splicing pattern toward the production of cd44s and results in suppression of lung metastasis . as shown in previous sections , alternative splicing variants of many cancer - related genes can directly contribute to the oncogenic phenotype and to the acquisition of resistance to therapeutic treatments [ 5 , 9 , 10 ] . alternative splicing isoforms selectively expressed by tumors and not by normal tissues may represent suitable targets for new therapeutical approaches . several highly sensitive methods allowed the identification of cancer - specific splicing isoforms [ 124128 ] . for example , the splicing profile of abcc1 , mdm2 , and fibronectin transcripts has been used to distinguish normal ovary from epithelial ovarian cancer , whereas altered splicing of med24 , mfi2 , srrt , cd44 , and clk1 has been associated with metastatic phenotype in breast cancer and poor prognosis in patients . notably , splicing of hmena may improve the early diagnosis of breast cancer and clinical decision , whereas the balance between splicing variants of klf6 and caspase-9 genes could be useful to predict the susceptibility of cancer cells to chemotherapy [ 129 , 130 ] . along this line , a splicedisease database ( http://cmbi.bjmu.edu.cn/sdisease ) , which provides information for relationships among gene mutations , cancer - specific splice variants may not only serve as diagnostic and prognostic tumor biomarkers but also provide potential targets for the development of new therapeutic strategies . promising avenues towards the development of more selective anticancer drugs are ( i ) antibodies against tumor - associated markers , ( ii ) small molecules targeting the spliceosome or trans - acting splicing regulatory factors , and ( iii ) antisense oligonucleotides that prevent the production of specific aberrant alternative splicing variants . alternative splicing in cancer can generate unique epitopes in the extracellular domain of cell membrane proteins . indeed , many receptors involved in cell - cell and cell - matrix interactions undergo alternative splicing and specific splicing isoforms are associated with human malignancies . binding of mabs to tumor - associated biomarkers can determine down - regulation or inhibit the function of the target ( figure 1(a ) ) . moreover , when conjugated with radioemitters or chemotherapics , the mabs can efficiently ensure in situ delivery of the bioactive molecule to cancer cells , sparing normal tissues . unfortunately , the expression of these isoforms is not confined to cancer cells , but it occurs also in normal tissues , as skin keratinocytes . endothelial cells of tumor vessels express splicing isoforms of matrix proteins such as the fibronectin ( fn ) [ 165 , 166 ] . all these molecules have been demonstrated to have selective toxicity and anti - cancer properties in human tumor xenografts . as a consequence , the correct selection of the 3 splice acceptor site fails to occur and alternatively spliced mrnas are generated . interestingly , the exposure of human colon carcinoma cells to tg003 has been described to determine accumulation of the tumor suppressor p53 , the most commonly mutated gene in human cancers . further studies are required to elucidate the role of p53 pathway as a sensor of alterations in the splicing machinery . interestingly , phosphorylation of srsf1 by srpk1 promotes the use of the proximal splice site within exon 8 of vegf pre - mrna and thus the production of the angiogenic isoform , while phosphorylation of srsf6 activates the distal splice site in exon 8 . in our recent study , we have used indole derivatives to modulate the splicing event that generates the cancer - associated ron variant . binding of srsf1 to an ese sequence within exon 12 leads to skipping of exon 11 and to the production of the constitutively active ron isoform . alternatively , transformed cells may differ from normal counterparts for the expression of modified version of tumor suppressors originated by aberrant splicing and drug treatment may reverse this defect . for example , treatment of melanoma cells , harbouring b - raf ( v600e ) mutation with b - raf inhibitors , determines over - expression of srsf6 that in turn regulates alternative splicing of the bim gene , a member of the bcl-2 family , promoting the production of the proapoptotic short isoform bims [ 177 , 178 ] . even though the use of these molecules in anti - cancer therapy is still at early stages , several recent works report therapeutically relevant and encouraging results . the first demonstration of in vivo anti - tumor efficacy of asos was reported by bauman and colleagues . importantly , the administration of the oligo coupled to nanoparticles produced a significant reduction of tumor burden in rapidly growing and highly tumorigenic lung metastases . the usage of an alternative acceptor site within exon 23 of stat3 pre - mrna leads to the production of the truncated stat3 isoform that promotes apoptosis and cell - cycle arrest . interestingly , by using a modified aso , targeting a splicing enhancer element that regulates alternative splicing of exon 23 , it was possible to promote a shift from stat3 to stat3 leading to tumor regression in a xenograft model of cancer . interestingly , in the case of the vascular endothelial growth factor receptor 2 ( vegfr2/kdr ) , the key molecule involved in the control of the vegf signaling , morpholino antisense oligonucleotides ( asos extremely stable within biological systems because they are resistant to a wide range of nucleases ) were used to induce the expression of dominant - negative secreted vegfr2/kdr and more importantly to inhibit the angiogenesis process . as described before , srsf1 over - expression produces skipping of ron exon 11 and the production of the oncogenic ron isoform . these features make survivin an attractive target for anti - cancer therapy and several efforts , so far unsuccessful , have been made along this line . the most important concept opened by the results reviewed here is that the rna - binding proteins are at the centre of the oncogenic alternative splicing switch that controls all the major aspects of cancer cell biology ( figure 1 ) . understanding the molecular basis and the effects of the splicing regulation on the transcriptome of cancer cells promises to identify key circuits that have a fundamental role in cell proliferation , apoptosis , and other aspects of tumor progression . in the era of the personalized medicine , each therapy would have to fit the combination of markers specific for each patient . powerful and cost - effective methods are required to evaluate cancer markers , including those generated by alternative splicing , not only to provide a diagnosis and a prognosis but also to suggest the right personalized therapy .

anatomical - clinical correspondence of swallowing disorder ( dysphagia ) has been poorly studied in acute phases of stroke . in addition , incomplete understanding of spatial and temporal features of cortical processing during swallowing have led to limited insight into the mechanisms explaining dysphagia after brain damage and hence it is hard to predict which cases are likely to develop swallowing dysfunction based on their neuroimaging . on the other hand , utilizing new rehabilitation techniques such as transcranial magnetic stimulation for dysphagia depends on knowledge about specific areas of the motor cortex supportive of swallowing functions . recent studies which have revealed parallel cortical networks functioning during volitional swallow need to clarify individual areas involved in swallowing . however , the information regarding these individual areas is not as solid . the most cited brain areas which are unfolded mainly through lesion studies include the primary sensorimotor cortex , sensorimotor integration areas , the insula and frontal operculum , the anterior cingulate cortex , parietooccipital region , basal ganglia , thalamus , cerebellum , and supplementary motor areas ( smas ) . in particular , the insula is shown to play a role in swallowing , due to the evidence that a lesion in the insular cortex produces profound dysphagia . some studies have revealed the bilateral involvement of the insula , while other researchers have reported activation of the right insula during swallow . there is also some evidence about the presence of anterior insula engagement in dysphagic patients . lowell and other researchers showed that the interactions of the left insula with other brain areas were more prominent than the right one during volitional swallowing . in addition , there is conflicting evidence about the involvement of other areas such as cerebellum in dysphagia problem , making a study of the issue necessary . the objective of current study was to determine the possible clinical - anatomical correspondence of dysphagia in acute conscious stroke patients based on the involvement of cortical brain lesion locations including primary sensory cortex , the primary motor cortex , sma , the insula , prefrontal cortex , inferior frontal cortex , operculum , and supramarginal gyrus ; and the subcortical brain lesion locations comprising cingulate cortex , basal ganglia , thalamus , and cerebellum . moreover , this research aimed at exploring other dysphagia - related brain lesions in neuroimaging of the patients . in a cross - sectional study , 113 conscious first stroke adult patients who satisfied the study inclusion criteria from april 2014 to september 2014 participated in our study . patients were selected using convenience sampling from two teaching hospitals clinics in isfahan , iran . all patients with a cerebrovascular accident who had consecutively admitted into the internal neurology units of teaching hospitals were assessed by a neurologist to confirm the diagnosis of stroke and assessed for evaluating the inclusion criteria . the inclusion criteria were age 18 years and over , stroke for the first time in the acute phase ; no previous history of swallowing disorder . the exclusion criteria were subject who could not cooperate in swallow examination because of low consciousness , low comprehension ( such as wernicke aphasia or mental retardation ) , using ventilator or orogastric tube , subject who suffered from transient ischemic attack or small vessel disease and did not have revealed a focal lesion in neuroimaging were excluded too . during the study course , the patients who revealed the clinical symptoms of vertebrobasilar ischemia , without neuroimaging evidence were excluded . after explaining the purposes of study for patients , a written informed signed consent was obtained participate in the study . the study was approved by the ethic committee of university of social welfare and rehabilitation sciences , tehran , iran ( the ethical code = uswr . checklists were prepared for recording both main study outcomes , i.e. , dysphasia and lesion as being acute in both hemorrhagic and ischemic lesions and involved brain region as well as demographic and medical conditions of the patients including stroke symptoms ( hemiplegia , dysarthria , low consciousness , vertigo , vomiting , headache , confusion , vision problems , and falling ) , stroke risk factors , and comorbidities ( hypertension , diabetes mellitus , hyperlipidemia , smoking , and kidney disease ) were obtained from their medical report . the patients who underwent magnetic resonance imaging ( mri ) ( preferable if accessible ) and computed tomography ( ct ) scan or both within 2472 h after stroke were included in the study ( ct : somatom definition as+ ; siemens healthcare ; 120 kv , 340 ma s , 5.0 mm slice reconstruction , 1.0 mm increment , 0.6 mm collimation , 0.8 pitch , and h30s soft kernel . mri : 1.5 t intera gyroscan , philips achieva , the netherlands ; diffusion - weighted images ( dwi ) with time to repetition ( tr ) 4.25 ms , echo time ( te ) 95 ms , matrix 256 9 256 , field of view 230 9 230 mm , transversal 5 mm thick slices , and b values 0 and 1.0 mm / s ; fluid - attenuated inversion recovery ( flair ) with tr 8.00 ms , te 120 ms , transversal 5 mm thick slices ) . at first , ct scan images were performed to confirm hemorrhagic or nonhemorrhagic stroke patients ; all ct scans were studied before discharge . flair images were studied to localize ischemic lesions . then , dwi images were used to confirm lesion as being acute in both hemorrhagic and ischemic lesions . eventually , flair and dwi images were correlated to demonstrate the exact anatomical location of the acute infarct areas based on the flair images . twelve brain regions which were cited from different authors as relevant areas of dysphagia were entered into a checklist . these regions were observed from both hemispheres which summed up to a total of 24 selected brain regions . the specified brain regions consisted of left and right primary sensory cortex , the primary motor cortex , sma , cingulate cortex , insula , prefrontal cortex , inferior frontal cortex , basal ganglia , thalamus , cerebellum , operculum , and supramarginal gyrus . the checklist from diagnostic of lesion as being acute in both hemorrhagic and ischemic lesions , as well as location of involved brain region aspects , was completed by three observers ( consisting of two neurologists and one radiologist ) independently ; inter - rater agreement was evaluated using kappa statistics leading to an acceptable to high levels of consistency ; ( range kappa : 0.21 ) . they scored the checklists based on a binary scoring system with ( 1 ) for the presence and ( 0 ) for the absence of the lesion in each region . an area was declared as impaired if at least two of three observers agreed on its impairment . the observers added additional areas to the above list according to the individual profiles of the patients as a heuristic aspect of the research . in the current study , the mann assessment of swallowing ability ( masa ) as a validated tool for diagnostics of dysphasia was used ; its sensitivity and specificity have been reported as 91% ( 95% confidence interval [ ci ] : 8295% ) and 74% ( 95% ci : 6480% ) , respectively , and positive predictive value of 95% and inter - rater reliability evaluated with cohen kappa k = 0.76 . masa was performed by an expert speech pathologist with good experience in dysphagia in the first 20 days from onset of stroke ; two dysphagia experts slps have scored the videos of 29 of patients . a high degree of agreement was found between two raters . the average measure intraclass correlation coefficient was obtained as 0.992 ( 95% ci : 0.9830.996 ) . the test quantifies each item according to a scoring system with 5 likert ordinal scale . for example , lip seal was scored from 1 to 5 as follows : ( 1 ) for no closure or unable to assess , ( 2 ) for incomplete seal , ( 3 ) for unilaterally weak or poor maintenance , ( 4 ) for mild impairment and occasional leakage , and ( 5 ) for no abnormality detected on screening . quantitative data were expressed as a mean standard deviation [ sd ] and qualitative variables as frequency ( percentage ) . the chi - square or fisher exact ( as appropriate ) independent sample 's t - test was used for comparing the quantitative data between groups . all statistical analysis was performed using statistical package for social sciences version ( spss , inc . in a cross - sectional study , 113 conscious first stroke adult patients who satisfied the study inclusion criteria from april 2014 to september 2014 participated in our study . patients were selected using convenience sampling from two teaching hospitals clinics in isfahan , iran . all patients with a cerebrovascular accident who had consecutively admitted into the internal neurology units of teaching hospitals were assessed by a neurologist to confirm the diagnosis of stroke and assessed for evaluating the inclusion criteria . the inclusion criteria were age 18 years and over , stroke for the first time in the acute phase ; no previous history of swallowing disorder . the exclusion criteria were subject who could not cooperate in swallow examination because of low consciousness , low comprehension ( such as wernicke aphasia or mental retardation ) , using ventilator or orogastric tube , subject who suffered from transient ischemic attack or small vessel disease and did not have revealed a focal lesion in neuroimaging were excluded too . during the study course , the patients who revealed the clinical symptoms of vertebrobasilar ischemia , without neuroimaging evidence were excluded . after explaining the purposes of study for patients , a written informed signed consent was obtained participate in the study . the study was approved by the ethic committee of university of social welfare and rehabilitation sciences , tehran , iran ( the ethical code = uswr . checklists were prepared for recording both main study outcomes , i.e. , dysphasia and lesion as being acute in both hemorrhagic and ischemic lesions and involved brain region as well as demographic and medical conditions of the patients including stroke symptoms ( hemiplegia , dysarthria , low consciousness , vertigo , vomiting , headache , confusion , vision problems , and falling ) , stroke risk factors , and comorbidities ( hypertension , diabetes mellitus , hyperlipidemia , smoking , and kidney disease ) were obtained from their medical report . the patients who underwent magnetic resonance imaging ( mri ) ( preferable if accessible ) and computed tomography ( ct ) scan or both within 2472 h after stroke were included in the study ( ct : somatom definition as+ ; siemens healthcare ; 120 kv , 340 ma s , 5.0 mm slice reconstruction , 1.0 mm increment , 0.6 mm collimation , 0.8 pitch , and h30s soft kernel . mri : 1.5 t intera gyroscan , philips achieva , the netherlands ; diffusion - weighted images ( dwi ) with time to repetition ( tr ) 4.25 ms , echo time ( te ) 95 ms , matrix 256 9 256 , field of view 230 9 230 mm , transversal 5 mm thick slices , and b values 0 and 1.0 mm / s ; fluid - attenuated inversion recovery ( flair ) with tr 8.00 ms , te 120 ms , transversal 5 mm thick slices ) . at first , ct scan images were performed to confirm hemorrhagic or nonhemorrhagic stroke patients ; all ct scans were studied before discharge . then , dwi images were used to confirm lesion as being acute in both hemorrhagic and ischemic lesions . eventually , flair and dwi images were correlated to demonstrate the exact anatomical location of the acute infarct areas based on the flair images . twelve brain regions which were cited from different authors as relevant areas of dysphagia were entered into a checklist . these regions were observed from both hemispheres which summed up to a total of 24 selected brain regions . the specified brain regions consisted of left and right primary sensory cortex , the primary motor cortex , sma , cingulate cortex , insula , prefrontal cortex , inferior frontal cortex , basal ganglia , thalamus , cerebellum , operculum , and supramarginal gyrus . the checklist from diagnostic of lesion as being acute in both hemorrhagic and ischemic lesions , as well as location of involved brain region aspects , was completed by three observers ( consisting of two neurologists and one radiologist ) independently ; inter - rater agreement was evaluated using kappa statistics leading to an acceptable to high levels of consistency ; ( range kappa : 0.21 ) . they scored the checklists based on a binary scoring system with ( 1 ) for the presence and ( 0 ) for the absence of the lesion in each region . an area was declared as impaired if at least two of three observers agreed on its impairment . the observers added additional areas to the above list according to the individual profiles of the patients as a heuristic aspect of the research . in the current study , the mann assessment of swallowing ability ( masa ) as a validated tool for diagnostics of dysphasia was used ; its sensitivity and specificity have been reported as 91% ( 95% confidence interval [ ci ] : 8295% ) and 74% ( 95% ci : 6480% ) , respectively , and positive predictive value of 95% and inter - rater reliability evaluated with cohen kappa k = 0.76 . masa was performed by an expert speech pathologist with good experience in dysphagia in the first 20 days from onset of stroke ; two dysphagia experts slps have scored the videos of 29 of patients the average measure intraclass correlation coefficient was obtained as 0.992 ( 95% ci : 0.9830.996 ) . the test quantifies each item according to a scoring system with 5 likert ordinal scale . for example , lip seal was scored from 1 to 5 as follows : ( 1 ) for no closure or unable to assess , ( 2 ) for incomplete seal , ( 3 ) for unilaterally weak or poor maintenance , ( 4 ) for mild impairment and occasional leakage , and ( 5 ) for no abnormality detected on screening . quantitative data were expressed as a mean standard deviation [ sd ] and qualitative variables as frequency ( percentage ) . the chi - square or fisher exact ( as appropriate ) independent sample 's t - test was used for comparing the quantitative data between groups . all statistical analysis was performed using statistical package for social sciences version ( spss , inc . twenty - three patients ( % 16.9 ) who suffered from small vessel ischemic stroke were excluded from the study . hence , the data of 113 first acute stroke patients ( 44 female and 69 male ) were finally analyzed . the type of stroke in 92 ( 81.4 ) patients was ischemic , and in other 21 ( 18.6 ) was hemorrhagic . of the 113 subjects , forty ( 35.39 ) were left hemisphere damaged , 59 ( 52.21 ) had right hemisphere stroke , and 15 ( 13.27 ) had brainstem lesion . the test was performed within a mean of 3.8 ( sd : 2.9 ) days postonset . table 1 presents the difference of main demographic and clinical characteristics of study participants in two dysphagic and nondysphagic patients . as can be seen , there are significant differences between two groups in terms of age and prevalence of aphasia . thirteen ( 9.3% ) participants were diagnosed as mild dysphagic ( masa score 168177 ) , moderate dysphagia ( masa score 139167 ) was observed in 16 ( 11.4% ) subjects , and 23 ( 16.4% ) patients were diagnosed as severe dysphagic ( the masa score < 138 ) . aphasia more prevalent among patient in severe category ( 20.2% ) than patients in categories of moderate ( 0% ) and mild ( 5.5% ) ( p = 0.024 ) . no significant differences were found among the patients in mentioned categories in terms of stroke type ( p = 0.59 ) . no statistically significant relationship was observed between the presence of dysphagia and the type of stroke 8 ( 8.7% ) for ischemic versus 1 ( 4.3% ) ( p = 0.47 ) . basic and clinical characteristics of study participants in dysphagic and nondysphagic patients table 2 present the frequency and corresponding percentages of dysphagia in different studied brain sites . as can be seen the presence of dysphasia more significantly higher among patients with involved right primary sensory area ( p = 0.03 ) , right insula ( p < 0.005 ) , and right internal capsule ( p < 0.05 ) . in the other hand , when midbrain , pons , and medulla were categorized as brainstem lesion , a significant relation between brainstem lesion and dysphagia was found ( p = 0.01 ) . the relationship between different variables such as site and type of lesion and the presence of dysphagia in the acute phase of stroke is controversial . yet , the lesion localization as a predictor of dysphagia has recently obtained more interest . while some studies have detected an association between lesion site and dysphagia , others have not found a significant relationship between the location of lesion and presence of dysphagia . the most important finding of this research was the higher frequency of dysphagia in the right hemisphere in all investigated areas than the left hemisphere . as shown by descriptive findings , in most of the proposed regions related to dysphagia , the right hemisphere lesions can predict swallowing disorder , which is in line with some other previous studies . as previously mentioned in the results section , there is a significant relationship between the presence of dysphagia and the right primary sensory cortex lesion . , hamdy et al . , toogood et al . , malandraki et al . , and gonzalez - fernandez et al . the higher occurrence of primary sensory cortex lesion in the dysphagic patients may be attributed to the role of sensory inputs in controlling complex swallowing movements . moreover , based on the association of sensory regions with motor cortex in the process of voluntary movement , it is necessary to study the function of primary motor cortex in future studies . some research studies have shown the role of primary motor cortex in controlling normal swallow in functional magnetic resonance imaging studies and presence of dysphagia in lesion studies . although some dysphagic patients with primary motor area impairment were observed ( left primary motor cortex : n = 4 , 3.5% ; right primary motor cortex : n = 6 , 5.3% ) , the number was not statistically significant , which may emphasize a more critical role of sensory versus motor inputs for controlling swallowing . insula integrates sensorimotor inputs and also is involved in speech and auditory process ( cited by ertekin , 2003 ) . in primates , swallowing was observed after stimulating insula ( cited by ertekin , 2003 ) . moreover , tasting a food stimulates primate insular neurons ( cited by ertekin , 2003 ) . hamdy and aziz believe that interaction of insula as a modulator of motor function with primary sensory cortex lead to control of swallowing ( cited by ertekin , 2003 ) . the result of this study also showed patients who suffer from lesions in primary sensory cortex or insula revealed dysphagia . further studies are necessary to clear the exact role of these two areas and their relation in controlling swallow . zald et al . and have found the dominancy of the right insula in swallow function . besides , martin et al . furthermore , lowell et al . pointed to activation of posterior part of the left insula . these results show that activation of anterior and posterior areas of insula in right and left hemispheres are different during swallow as observed in a previous case study . in this study , insula in each hemisphere was explored , and anterior and posterior insula were not studied separately . further studies need to focus on the role of posterior and anterior insula in swallow . in line with the findings of previous studies about the dysphagia diagnosed 34 weeks after onset of cva following basal ganglia and internal capsule stroke by videofluoroscopy ( vf ) , results of our study showed a significant correlation between the right internal capsule and swallowing disorder . it should be noted our findings was based on diagnostics of within a mean of 3 days after stroke by a bedside examination . this outcome strongly suggests that basal ganglia and left internal capsule stroke patients need to be assessed about 1 month after stroke instrumentally . it differs from other researchers opinion which suggests that regardless of lesion site , all stroke patients need to be screened about dysphagia at the onset of stroke . on the other hand , significant relation of right internal capsule and dysphagia shows the necessity of further studies about the different role of right and left internal capsule in swallowing . some areas have been listed as not significant in the results section . from one point of view , the low number of cases in each area may have led to no significant relationship . for instance , when we considered areas of the brainstem ( midbrain , pons , and medulla oblongata ) individually , the relationship was not significant , but when brainstem was considered as a category , the results became significant . regarding cerebellum and diencephalon which our study did not show a significant relationship with dysphagia , due to low number of patients because of high mortality of them in the acute phase . but it is noteworthy that some researchers have shown bilateral activation of cerebellum during swallow . hence , our results may not interpreted that bilateral diencephalon and cerebellum stroke patients would not suffer from dysphagia . it seems that lesion study is not a fruitful method for studying regions of posterior cerebral artery bloodshed . these areas activities are better examined by functional neuroimaging technologies . swallowing disorder after stroke shows a spontaneous recovery . in this study , the mri was done in the first 3 days after stroke and three observers ( two neurologists and one radiologist ) were reported the images . to our knowledge , the rare lesion studies have explored brain controlling of swallowing in the acute phase of stroke in such a large sample . dysphagia in 105 ( % 93/8 ) was evaluated within 8 days after the stroke . eight other subjects had low consciousness and have been assessed 919 days after stroke ( when they could cooperate in masa ) . these setting made it possible to research these subjects in a prospective study with minimal changes due to spontaneous recovery or treatment . swallowing assessment is a problematic issue in the acute phase of stroke . performing vf , as the gold standard of dysphagia , some researchers were used other instrumental assessments such as fiberoptic endoscopic evaluation of swallowing ( fees ) in acute phase , but fees only assesses pharyngeal phase of swallowing which leads to biased results . in the present work , dysphagia in all cases was evaluated by masa which assesses both oral and pharyngeal phases to control biased previous results . dealing with feeding and respiration problems of acute stroke patients requires prediction of likelihood of developing swallowing dysfunction based on clinical and paraclinical data . this study showed the relation between the right insula , right internal capsule , right primary sensory cortex lesions , and the presence of dysphagia . it also found that in all statistically significant and not significant areas , right hemisphere was involved more than left hemisphere in dysphagic patients . sed contributed in the conception of the work , designing the study , definition of intellectual content , literature review , data gathering , data analysis , manuscript preparation , and agreed for all aspects of the workfy contributed in the conception of the work , designing the study , definition of intellectual content , manuscript editing , manuscript review , and agreed for all aspects of the workaa contributed in the design of the work , definition of intellectual content , statistical model development , data analysis , and agreed for all aspects of the workac and mk contributed in the data acquisition and agreed for all aspects of the work . sed contributed in the conception of the work , designing the study , definition of intellectual content , literature review , data gathering , data analysis , manuscript preparation , and agreed for all aspects of the work fy contributed in the conception of the work , designing the study , definition of intellectual content , manuscript editing , manuscript review , and agreed for all aspects of the work aa contributed in the design of the work , definition of intellectual content , statistical model development , data analysis , and agreed for all aspects of the work ac and mk contributed in the data acquisition and agreed for all aspects of the work .

background : limited data available about the mechanisms of dysphagia and areas involving swallow after brain damage ; accordingly it is hard to predict which cases are more likely to develop swallowing dysfunction based on the neuroimaging . the aim of this study was to investigate the relationship between brain lesions and dysphagia in a sample of acute conscious stroke patients.materials and methods : in a cross - sectional study , 113 acute conscious stroke patients ( 69 male mean [ standard deviation ( sd ) ] age 64.37 [ 15.1 ] ) , participated in this study . two neurologists and one radiologist localized brain lesions according to neuroimaging of the patients . swallowing functions were assessed clinically by an expert speech pathologist with the mann assessment of swallowing ability ( masa ) . the association of brain region and swallowing problem was statistically evaluated using chi - square test.results:mean ( sd ) masa score for the dysphagic patients was 139.61 ( 29.77 ) . swallowing problem was significantly more prevalent in the right primary sensory ( p = 0.03 ) , right insula ( p = 0.005 ) , and right internal capsule ( p = 0.05).conclusion : it may be concluded from these findings that the right hemisphere lesions associated with occurring dysphagia . further studies using more advanced diagnostic tools on big samples particularly in a perspective structure are needed .

INTRODUCTION MATERIALS AND METHODS Study design and participants Outcome evaluation: Neuroimaging acquisition and interpretation Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest AUTHORS CONTRIBUTION

in addition , incomplete understanding of spatial and temporal features of cortical processing during swallowing have led to limited insight into the mechanisms explaining dysphagia after brain damage and hence it is hard to predict which cases are likely to develop swallowing dysfunction based on their neuroimaging . on the other hand , utilizing new rehabilitation techniques such as transcranial magnetic stimulation for dysphagia depends on knowledge about specific areas of the motor cortex supportive of swallowing functions . the most cited brain areas which are unfolded mainly through lesion studies include the primary sensorimotor cortex , sensorimotor integration areas , the insula and frontal operculum , the anterior cingulate cortex , parietooccipital region , basal ganglia , thalamus , cerebellum , and supplementary motor areas ( smas ) . some studies have revealed the bilateral involvement of the insula , while other researchers have reported activation of the right insula during swallow . there is also some evidence about the presence of anterior insula engagement in dysphagic patients . lowell and other researchers showed that the interactions of the left insula with other brain areas were more prominent than the right one during volitional swallowing . in addition , there is conflicting evidence about the involvement of other areas such as cerebellum in dysphagia problem , making a study of the issue necessary . the objective of current study was to determine the possible clinical - anatomical correspondence of dysphagia in acute conscious stroke patients based on the involvement of cortical brain lesion locations including primary sensory cortex , the primary motor cortex , sma , the insula , prefrontal cortex , inferior frontal cortex , operculum , and supramarginal gyrus ; and the subcortical brain lesion locations comprising cingulate cortex , basal ganglia , thalamus , and cerebellum . moreover , this research aimed at exploring other dysphagia - related brain lesions in neuroimaging of the patients . in a cross - sectional study , 113 conscious first stroke adult patients who satisfied the study inclusion criteria from april 2014 to september 2014 participated in our study . the inclusion criteria were age 18 years and over , stroke for the first time in the acute phase ; no previous history of swallowing disorder . the exclusion criteria were subject who could not cooperate in swallow examination because of low consciousness , low comprehension ( such as wernicke aphasia or mental retardation ) , using ventilator or orogastric tube , subject who suffered from transient ischemic attack or small vessel disease and did not have revealed a focal lesion in neuroimaging were excluded too . during the study course , the patients who revealed the clinical symptoms of vertebrobasilar ischemia , without neuroimaging evidence were excluded . after explaining the purposes of study for patients , a written informed signed consent was obtained participate in the study . the study was approved by the ethic committee of university of social welfare and rehabilitation sciences , tehran , iran ( the ethical code = uswr . , dysphasia and lesion as being acute in both hemorrhagic and ischemic lesions and involved brain region as well as demographic and medical conditions of the patients including stroke symptoms ( hemiplegia , dysarthria , low consciousness , vertigo , vomiting , headache , confusion , vision problems , and falling ) , stroke risk factors , and comorbidities ( hypertension , diabetes mellitus , hyperlipidemia , smoking , and kidney disease ) were obtained from their medical report . the patients who underwent magnetic resonance imaging ( mri ) ( preferable if accessible ) and computed tomography ( ct ) scan or both within 2472 h after stroke were included in the study ( ct : somatom definition as+ ; siemens healthcare ; 120 kv , 340 ma s , 5.0 mm slice reconstruction , 1.0 mm increment , 0.6 mm collimation , 0.8 pitch , and h30s soft kernel . mri : 1.5 t intera gyroscan , philips achieva , the netherlands ; diffusion - weighted images ( dwi ) with time to repetition ( tr ) 4.25 ms , echo time ( te ) 95 ms , matrix 256 9 256 , field of view 230 9 230 mm , transversal 5 mm thick slices , and b values 0 and 1.0 mm / s ; fluid - attenuated inversion recovery ( flair ) with tr 8.00 ms , te 120 ms , transversal 5 mm thick slices ) . eventually , flair and dwi images were correlated to demonstrate the exact anatomical location of the acute infarct areas based on the flair images . the specified brain regions consisted of left and right primary sensory cortex , the primary motor cortex , sma , cingulate cortex , insula , prefrontal cortex , inferior frontal cortex , basal ganglia , thalamus , cerebellum , operculum , and supramarginal gyrus . the checklist from diagnostic of lesion as being acute in both hemorrhagic and ischemic lesions , as well as location of involved brain region aspects , was completed by three observers ( consisting of two neurologists and one radiologist ) independently ; inter - rater agreement was evaluated using kappa statistics leading to an acceptable to high levels of consistency ; ( range kappa : 0.21 ) . they scored the checklists based on a binary scoring system with ( 1 ) for the presence and ( 0 ) for the absence of the lesion in each region . the observers added additional areas to the above list according to the individual profiles of the patients as a heuristic aspect of the research . in the current study , the mann assessment of swallowing ability ( masa ) as a validated tool for diagnostics of dysphasia was used ; its sensitivity and specificity have been reported as 91% ( 95% confidence interval [ ci ] : 8295% ) and 74% ( 95% ci : 6480% ) , respectively , and positive predictive value of 95% and inter - rater reliability evaluated with cohen kappa k = 0.76 . masa was performed by an expert speech pathologist with good experience in dysphagia in the first 20 days from onset of stroke ; two dysphagia experts slps have scored the videos of 29 of patients . the test quantifies each item according to a scoring system with 5 likert ordinal scale . for example , lip seal was scored from 1 to 5 as follows : ( 1 ) for no closure or unable to assess , ( 2 ) for incomplete seal , ( 3 ) for unilaterally weak or poor maintenance , ( 4 ) for mild impairment and occasional leakage , and ( 5 ) for no abnormality detected on screening . quantitative data were expressed as a mean standard deviation [ sd ] and qualitative variables as frequency ( percentage ) . the chi - square or fisher exact ( as appropriate ) independent sample 's t - test was used for comparing the quantitative data between groups . in a cross - sectional study , 113 conscious first stroke adult patients who satisfied the study inclusion criteria from april 2014 to september 2014 participated in our study . all patients with a cerebrovascular accident who had consecutively admitted into the internal neurology units of teaching hospitals were assessed by a neurologist to confirm the diagnosis of stroke and assessed for evaluating the inclusion criteria . the inclusion criteria were age 18 years and over , stroke for the first time in the acute phase ; no previous history of swallowing disorder . the exclusion criteria were subject who could not cooperate in swallow examination because of low consciousness , low comprehension ( such as wernicke aphasia or mental retardation ) , using ventilator or orogastric tube , subject who suffered from transient ischemic attack or small vessel disease and did not have revealed a focal lesion in neuroimaging were excluded too . after explaining the purposes of study for patients , a written informed signed consent was obtained participate in the study . the study was approved by the ethic committee of university of social welfare and rehabilitation sciences , tehran , iran ( the ethical code = uswr . , dysphasia and lesion as being acute in both hemorrhagic and ischemic lesions and involved brain region as well as demographic and medical conditions of the patients including stroke symptoms ( hemiplegia , dysarthria , low consciousness , vertigo , vomiting , headache , confusion , vision problems , and falling ) , stroke risk factors , and comorbidities ( hypertension , diabetes mellitus , hyperlipidemia , smoking , and kidney disease ) were obtained from their medical report . the patients who underwent magnetic resonance imaging ( mri ) ( preferable if accessible ) and computed tomography ( ct ) scan or both within 2472 h after stroke were included in the study ( ct : somatom definition as+ ; siemens healthcare ; 120 kv , 340 ma s , 5.0 mm slice reconstruction , 1.0 mm increment , 0.6 mm collimation , 0.8 pitch , and h30s soft kernel . mri : 1.5 t intera gyroscan , philips achieva , the netherlands ; diffusion - weighted images ( dwi ) with time to repetition ( tr ) 4.25 ms , echo time ( te ) 95 ms , matrix 256 9 256 , field of view 230 9 230 mm , transversal 5 mm thick slices , and b values 0 and 1.0 mm / s ; fluid - attenuated inversion recovery ( flair ) with tr 8.00 ms , te 120 ms , transversal 5 mm thick slices ) . eventually , flair and dwi images were correlated to demonstrate the exact anatomical location of the acute infarct areas based on the flair images . the specified brain regions consisted of left and right primary sensory cortex , the primary motor cortex , sma , cingulate cortex , insula , prefrontal cortex , inferior frontal cortex , basal ganglia , thalamus , cerebellum , operculum , and supramarginal gyrus . the checklist from diagnostic of lesion as being acute in both hemorrhagic and ischemic lesions , as well as location of involved brain region aspects , was completed by three observers ( consisting of two neurologists and one radiologist ) independently ; inter - rater agreement was evaluated using kappa statistics leading to an acceptable to high levels of consistency ; ( range kappa : 0.21 ) . they scored the checklists based on a binary scoring system with ( 1 ) for the presence and ( 0 ) for the absence of the lesion in each region . the observers added additional areas to the above list according to the individual profiles of the patients as a heuristic aspect of the research . in the current study , the mann assessment of swallowing ability ( masa ) as a validated tool for diagnostics of dysphasia was used ; its sensitivity and specificity have been reported as 91% ( 95% confidence interval [ ci ] : 8295% ) and 74% ( 95% ci : 6480% ) , respectively , and positive predictive value of 95% and inter - rater reliability evaluated with cohen kappa k = 0.76 . masa was performed by an expert speech pathologist with good experience in dysphagia in the first 20 days from onset of stroke ; two dysphagia experts slps have scored the videos of 29 of patients the average measure intraclass correlation coefficient was obtained as 0.992 ( 95% ci : 0.9830.996 ) . for example , lip seal was scored from 1 to 5 as follows : ( 1 ) for no closure or unable to assess , ( 2 ) for incomplete seal , ( 3 ) for unilaterally weak or poor maintenance , ( 4 ) for mild impairment and occasional leakage , and ( 5 ) for no abnormality detected on screening . quantitative data were expressed as a mean standard deviation [ sd ] and qualitative variables as frequency ( percentage ) . the chi - square or fisher exact ( as appropriate ) independent sample 's t - test was used for comparing the quantitative data between groups . twenty - three patients ( % 16.9 ) who suffered from small vessel ischemic stroke were excluded from the study . hence , the data of 113 first acute stroke patients ( 44 female and 69 male ) were finally analyzed . the type of stroke in 92 ( 81.4 ) patients was ischemic , and in other 21 ( 18.6 ) was hemorrhagic . of the 113 subjects , forty ( 35.39 ) were left hemisphere damaged , 59 ( 52.21 ) had right hemisphere stroke , and 15 ( 13.27 ) had brainstem lesion . thirteen ( 9.3% ) participants were diagnosed as mild dysphagic ( masa score 168177 ) , moderate dysphagia ( masa score 139167 ) was observed in 16 ( 11.4% ) subjects , and 23 ( 16.4% ) patients were diagnosed as severe dysphagic ( the masa score < 138 ) . aphasia more prevalent among patient in severe category ( 20.2% ) than patients in categories of moderate ( 0% ) and mild ( 5.5% ) ( p = 0.024 ) . no significant differences were found among the patients in mentioned categories in terms of stroke type ( p = 0.59 ) . no statistically significant relationship was observed between the presence of dysphagia and the type of stroke 8 ( 8.7% ) for ischemic versus 1 ( 4.3% ) ( p = 0.47 ) . basic and clinical characteristics of study participants in dysphagic and nondysphagic patients table 2 present the frequency and corresponding percentages of dysphagia in different studied brain sites . as can be seen the presence of dysphasia more significantly higher among patients with involved right primary sensory area ( p = 0.03 ) , right insula ( p < 0.005 ) , and right internal capsule ( p < 0.05 ) . in the other hand , when midbrain , pons , and medulla were categorized as brainstem lesion , a significant relation between brainstem lesion and dysphagia was found ( p = 0.01 ) . the relationship between different variables such as site and type of lesion and the presence of dysphagia in the acute phase of stroke is controversial . while some studies have detected an association between lesion site and dysphagia , others have not found a significant relationship between the location of lesion and presence of dysphagia . the most important finding of this research was the higher frequency of dysphagia in the right hemisphere in all investigated areas than the left hemisphere . as shown by descriptive findings , in most of the proposed regions related to dysphagia , the right hemisphere lesions can predict swallowing disorder , which is in line with some other previous studies . as previously mentioned in the results section , there is a significant relationship between the presence of dysphagia and the right primary sensory cortex lesion . the higher occurrence of primary sensory cortex lesion in the dysphagic patients may be attributed to the role of sensory inputs in controlling complex swallowing movements . moreover , based on the association of sensory regions with motor cortex in the process of voluntary movement , it is necessary to study the function of primary motor cortex in future studies . some research studies have shown the role of primary motor cortex in controlling normal swallow in functional magnetic resonance imaging studies and presence of dysphagia in lesion studies . although some dysphagic patients with primary motor area impairment were observed ( left primary motor cortex : n = 4 , 3.5% ; right primary motor cortex : n = 6 , 5.3% ) , the number was not statistically significant , which may emphasize a more critical role of sensory versus motor inputs for controlling swallowing . in primates , swallowing was observed after stimulating insula ( cited by ertekin , 2003 ) . hamdy and aziz believe that interaction of insula as a modulator of motor function with primary sensory cortex lead to control of swallowing ( cited by ertekin , 2003 ) . the result of this study also showed patients who suffer from lesions in primary sensory cortex or insula revealed dysphagia . and have found the dominancy of the right insula in swallow function . these results show that activation of anterior and posterior areas of insula in right and left hemispheres are different during swallow as observed in a previous case study . in this study , insula in each hemisphere was explored , and anterior and posterior insula were not studied separately . further studies need to focus on the role of posterior and anterior insula in swallow . in line with the findings of previous studies about the dysphagia diagnosed 34 weeks after onset of cva following basal ganglia and internal capsule stroke by videofluoroscopy ( vf ) , results of our study showed a significant correlation between the right internal capsule and swallowing disorder . it should be noted our findings was based on diagnostics of within a mean of 3 days after stroke by a bedside examination . this outcome strongly suggests that basal ganglia and left internal capsule stroke patients need to be assessed about 1 month after stroke instrumentally . it differs from other researchers opinion which suggests that regardless of lesion site , all stroke patients need to be screened about dysphagia at the onset of stroke . on the other hand , significant relation of right internal capsule and dysphagia shows the necessity of further studies about the different role of right and left internal capsule in swallowing . for instance , when we considered areas of the brainstem ( midbrain , pons , and medulla oblongata ) individually , the relationship was not significant , but when brainstem was considered as a category , the results became significant . in this study , the mri was done in the first 3 days after stroke and three observers ( two neurologists and one radiologist ) were reported the images . to our knowledge , the rare lesion studies have explored brain controlling of swallowing in the acute phase of stroke in such a large sample . these setting made it possible to research these subjects in a prospective study with minimal changes due to spontaneous recovery or treatment . performing vf , as the gold standard of dysphagia , some researchers were used other instrumental assessments such as fiberoptic endoscopic evaluation of swallowing ( fees ) in acute phase , but fees only assesses pharyngeal phase of swallowing which leads to biased results . in the present work , dysphagia in all cases was evaluated by masa which assesses both oral and pharyngeal phases to control biased previous results . dealing with feeding and respiration problems of acute stroke patients requires prediction of likelihood of developing swallowing dysfunction based on clinical and paraclinical data . this study showed the relation between the right insula , right internal capsule , right primary sensory cortex lesions , and the presence of dysphagia . it also found that in all statistically significant and not significant areas , right hemisphere was involved more than left hemisphere in dysphagic patients . sed contributed in the conception of the work , designing the study , definition of intellectual content , literature review , data gathering , data analysis , manuscript preparation , and agreed for all aspects of the workfy contributed in the conception of the work , designing the study , definition of intellectual content , manuscript editing , manuscript review , and agreed for all aspects of the workaa contributed in the design of the work , definition of intellectual content , statistical model development , data analysis , and agreed for all aspects of the workac and mk contributed in the data acquisition and agreed for all aspects of the work . sed contributed in the conception of the work , designing the study , definition of intellectual content , literature review , data gathering , data analysis , manuscript preparation , and agreed for all aspects of the work fy contributed in the conception of the work , designing the study , definition of intellectual content , manuscript editing , manuscript review , and agreed for all aspects of the work aa contributed in the design of the work , definition of intellectual content , statistical model development , data analysis , and agreed for all aspects of the work ac and mk contributed in the data acquisition and agreed for all aspects of the work .

several corrective procedures have been described , with the goal to provide a pain free , plantigrade foot . the ilizarov method of external fixation and gradual distraction has been reported as an alternative to conventional techniques . encouraging results are reported with this method.1 the ilizarov 's external fixator allows simultaneous correction of all the severe foot deformities associated with neglected clubfoot with minimal surgery , reducing risks of cutaneous or neurovascular complications and avoiding excessive shortening of the foot.2 the external fixation remains an essential tool for future orthopedic surgeons.3 the ilizarov method is being used increasingly to correct many orthopedic deformities . the frames required for ankle and foot deformity correction are among the most difficult to construct owing to the complexity of the deformities which must be corrected.4 the application of ilizarov frame is in two parts , one to the leg and one to the foot . the foot construct consists of a half ring posteriorly and half ring anteriorly.5 a half ring is fixed to the forefoot with 2 crossed olive wires , 1 traversing the 5 metatarsals and the other fixed to the medial 3 metatarsals medially to laterally . a half ring was applied to the hind foot with 2 crossed wires through the calcaneus , with a shanz pin fixed to the calcaneus from posterior to anterior . the forefoot half ring was fixed to the hindfoot half ring by a medial rod on a hinge and connected to the distal tibial ring by a central dorsal rod on a universal hinge . two dorsal rods were used on each side of the central hole of the forefoot half ring in patients with forefoot supination.6 the ilizarov technique has been suggested as an alternative to major surgery for the treatment of neglected or recurrent deformities in clubfoot.7 even in those patients in whom final corrective arthrodesis is necessary , this may be carried out with minimal bone resection , since the severe deformities of the foot and ankle have been corrected.8 we report a series of neglected resistant clubfoot deformities treated by limited surgery and ilizarov distraction histogenesis . we evaluated the results of this prospective study with ilizarov 's external fixation system in 21 patients with 27 feet without corrective osteotomies as treatment of neglected resistant clubfoot deformity , between april 2005 and may 2008 . this study includes a resistant clubfeet ( n=13 ) which were treated for the first time , already operated clubfeet by other methods ( n=14 ) , and also presented with multiple surgical scars ( n=5 ) . all resistant clubfeet with common deformities like adduction or supination , or both , of the forefoot , short medial column and long lateral column , varus of the hind foot , and equinus of the ankle were included to decide the same surgical planning with ilizarov in all patients because it permits simultaneous correction of these multiplanar deformities . resistant clubfeet with non healing ulcers over the callosities on the dorsum of lateral column due to weight bearing were excluded from the study . minimal soft tissue procedures for those were done below the age of 12 years depending upon their flexibility to correct the deformities without stress over the soft tissues with ilizarov method during distraction and compression . gait of all patients like their walking pattern were assessed clinically through functional mobility scale9 at the time of admission and every step was planned like physical assistance , bracing , and assistive devices to improve the speed , distance and efficiency while walking . they were also assessed for skin and soft tissue conditions , range of ankle and subtalar joint movement , levels of the particular deformity , limb shortening . the radiographs were obtained for standard anteroposterior and lateral views with and without weight bearing . the condition of ankle and other joints of foot , angles of individual deformities were assessed . ilizarov frame was preassembled according to the deformity of foot as well as radiographs to correct without any stress over the related soft tissues . two full rings , mostly of 16 and 18 hole sizes , were assembled and connected with two rods and mounted over the tibia with one transverse wire and two 5 mm half pins with rancho cube system in each ring . one half ring was mounted to the calcaneus , using one plain wire and two shanz screws with rancho system . another half ring with two plain crossed wires was applied through 1 to 3 metatarsal and from 5 to 2 or 3 metatarsal and connected with the rings applied over the tibia through the hinges , plates , and conical washers were also used wherever required . the calcaneal half ring had three connections to the lower ring of the tibia frame ( posteriorly , medially , and laterally ) , while the forefoot half ring had two connections situated medially and laterally and sometimes only one connection situated centrally to the long axis of the foot . the hinges allow the rods to move without bending and also prevent the subluxation of joints during distraction . half ring over the calcaneus was used as a distractor or pusher and compression or pulling was achieved through the half ring over dorsum of the fore foot . varus deformities of the heel were corrected through medial and lateral rods by compression and distraction method . these rods with hinges were applied in the rings fastened over the calcaneus and forefoot to increase or decrease the medial or lateral column according to the need of deformity correction . for correction of rotational like supination and pronation deformities , two frontal rods with hinges were applied in oblique directions connected with the ring in the forefoot and assembly over the leg and corrected accordingly . the sequence of deformities correction was gradual distraction of medial column and compression of lateral column to correct the forefoot adduction , 12 mm / day , which was started on the 2 postoperative day . after correction of forefoot adduction supination , hind foot varus and finally equinus were corrected through the setting of the ilizarov 's external fixator . during distraction , tension over the soft tissues , neurovascular status , and improvement in the correction of the deformities were observed clinically , and radiologically dislocation or subluxation of tarsal and ankle joints was observed [ figure 1 ] . ilizarov frame for ankle and foot with two full rings for leg , one half for calcaneum and one half for forefoot over the metatarsals after application of ilizarov , a limited soft tissue dissection , percutaneous achilles sheath tenotomy , and plantar fasciotomy were done for patients under 12 years and achilles tendon lengthening was done for those above 12 years of age . achilles tendon lengthening was performed in 9 ( 59.93% ) feet , achilles tendon sheath tenotomy in 8 ( 29.62% ) feet , and planter fasciotomy in 6 ( 22.22% ) feet . approximately 810 deformity was over corrected and this over lengthening of soft tissues avoided the recoil that could occur in the soft tissue after frame removal . all feet were also assessed radiologically as well as clinically every week during the followup period . all patients were encouraged and weight bearing was allowed according to tolerance of the patients and their degree of correction . adjustments were done by the surgeon and postgraduate students during stay in the hospital , and after discharge by either the patient himself / herself , and if the patient was a child , the family members were trained to move the nuts for compression , distraction , and lengthening through the markings . care of pins was taken during their stay in the hospital , and patients and their attendants were trained to take care of pins by themselves after discharge . all the pins were cleaned with normal saline and dressed with sterilized dry gauzes initially after 24 hours than after 72 hours . loose clamps , nuts , and bolts were tightened regularly in outpatient clinic initially weekly up to deformity correction , then fortnightly up to removal of the fixator . the fixator removal was decided after correction of all required deformities and the time needed for correction of the deformities was variable depending on the resistance and rigidity of the deformities . after complete correction , the distraction stopped and the ilizarov was retained in a fixed position for a further period of 46 weeks . during this period , up to the removal , the patients were encouraged and allowed to bear full weight and improve the gait . fixator was removed , all the pin wounds were washed , and aseptic dressings were done ; a short leg walking plaster of paris ( pop ) cast was applied for 2 weeks , the patients were reviewed at the outpatient clinic after 2 weeks , and the pop cast was removed . the status of wounds was checked and again short leg walking cast was applied for 4 more weeks . the total time of the pop cast was 6 weeks after removal of the fixator , followed by an ankle foot orthrosis ( afo ) for 3 months . ilizarov frame was preassembled according to the deformity of foot as well as radiographs to correct without any stress over the related soft tissues . two full rings , mostly of 16 and 18 hole sizes , were assembled and connected with two rods and mounted over the tibia with one transverse wire and two 5 mm half pins with rancho cube system in each ring . one half ring was mounted to the calcaneus , using one plain wire and two shanz screws with rancho system . another half ring with two plain crossed wires was applied through 1 to 3 metatarsal and from 5 to 2 or 3 metatarsal and connected with the rings applied over the tibia through the hinges , plates , and conical washers were also used wherever required . the calcaneal half ring had three connections to the lower ring of the tibia frame ( posteriorly , medially , and laterally ) , while the forefoot half ring had two connections situated medially and laterally and sometimes only one connection situated centrally to the long axis of the foot . the hinges allow the rods to move without bending and also prevent the subluxation of joints during distraction . half ring over the calcaneus was used as a distractor or pusher and compression or pulling was achieved through the half ring over dorsum of the fore foot . varus deformities of the heel were corrected through medial and lateral rods by compression and distraction method . these rods with hinges were applied in the rings fastened over the calcaneus and forefoot to increase or decrease the medial or lateral column according to the need of deformity correction . for correction of rotational like supination and pronation deformities , two frontal rods with hinges were applied in oblique directions connected with the ring in the forefoot and assembly over the leg and corrected accordingly . the sequence of deformities correction was gradual distraction of medial column and compression of lateral column to correct the forefoot adduction , 12 mm / day , which was started on the 2 postoperative day . after correction of forefoot adduction supination , hind foot varus and finally equinus were corrected through the setting of the ilizarov 's external fixator . during distraction , tension over the soft tissues , neurovascular status , and improvement in the correction of the deformities were observed clinically , and radiologically dislocation or subluxation of tarsal and ankle joints was observed [ figure 1 ] . ilizarov frame for ankle and foot with two full rings for leg , one half for calcaneum and one half for forefoot over the metatarsals after application of ilizarov , a limited soft tissue dissection , percutaneous achilles sheath tenotomy , and plantar fasciotomy were done for patients under 12 years and achilles tendon lengthening was done for those above 12 years of age . achilles tendon lengthening was performed in 9 ( 59.93% ) feet , achilles tendon sheath tenotomy in 8 ( 29.62% ) feet , and planter fasciotomy in 6 ( 22.22% ) feet . approximately 810 deformity was over corrected and this over lengthening of soft tissues avoided the recoil that could occur in the soft tissue after frame removal . all feet were also assessed radiologically as well as clinically every week during the followup period . all patients were encouraged and weight bearing was allowed according to tolerance of the patients and their degree of correction . adjustments were done by the surgeon and postgraduate students during stay in the hospital , and after discharge by either the patient himself / herself , and if the patient was a child , the family members were trained to move the nuts for compression , distraction , and lengthening through the markings . care of pins was taken during their stay in the hospital , and patients and their attendants were trained to take care of pins by themselves after discharge . all the pins were cleaned with normal saline and dressed with sterilized dry gauzes initially after 24 hours than after 72 hours . loose clamps , nuts , and bolts were tightened regularly in outpatient clinic initially weekly up to deformity correction , then fortnightly up to removal of the fixator . the fixator removal was decided after correction of all required deformities and the time needed for correction of the deformities was variable depending on the resistance and rigidity of the deformities . after complete correction , the distraction stopped and the ilizarov was retained in a fixed position for a further period of 46 weeks . during this period , up to the removal , the patients were encouraged and allowed to bear full weight and improve the gait . fixator was removed , all the pin wounds were washed , and aseptic dressings were done ; a short leg walking plaster of paris ( pop ) cast was applied for 2 weeks , the patients were reviewed at the outpatient clinic after 2 weeks , and the pop cast was removed . the status of wounds was checked and again short leg walking cast was applied for 4 more weeks . the total time of the pop cast was 6 weeks after removal of the fixator , followed by an ankle foot orthrosis ( afo ) for 3 months . the mean distraction time for deformity correction was 7.2 weeks ( range 4 - 12 weeks ) . ilizarov was removed after an average of 2.5 weeks ( range 4 - 6 weeks ) after correction of deformities . at the time of removal of fixator , a plantigrade foot was achieved in 25 feet and gait was improved in all patients . there was residual varus hind foot deformity in two patients the correction of the deformity and function were assessed and the results were graded based on the reinker and carpenter scale as follows : excellent : painless , plantigrade foot with no functional limitations ; good : plantigrade foot in a patient able to ambulate long distances with mild pain ; fair : mild residual deformity , required bracing , and/or some functional limitations , but the patient leading an active life ; and poor : significant residual deformity , pain , and activity limitations . out of 27 feet , 3 ( 11.11% ) were rated as excellent , 17 ( 62.96% ) as good , 5 ( 18.51% ) as fair , and 2 ( 7.40% ) as poor . excellent and good results ( 74.07% ) were considered satisfactory , while fair and poor results ( 25.92% ) were considered unsatisfactory . thus , the ilizarov technique gave satisfactory results in resistant club foot deformities that were difficult to treat by other conventional methods . superficial pin tract infection with hypergranulation over the schanz screws were in 4 feet , and only superficial pin tract infection with watery discharge from the wires noted in 2 feet and were managed with regular dressings and short term 4 generation oral cephalosporin . kinking of the skin with temporary edema over the callosities was noticed in 6 feet above the age of 15 years due to shortening of lateral column and resolved after 46 weeks after the deformity correction . flexion deformities of toes developed due to increasing tension on the long flexor tendons during deformity correction and was controlled by toe slings , supports over forefoot , and insertion of kwires . there were 8 feet above the age of 15 years , and the overall outcome was good in 5 , fair in 2 , and poor in 1 foot [ figures 2 and 3 ] . ( a ) preoperative clinical photograph of the feet , from the front of neglected congenital telepesequinovarus ; ( b ) preoperative clinical photograph of the same feet , from behind ; ( c ) preoperative lateral radiographs of both feet showing the deformity ( d ) preoperative anteroposterior radiographs of both feet showing the deformity ( a , b ) clinical photograph of the same patient showing application of ilizarov and deformity correction ; ( c ) postoperative anteroposterior radiographs of the same patient showing fixator in situ ( a , b ) clinical photograph of the same patient after removal of ilizarov showing deformity correction ( c , d ) followup photographs showing maintenance of correction a 20-year old girl with right sided clubfoot ( a ) preoperative clinical photograph of the feet from the front ; ( b ) preoperative clinical photograph of the feet from the side ; ( c ) preoperative clinical photograph from back ; ( d , e ) preoperative lateral and anteroposterior radiograph of the right foot showing deformity ( a , b ) clinical photograph of right foot from medial and lateral side showing ilizarov fixator in situ ( c , d ) lateral and antero posterior view of right leg and ( e ) antero posterior view of right foot showing ilizarov fixator in situ ( a , b , c , d ) clinical photographs showing ilizarov in situ ( e ) anteroposterior and lateral radiograph showing limb lengthening ( a , b , c , d ) clinical photographs showing good correction ( e , f ) lateral and antero posterior radiograph of foot at final followup showing correction it also includes deformed feet with poor soft tissue coverage , relapsed or neglected cases.10 the introduction of the ilizarov technique to italy in 1981 changed the concepts of the management of congenital and posttraumatic foot deformities . the technique allowed for a three dimensional approach to the foot without the need for additional wide surgical exposure11 [ table 1 ] . professor ilizarov recognized that his technique could be used to treat foot pathologies such as clubfoot ; physicians continue to expand the applications of the ilizarov procedure in treating complex foot pathologies.12 complications were numerous but manageable , and for the most part , did not compromise overall patient satisfaction in this very difficult to treat clinical condition.6 the technique , however , is difficult and should be performed by surgeons who are familiar with correction of pediatric foot and ankle deformities and are versed fully in ilizarov fixation techniques.13 types of foot deformities ( catagni et al.)5 our study included 27 resistant clubfeet in patients with ages ranging from 8 to 20 years ( mean 12 years ) who underwent limited soft tissue dissection with ilizarov distraction . achilles tendon sheath tenotomy was performed in 8 ( 29.62% ) , plantar fasciotomy in 6 ( 22.22% ) , and achilles tendon lengthening in 9 ( 59.93% ) feet . of the patients , 74.07% considered it as satisfactory , 3 ( 11.11% ) as excellent , and 17 ( 62.96% ) as good , while 25.92% considered it as unsatisfactory , 5 ( 18.51% ) as fair , and 2 ( 7.40% ) as poor . gradual soft tissue compression and distraction with the setting of ilizarov frame was used for correction of the deformities in all patients . the same evaluation criteria were used in all patients as the same principle was used in them . our results were compared with those of ferreira et al . , who reviewed 30 patients ( 38 feet ) with a mean age of 19 years ( range 539 years ) with severe deformities and studied stiff feet associated with neglected clubfoot . the device was used for 16 weeks on average , and after removal , a shortleg walking cast was used for an additional 6 weeks , followed by an afo for 6 months . freedman in 20061 conducted a review of 21 resistant clubfeet in 17 patients , who had undergone previous surgery , and were treated with ilizarov 's external fixation and gradual distraction by one of the two surgeons . amin abdel - razek youssef ahmed ( 2010)12 included 18 feet in 13 children with ages ranging from 3 to 12 years ( mean 5.5 years).soft tissue distraction by ilizarov was done in 12 feet , with elongation of the tendo achilles in 3 patients and midtarsal osteotomy in 3 patients , and the average followup period was 15.8 months . of the patients , 72.2% considered it as satisfactory ( 2 excellent and 11 good ) while 27.8% considered it to be unsatisfactory ( 4 fair and 1 poor ) . franke et al.13 succeeded to achieve plantigrade feet in all the feet with relapses in 3 feet in 815 years age group . twenty two feet were plantigrade and three feet showed recurrence after 18 months of followup . el barbary et al.15 achieved satisfactory correction using an ilizarov fixator for treatment of 66 relapsed or neglected club feet ( 40 months followup ) . prem et al.16 followed 19 feet managed by ilizarov soft tissue distraction for 510 years postoperatively . they reported on 14 of 19 feet graded good or excellent and 13 of 14 children satisfied with the results of the treatment . they reported unsatisfactory results of soft tissue and bony distraction with a recurrence rate of 70% for soft tissue distraction and 55% for bony distraction after a longer period of followup ( 47 months ) , but found that functional results ( patient based outcomes ) were better despite a poor surgical outcome . reinker and carpenter18 achieved excellent and good results in 21 of 23 feet treated by ilizarov 's external fixation . nineteen feet had received one or more osteotomies at the time of ilizarov 's external fixation application ; additional procedures were required during the course of treatment , including four percutaneous tendo achilles lengthening , two first metatarso phalangeal joint fusions , and talectomy , ankle arthrodesis , achilles tenodesis , and plantar arthrodesis in onecase . hosny19 used the bloodless technique in treating 23 foot deformities in 22 patients without any real surgical incision ; there was no need for soft tissue release or osteotomy , all patients had a plantigrade foot , and the results were rated as good in 20 and fair in 3 cases . de la huerta20 reported on 12 feet in seven adults with the achievement of complete correction in all patients except three in whom mild adduction of the forefoot reoccurred17 [ table 2 ] . comparison of surgical outcome in different studies surgeries with present study the average followup in our study was 18.7 months , and in situations like these , where the original foot and ankle deformities are so severe that the treatment option involves the use of an external fixator , minor or major recurrence of the deformities can be expected over time . as the followup time increases , further re - evaluation can reveal higher incidence of complications , some minor ( stiff toes , callus , mild deformity recurrence ) and others major ( severe recurrence of the deformities , painful arthritis ) . it is easier to deal with the late complications than it was before the correction . conventional surgical procedures , like osteotomies , limited arthrodesis , or soft tissue procedures , can be useful in order to correct residual mild deformities under safer conditions . we conclude that the short term clinical and functional results of treating resistant clubfoot deformities with ilizarov 's external fixator are promising and apparently it is a good option .

background : resistant clubfoot deformities of the foot and ankle remain a difficult problem even for the most experienced surgeon . we report a series of neglected resistant clubfoot deformities treated by limited surgery and ilizarov distraction histogenesis.materials and methods : twenty one patients with 27 feet having resistant clubfoot deformities were managed by ilizarov distraction histogenesis from april 2005 to may 2008 . the mean age was 12 years ( range 820 years ) . a limited soft tissue dissection like percutaneous achilles sheath tenotomy and plantar fasciotomy were done . progressive correction of the deformities was achieved through the standard and simple ilizarov frame construct setting . after removal of ilizarov frame , a short leg walking cast was used for an additional 6 weeks , followed by an ankle foot orthrosis for 3 months.results:the mean followup period was 18.7 months ( range 20 - 36 months ) . the mean duration of fixator application was 3.6 months ( range 35 months ) . at the time of removal of the fixator , a plantigrade foot was achieved in 25 feet and gait was improved in all patients . there was residual varus hind foot deformity in two patients . out of 27 feet , 3 ( 11.11% ) were rated as excellent , 17 ( 62.96% ) as good , 5 ( 18.51% ) as fair , and 2 ( 7.40% ) as poor according to reinkerand carpenter scale . excellent and good results ( 74.07% ) were considered satisfactory , while fair and poor results ( 25.92% ) were considered unsatisfactory.conclusion:the short term clinical and functional results of resistant clubfoot deformities with ilizarov 's external fixator is promising and apparently a good option .

I M Operative procedure R D

several corrective procedures have been described , with the goal to provide a pain free , plantigrade foot . encouraging results are reported with this method.1 the ilizarov 's external fixator allows simultaneous correction of all the severe foot deformities associated with neglected clubfoot with minimal surgery , reducing risks of cutaneous or neurovascular complications and avoiding excessive shortening of the foot.2 the external fixation remains an essential tool for future orthopedic surgeons.3 the ilizarov method is being used increasingly to correct many orthopedic deformities . the frames required for ankle and foot deformity correction are among the most difficult to construct owing to the complexity of the deformities which must be corrected.4 the application of ilizarov frame is in two parts , one to the leg and one to the foot . the foot construct consists of a half ring posteriorly and half ring anteriorly.5 a half ring is fixed to the forefoot with 2 crossed olive wires , 1 traversing the 5 metatarsals and the other fixed to the medial 3 metatarsals medially to laterally . a half ring was applied to the hind foot with 2 crossed wires through the calcaneus , with a shanz pin fixed to the calcaneus from posterior to anterior . two dorsal rods were used on each side of the central hole of the forefoot half ring in patients with forefoot supination.6 the ilizarov technique has been suggested as an alternative to major surgery for the treatment of neglected or recurrent deformities in clubfoot.7 even in those patients in whom final corrective arthrodesis is necessary , this may be carried out with minimal bone resection , since the severe deformities of the foot and ankle have been corrected.8 we report a series of neglected resistant clubfoot deformities treated by limited surgery and ilizarov distraction histogenesis . we evaluated the results of this prospective study with ilizarov 's external fixation system in 21 patients with 27 feet without corrective osteotomies as treatment of neglected resistant clubfoot deformity , between april 2005 and may 2008 . this study includes a resistant clubfeet ( n=13 ) which were treated for the first time , already operated clubfeet by other methods ( n=14 ) , and also presented with multiple surgical scars ( n=5 ) . all resistant clubfeet with common deformities like adduction or supination , or both , of the forefoot , short medial column and long lateral column , varus of the hind foot , and equinus of the ankle were included to decide the same surgical planning with ilizarov in all patients because it permits simultaneous correction of these multiplanar deformities . minimal soft tissue procedures for those were done below the age of 12 years depending upon their flexibility to correct the deformities without stress over the soft tissues with ilizarov method during distraction and compression . gait of all patients like their walking pattern were assessed clinically through functional mobility scale9 at the time of admission and every step was planned like physical assistance , bracing , and assistive devices to improve the speed , distance and efficiency while walking . they were also assessed for skin and soft tissue conditions , range of ankle and subtalar joint movement , levels of the particular deformity , limb shortening . the condition of ankle and other joints of foot , angles of individual deformities were assessed . ilizarov frame was preassembled according to the deformity of foot as well as radiographs to correct without any stress over the related soft tissues . another half ring with two plain crossed wires was applied through 1 to 3 metatarsal and from 5 to 2 or 3 metatarsal and connected with the rings applied over the tibia through the hinges , plates , and conical washers were also used wherever required . the calcaneal half ring had three connections to the lower ring of the tibia frame ( posteriorly , medially , and laterally ) , while the forefoot half ring had two connections situated medially and laterally and sometimes only one connection situated centrally to the long axis of the foot . half ring over the calcaneus was used as a distractor or pusher and compression or pulling was achieved through the half ring over dorsum of the fore foot . varus deformities of the heel were corrected through medial and lateral rods by compression and distraction method . for correction of rotational like supination and pronation deformities , two frontal rods with hinges were applied in oblique directions connected with the ring in the forefoot and assembly over the leg and corrected accordingly . after correction of forefoot adduction supination , hind foot varus and finally equinus were corrected through the setting of the ilizarov 's external fixator . during distraction , tension over the soft tissues , neurovascular status , and improvement in the correction of the deformities were observed clinically , and radiologically dislocation or subluxation of tarsal and ankle joints was observed [ figure 1 ] . ilizarov frame for ankle and foot with two full rings for leg , one half for calcaneum and one half for forefoot over the metatarsals after application of ilizarov , a limited soft tissue dissection , percutaneous achilles sheath tenotomy , and plantar fasciotomy were done for patients under 12 years and achilles tendon lengthening was done for those above 12 years of age . achilles tendon lengthening was performed in 9 ( 59.93% ) feet , achilles tendon sheath tenotomy in 8 ( 29.62% ) feet , and planter fasciotomy in 6 ( 22.22% ) feet . approximately 810 deformity was over corrected and this over lengthening of soft tissues avoided the recoil that could occur in the soft tissue after frame removal . all feet were also assessed radiologically as well as clinically every week during the followup period . all patients were encouraged and weight bearing was allowed according to tolerance of the patients and their degree of correction . adjustments were done by the surgeon and postgraduate students during stay in the hospital , and after discharge by either the patient himself / herself , and if the patient was a child , the family members were trained to move the nuts for compression , distraction , and lengthening through the markings . care of pins was taken during their stay in the hospital , and patients and their attendants were trained to take care of pins by themselves after discharge . loose clamps , nuts , and bolts were tightened regularly in outpatient clinic initially weekly up to deformity correction , then fortnightly up to removal of the fixator . the fixator removal was decided after correction of all required deformities and the time needed for correction of the deformities was variable depending on the resistance and rigidity of the deformities . fixator was removed , all the pin wounds were washed , and aseptic dressings were done ; a short leg walking plaster of paris ( pop ) cast was applied for 2 weeks , the patients were reviewed at the outpatient clinic after 2 weeks , and the pop cast was removed . the status of wounds was checked and again short leg walking cast was applied for 4 more weeks . the total time of the pop cast was 6 weeks after removal of the fixator , followed by an ankle foot orthrosis ( afo ) for 3 months . ilizarov frame was preassembled according to the deformity of foot as well as radiographs to correct without any stress over the related soft tissues . another half ring with two plain crossed wires was applied through 1 to 3 metatarsal and from 5 to 2 or 3 metatarsal and connected with the rings applied over the tibia through the hinges , plates , and conical washers were also used wherever required . the calcaneal half ring had three connections to the lower ring of the tibia frame ( posteriorly , medially , and laterally ) , while the forefoot half ring had two connections situated medially and laterally and sometimes only one connection situated centrally to the long axis of the foot . half ring over the calcaneus was used as a distractor or pusher and compression or pulling was achieved through the half ring over dorsum of the fore foot . varus deformities of the heel were corrected through medial and lateral rods by compression and distraction method . for correction of rotational like supination and pronation deformities , two frontal rods with hinges were applied in oblique directions connected with the ring in the forefoot and assembly over the leg and corrected accordingly . after correction of forefoot adduction supination , hind foot varus and finally equinus were corrected through the setting of the ilizarov 's external fixator . during distraction , tension over the soft tissues , neurovascular status , and improvement in the correction of the deformities were observed clinically , and radiologically dislocation or subluxation of tarsal and ankle joints was observed [ figure 1 ] . ilizarov frame for ankle and foot with two full rings for leg , one half for calcaneum and one half for forefoot over the metatarsals after application of ilizarov , a limited soft tissue dissection , percutaneous achilles sheath tenotomy , and plantar fasciotomy were done for patients under 12 years and achilles tendon lengthening was done for those above 12 years of age . achilles tendon lengthening was performed in 9 ( 59.93% ) feet , achilles tendon sheath tenotomy in 8 ( 29.62% ) feet , and planter fasciotomy in 6 ( 22.22% ) feet . all feet were also assessed radiologically as well as clinically every week during the followup period . all patients were encouraged and weight bearing was allowed according to tolerance of the patients and their degree of correction . adjustments were done by the surgeon and postgraduate students during stay in the hospital , and after discharge by either the patient himself / herself , and if the patient was a child , the family members were trained to move the nuts for compression , distraction , and lengthening through the markings . loose clamps , nuts , and bolts were tightened regularly in outpatient clinic initially weekly up to deformity correction , then fortnightly up to removal of the fixator . the fixator removal was decided after correction of all required deformities and the time needed for correction of the deformities was variable depending on the resistance and rigidity of the deformities . fixator was removed , all the pin wounds were washed , and aseptic dressings were done ; a short leg walking plaster of paris ( pop ) cast was applied for 2 weeks , the patients were reviewed at the outpatient clinic after 2 weeks , and the pop cast was removed . the status of wounds was checked and again short leg walking cast was applied for 4 more weeks . the total time of the pop cast was 6 weeks after removal of the fixator , followed by an ankle foot orthrosis ( afo ) for 3 months . the mean distraction time for deformity correction was 7.2 weeks ( range 4 - 12 weeks ) . ilizarov was removed after an average of 2.5 weeks ( range 4 - 6 weeks ) after correction of deformities . at the time of removal of fixator , a plantigrade foot was achieved in 25 feet and gait was improved in all patients . there was residual varus hind foot deformity in two patients the correction of the deformity and function were assessed and the results were graded based on the reinker and carpenter scale as follows : excellent : painless , plantigrade foot with no functional limitations ; good : plantigrade foot in a patient able to ambulate long distances with mild pain ; fair : mild residual deformity , required bracing , and/or some functional limitations , but the patient leading an active life ; and poor : significant residual deformity , pain , and activity limitations . out of 27 feet , 3 ( 11.11% ) were rated as excellent , 17 ( 62.96% ) as good , 5 ( 18.51% ) as fair , and 2 ( 7.40% ) as poor . excellent and good results ( 74.07% ) were considered satisfactory , while fair and poor results ( 25.92% ) were considered unsatisfactory . superficial pin tract infection with hypergranulation over the schanz screws were in 4 feet , and only superficial pin tract infection with watery discharge from the wires noted in 2 feet and were managed with regular dressings and short term 4 generation oral cephalosporin . flexion deformities of toes developed due to increasing tension on the long flexor tendons during deformity correction and was controlled by toe slings , supports over forefoot , and insertion of kwires . there were 8 feet above the age of 15 years , and the overall outcome was good in 5 , fair in 2 , and poor in 1 foot [ figures 2 and 3 ] . ( a ) preoperative clinical photograph of the feet , from the front of neglected congenital telepesequinovarus ; ( b ) preoperative clinical photograph of the same feet , from behind ; ( c ) preoperative lateral radiographs of both feet showing the deformity ( d ) preoperative anteroposterior radiographs of both feet showing the deformity ( a , b ) clinical photograph of the same patient showing application of ilizarov and deformity correction ; ( c ) postoperative anteroposterior radiographs of the same patient showing fixator in situ ( a , b ) clinical photograph of the same patient after removal of ilizarov showing deformity correction ( c , d ) followup photographs showing maintenance of correction a 20-year old girl with right sided clubfoot ( a ) preoperative clinical photograph of the feet from the front ; ( b ) preoperative clinical photograph of the feet from the side ; ( c ) preoperative clinical photograph from back ; ( d , e ) preoperative lateral and anteroposterior radiograph of the right foot showing deformity ( a , b ) clinical photograph of right foot from medial and lateral side showing ilizarov fixator in situ ( c , d ) lateral and antero posterior view of right leg and ( e ) antero posterior view of right foot showing ilizarov fixator in situ ( a , b , c , d ) clinical photographs showing ilizarov in situ ( e ) anteroposterior and lateral radiograph showing limb lengthening ( a , b , c , d ) clinical photographs showing good correction ( e , f ) lateral and antero posterior radiograph of foot at final followup showing correction it also includes deformed feet with poor soft tissue coverage , relapsed or neglected cases.10 the introduction of the ilizarov technique to italy in 1981 changed the concepts of the management of congenital and posttraumatic foot deformities . professor ilizarov recognized that his technique could be used to treat foot pathologies such as clubfoot ; physicians continue to expand the applications of the ilizarov procedure in treating complex foot pathologies.12 complications were numerous but manageable , and for the most part , did not compromise overall patient satisfaction in this very difficult to treat clinical condition.6 the technique , however , is difficult and should be performed by surgeons who are familiar with correction of pediatric foot and ankle deformities and are versed fully in ilizarov fixation techniques.13 types of foot deformities ( catagni et al. )5 our study included 27 resistant clubfeet in patients with ages ranging from 8 to 20 years ( mean 12 years ) who underwent limited soft tissue dissection with ilizarov distraction . achilles tendon sheath tenotomy was performed in 8 ( 29.62% ) , plantar fasciotomy in 6 ( 22.22% ) , and achilles tendon lengthening in 9 ( 59.93% ) feet . of the patients , 74.07% considered it as satisfactory , 3 ( 11.11% ) as excellent , and 17 ( 62.96% ) as good , while 25.92% considered it as unsatisfactory , 5 ( 18.51% ) as fair , and 2 ( 7.40% ) as poor . gradual soft tissue compression and distraction with the setting of ilizarov frame was used for correction of the deformities in all patients . the same evaluation criteria were used in all patients as the same principle was used in them . , who reviewed 30 patients ( 38 feet ) with a mean age of 19 years ( range 539 years ) with severe deformities and studied stiff feet associated with neglected clubfoot . the device was used for 16 weeks on average , and after removal , a shortleg walking cast was used for an additional 6 weeks , followed by an afo for 6 months . freedman in 20061 conducted a review of 21 resistant clubfeet in 17 patients , who had undergone previous surgery , and were treated with ilizarov 's external fixation and gradual distraction by one of the two surgeons . amin abdel - razek youssef ahmed ( 2010)12 included 18 feet in 13 children with ages ranging from 3 to 12 years ( mean 5.5 years).soft tissue distraction by ilizarov was done in 12 feet , with elongation of the tendo achilles in 3 patients and midtarsal osteotomy in 3 patients , and the average followup period was 15.8 months . of the patients , 72.2% considered it as satisfactory ( 2 excellent and 11 good ) while 27.8% considered it to be unsatisfactory ( 4 fair and 1 poor ) . prem et al.16 followed 19 feet managed by ilizarov soft tissue distraction for 510 years postoperatively . they reported on 14 of 19 feet graded good or excellent and 13 of 14 children satisfied with the results of the treatment . they reported unsatisfactory results of soft tissue and bony distraction with a recurrence rate of 70% for soft tissue distraction and 55% for bony distraction after a longer period of followup ( 47 months ) , but found that functional results ( patient based outcomes ) were better despite a poor surgical outcome . reinker and carpenter18 achieved excellent and good results in 21 of 23 feet treated by ilizarov 's external fixation . nineteen feet had received one or more osteotomies at the time of ilizarov 's external fixation application ; additional procedures were required during the course of treatment , including four percutaneous tendo achilles lengthening , two first metatarso phalangeal joint fusions , and talectomy , ankle arthrodesis , achilles tenodesis , and plantar arthrodesis in onecase . hosny19 used the bloodless technique in treating 23 foot deformities in 22 patients without any real surgical incision ; there was no need for soft tissue release or osteotomy , all patients had a plantigrade foot , and the results were rated as good in 20 and fair in 3 cases . de la huerta20 reported on 12 feet in seven adults with the achievement of complete correction in all patients except three in whom mild adduction of the forefoot reoccurred17 [ table 2 ] . comparison of surgical outcome in different studies surgeries with present study the average followup in our study was 18.7 months , and in situations like these , where the original foot and ankle deformities are so severe that the treatment option involves the use of an external fixator , minor or major recurrence of the deformities can be expected over time . as the followup time increases , further re - evaluation can reveal higher incidence of complications , some minor ( stiff toes , callus , mild deformity recurrence ) and others major ( severe recurrence of the deformities , painful arthritis ) . conventional surgical procedures , like osteotomies , limited arthrodesis , or soft tissue procedures , can be useful in order to correct residual mild deformities under safer conditions . we conclude that the short term clinical and functional results of treating resistant clubfoot deformities with ilizarov 's external fixator are promising and apparently it is a good option .

l. monocytogenes is an intracellular pathogen which , especially if foodborne , may induce what is known as listeriosis . once ingested , l. monocytogenes can penetrate the intestinal endothelial barrier , the placental or the hematoencephalic barrier [ 1 , 2 ] . the groups at higher risk of contracting the disease are young people , the old , ( over 65 ) , pregnant women , and immune compromised people , the yopi , an acronym coined by de cesare et al . , l. monocytogenes can lead to episodes of gastroenteritis and fever [ 5 , 6 ] . listeriosis is considered a rare disease , its incidence in humans ranges between 0.1 and 11.3 cases / million , with a high mortality rate , up to 30% in the categories most at risk ( yopi ) . based on an efsa 2010 report , the incidence in europe was of 3 cases / million of inhabitants . because the incubation period can span from 3 up to 60 days , this disease is often difficult to trace because it is not easy to isolate the food that is responsible for the infection . europe has some well - documented episodes particularly in france , finland , switzerland , the uk , belgium , and ireland . distinct psychrotolerant characteristics allow l. monocytogenes to adapt to acidic conditions and to low water activity environments , making it an insidious threat to some kinds of food as ready - to - eat food ( rte ) that is characterized by mild treatments and a medium - to - long shelf - life a highly sought - after quality by today 's consumer [ 1522 ] . the fish products that pose potential risks include mainly cold smoked fish , raw carpaccio , and marinated fish . smoked fish products in particular were reported to cause human infections [ 2325 ] . in europe , smoked salmon , more than all the other products , was reported to have surpassed the maximum threshold limits allowed for l. monocytogenes contamination . the risk of consuming rte seafood does not so much entail the contamination of the raw product , which will often have l. monocytogenes , but at low concentrations , as much as the product 's characteristics which in time encourage its growth [ 2628 ] . inadequate consumer knowledge on how to store rte food at home , at the right refrigerated temperature , has led to higher risks of l. monocytogenes growth [ 29 , 30 ] . the goal of this study is to determine the distribution of l. monocytogenes in rte fish semipreserves , as set out by ec regulations 2073/2005 related to this category which distinguishes the foods that favour l. monocytogenes growth from those that do not . different types of repfed products were examined , these include marinated seafood salads ( with cephalopods , surimi , crustaceans , bivalves ) , marinated shrimps , cephalopods and salmon carpaccio , marinated mackerel , smoked herrings , and cold smoked salmon . packaged products marketed in the veneto region ( italy ) were sampled to determine the levels of l. monocytogenes . the products inspected included ones with intrinsically favourable characteristics , thus ideal for l. monocytogenes growth , and those with unfavourable characteristics ( ph 4.4 , aw 0.92 ; ph 5.0 and aw 0.94 ) . tests were carried on both these types of products stored , at 4c and at 10c , the latter being a more realistic simulation of household conditions which may experience thermal abuse . each sample unit , aside from examining l. monocytogenes ( qualitative and quantitative tests ) also analysed the following : total aerobic mesophilic count , total psychrophiles count , total psychrophiles h2s producers , moulds , yeasts , and lactic bacteria to establish whether correlation exists in relation to the presence of l. monocytogenes furthermore , enrichment broths were also tested for l. monocytogenes using pcr and serotyping and ribotyping of isolated strains and culture tests . the samples , each consisting of 3 sample units coming from a production batch , upon reaching the laboratory , were stored in a refrigerator in their original package at a temperature of 4c and 10c until their shelf - life expiry ( storage at 10c in the last third of their shelf - life expiry ) . product shelf - life ranged from 8 days ( raw salmon carpaccio ) to 70 days ( seafood salads ) . during the collection phase at the various traders , only the samples which had not already surpassed half of their expiry period were considered . the products selected were both national and international products taken from 9 different stores . upon reaching the laboratory , one sample unit was examined immediately , the other two were on the last day of expiry . in total 38 samples were analysed , amounting to 114 sample units . total aerobic mesophilic count on agar plates with incubation in aerobiosis at 30c for 72 hours ( iso 4833:2003 ) . total psychrotolerant count on iron agar plates ( lyngby ) with incubation in aerobiosis at 15c for 7 days.total psychrophiles producing hydrogen sulphide count on iron plates ( lyngby ) with incubation in aerobiosis at 15c for 7 days.count of the moulds and yeasts on rose bengal chloramphenicol agar plates with incubation in aerobiosis at 25c for 5 days.lactic bacteria count on mrs agar plates ( final ph 6.4 ) with incubation in aerobiosis at 30c for 72 hours . l. monocytogenes count on aloa agar at 37c for 48 hours ( iso 11290 - 2:1998/amd 1 2004).detection of l. monocytogenes on aloa agar and palcam agar plates at 37c for 48 hours following selective enrichment in half fraser and fraser broth ( iso 11290 - 1:1996/amd 1 2004 ) . total aerobic mesophilic count on agar plates with incubation in aerobiosis at 30c for 72 hours ( iso 4833:2003 ) . total psychrotolerant count on iron agar plates ( lyngby ) with incubation in aerobiosis at 15c for 7 days . total psychrophiles producing hydrogen sulphide count on iron plates ( lyngby ) with incubation in aerobiosis at 15c for 7 days . count of the moulds and yeasts on rose bengal chloramphenicol agar plates with incubation in aerobiosis at 25c for 5 days . lactic bacteria count on mrs agar plates ( final ph 6.4 ) with incubation in aerobiosis at 30c for 72 hours . l. monocytogenes count on aloa agar at 37c for 48 hours ( iso 11290 - 2:1998/amd 1 2004 ) . detection of l. monocytogenes on aloa agar and palcam agar plates at 37c for 48 hours following selective enrichment in half fraser and fraser broth ( iso 11290 - 1:1996/amd 1 2004 ) . the quantitative and qualitative methods were carried out at the same time , the same day . ph measure using the mettler toledo mp 220 instrument , with temperature autocompensation.water activity ( aw ) using rotronic 29539 instrument ( iso 21807:2004 ) . detection of l. monocytogenes consisted in taking 1 ml of enrichment broth ( half fraser ) , after 24 hours of incubation , to extract dna and carry out subsequent pcr tests for listeriaspp and l. monocytogenes . dna extraction was carried out on pellet , obtained after centrifugation of the enrichment broth ( 2,000 g for 2 minutes , followed by 12,000 g for 5 minutes on the surnatant ) . once the surnatant was removed , pbs was added and 16,000 g underwent centrifugation for another 2 minutes . genelute bacterial genomic dna mini kit ( sigma ) was used , following the manufacturer 's instructions , protocol for gram positive bacteria . nested pcr was the method used to target the codifying gene for 16s rrna in which the amplified product of the first reaction becomes the template for subsequent nested reactions . the primers used in the first reaction were the forward primer li1 5-ctc cat aaa ggt gac cct-3 and the reverse primer u1 5-cag cmg ccg cgg taa twc-3 . the reaction took place in a final volume of 25 l with concentrations of 1x geneamp pcr buffer ii ( applied biosystems ) , 1.5 mm of mgcl2 , 0.2 mm of each dntp , 0.2 m of both primers , 1.25 u of ampli taq dna polymerase ( applied biosystems ) , and 5 l of extracted dna . for amplification , the thermal cyler geneamp pcr system 9700 ( applied biosystem ) was used with temperatures set at initial denaturation at 95c for 3 min , followed by 25 cycles , each with a denaturation phase at 95c for 90 sec , and an annealing phase at 50c for 90 sec and an extension phase at 72c for 2 min , followed by a final extension phase at 72c for 10 min . the primers used for the nested reactions were forward primer ls1 5-acg acc gca adg ttg aaa ct-3 and reverse primer ls2 5-gac gtc atc ccc acc ttc ct-3 manufactured at the nucleic acids technology laboratory applied to foods at the istituto zooprofilattico sperimentale of brescia . the reaction was prepared in a final volume of 25 l with concentrations of 1x geneamp pcr buffer ii ( applied biosystems ) , 1.5 mm of mgcl2 , 0.2 mm of each dntp , 0.2 m of both primer , with 0.75 u of ampli taq dna polymerase ( applied biosystems ) and 2.5 l of extracted dna . for amplification the thermal cycler geneamp pcr system 9700 ( applied biosystem ) was used with temperatures for initial denaturation set at 95c for 3 min , followed by 35 cycles , each comprising a denaturation phase at 95c for 30 sec , an annealing phase at 59c for 30 sec , an extension phase at 72c for 30 sec , followed by a final extension phase at 72c for 5 min . analysis of foreseen amplification , of 301 bp , was carried out after electrophoresis in agarose gel at 2.5% stained with ethidium bromide ( final concentration on gel : 0.5 g / ml ) , by means of exposure to uv radiation . a method to detect the hly gene target ( haemolytic secreted pathogenic factor or hemolysin ) was used with forward primer lis1 5-cgg agg ttc cgc aaa aga tg-3 and reverse primer lis2 5-cct cca gag tga tcg atg tt-3 . reaction was prepared using an end volume of 25 l with concentrations of 1x geneamp pcr buffer ii ( applied biosystems ) , 1.5 mm of mgcl2 , 0.2 mm for each dntp , 0.2 m of both primers , 0.75 u of ampli taq dna polymerase ( applied biosystems ) and 5 l of extracted dna . amplification was carried out in the thermal cycler geneamp pcr system 9700 ( applied biosystem ) with a temperature profile for initial denaturation set at 95c for 3 min , followed by 35 cycles , each comprising a denaturation phase at 95c for 30 sec , an annealing phase at 58c for 30 sec and an extension phase at 72c for 30 sec , followed by a final extension phase at 72c for 5 min . analysis of foreseen amplification , of 234 bp , was then conducted after electrophoresis in agarose gel at 2.5% stained with ethidium bromide ( final concentration on gel : 0,5 g / ml ) , by means of exposure to uv radiation . serotyping by m - pcr was performed using primers as described by doumith et al .. primers enable the identification of listeria and the subdivision of strains belonging only to the l. monocytogenes species into four distinct serogroups . serogroup 1 comprises serotype 1/2a and 3a ; serogroup 2 of serotypes 1/2c and 3c ; serogroup 3 serotypes 1/2b and 3b ; and serogroup 4 , serotypes 4b , 4d and 4e . the pcr mix included pcr master mix 1x ( qiagen , milan , italy ) , mix of primers ( lmo0737 , orf2819 , orf2110 , lmo1118 and prs ) , sterile distilled penta - h2o and the dna extracted . pcr reaction conditions included an initial step of denaturation at 94c for 3 minutes , 35 cycles at 94c for 0.40 minutes , 53c for 1.15 minutes , 72c for 1.15 minutes and a final step at 72c for 7 minutes . pcr products underwent 2% agarose gel electrophoretic separation at 90 v for 90 minutes . then , after being stained with ethidium bromide ( 10 ng / ml ) , they were visualised on uv transilluminator . serotyping with antisera yielded 12 different serotypes of l. monocytogenes in terms of cellular surface , and the somatic seroagglutination was carried out to confirm the serotypes and serogroups obtained using the molecular method . listeria antisera kit ( denka seiken co. ltd , tokyo , japan ) was used according to a modified method outlined by seeliger and hohne . the solution was lysed at 85c for 20 minutes , and two specific lysing agents were added . subsequently , a batch containing eight containers to hold eight samples was inserted in the automated ribotyping instrument . in brief , riboprinter generates an enzymatic digestion of the solutions using the ecori restriction enzyme , and electrophoresis of dna fragments was transferred onto a membrane . finally , the instrument detects the signal emitted by a ccd camera and software converts the images in ribotyping patterns . ribotyping ( of the species ) is an automated process if more than 85% of the sample patterns resemble the reference patterns of the instrument 's database . the latter were obtained from various international collections ( e.g. , atcc , dsmz , or jmc ) and identified with a dup - id code ( dupont identification ) . genotypic characterization of bacterial strains consisted of comparing the strains of a batch , assigning to them what is called a ribogroup code ( rg ) . each ribotyped strain was compared with all the patterns of the profiles contained in the database . in general , if there was a similarity 93% between the profile of the strain investigated and that of the databank , the strain was assigned to the corresponding ribogroup of that profile . ribogroup attribution is only possible if strains undergo simultaneous analysis or if analysis is conducted a few days later since the database has to be updated on an ongoing basis , accessing international databanks available on the internet . to obtain phylogenetic trees the results were extracted from a pearson correlation ( upgma method ) with bionumerics software version 6.1 . the analyses were carried out on a range of 38 different commercial products 28 ( 73,68% ) of which had aw and ph values favourable for growth of l. monocytogenes , while 10 ( 26,32% ) had unfavourable values instead ( table 1 ) . of the 28 samples with favourable characteristics for growth of the pathogen conditions , nine were positive for l. monocytogenes ( 32,14% ) , according to the standard culture methods ( iso 11290 - 1:1996/amd 1 2004 ) ; of these , 3 ( 10,71% ) had values exceeding the limit ( 100 cfu / g ) established by ec reg . no sample with unfavourable characteristics was found positive for viable l. monocytogenes in microbiological testing . table 2 illustrates the corresponding values of l. monocytogenes qualitative test , quantitative test , and pcr results . listerial genus was present in all 38 samples analysed according to the amplification results of the genus specific pcr reaction . nonetheless , the species specific pcr demonstrated the presence of l. monocytogenes after enrichment in 12 samples only , of which 9 were also found to contain the pathogen by classical culture tests . in general they rose when thermal abuse at 10c occurred . in the 3 samples with a charge of > 100 cfu / g , l. monocytogenes grew despite the presence of competitive flora , both lactic and alternating . despite the increase of microbial values at the end of shelf - life , there were no significant changes in the product ph and aw values . in evidence that of three samples with a higher than 100 cfu / g value ( sample no . 2 , 8 and 10 ) , two were analysed after thermal abuse at 10c ( last third of its shelf - life ) , and one proved unfit at the time it was being taken . 2 , at the end of its shelf - life , had bulging but no unpleasant odours . other samples , when opened , had no significant organoleptic alterations . serotyping of 15 isolated l. monocytogenes strains coming from rte fish semipreserves was carried out using multiplex pcr to separate the 4 main serotypes ( 1/2a , 1/2b , 1/2c and 4b ) into 4 distinct groups . confirmation of the serotype of single serogroups was achieved with the seroagglutination method ( table 4 and figure 1 ) . the 15 strains of l. monocytogenes analysed came from 4 different serotypes : the highest percentage was related to serotype 1/2a ( 73,33% ) , followed by serotype 4b ( 13,33% ) , 1/2b ( 6,67% ) and 4d ( 6,67% ) . ribotyping of l.monocytogenes colonies in microbiological tests ( see table 4 ) has yielded the broad variation of isolated strains , identifying 15 particular strains on the basis of species . dup-20243 , dup-1043 , and -dup1052 were characterised in the strains examined , in 3 ( 20% ) , 2 ( 13.33% ) and 1 ( 6.66% ) sample , respectively . in terms of ribogroup distribution , rg-568 was the most diffuse , found in 9 of the 15 samples ( 60% ) , and the other ribogroups were each found in only one sample , except rg-1296 which was found in 2 samples . data from phylogenetic analysis on the restriction pattern ( figure 2 ) yielded a classification of three main strain groups identified with ribogroup 568 , being highly phylogenetically related , in turn distant , though still phylogenetically related to the 4 strains grouped in the upper part of the dendrogram . it was analysed after a shelf - life at a temperature of 4c being very different from rest of the l. monocytogenes that were characterized . the data demonstrate that there is a rather low probability of l.monocytogenes exceeding the 100 cfu / g limit in the rte seafood distributed in the veneto region , which would at any rate be associated with improper storage of the product or with thermal abuse . in fact , of the 3 positive samples out of the 38 , 2 had been stored at 10c in the last third period of their lifecycle and only one was defective at the time it was collected . analysis conducted at the end of shelf - life provides additional confirmation of the above - mentioned data . the total charge of psychrophiles and of lactic bacteria found in the three positive samples was high , differently from jameson 's theory which affirms that the lactic charge should develop a sort of microbial competition against l. monocytogenes . it is worth noting that 9 semi - preserves resulted positive at the qualitative microbiological test for l. monocytogenes ( found in 25 g ) . if at the end of shelf - life the above mentioned 9 samples had a < 5 cfu level , the producer would still have to demonstrate , with appropriate studies and challenge tests , that l. monocytogenes will not grow and multiply . our report reiterates that smoked and fresh salmon are particularly hazardous products . in case of marinated products , just three samples had viable l. monocytogenes . at the end of shelf - life differently from salmon , these products have much lower aw and ph values , a factor that no doubt affects the growth of l. monocytogenes . moreover , smoked salmon production will have l. monocytogenes in the raw ingredient which can not be eliminated in the phases leading to final packaging but can only be contained . seafood salads instead , since they are made with precooked raw ingredients , do not have l. monocytogenes . if there is any l. monocytogenes present in the end product , it implies that contamination occurred after the processing phases , after marinating . the higher number of positive samples in the molecular biology tests and the inability to isolate culture underscore the presence of viable not culturable ( vnc ) organisms or no longer viable cells . the pcr test compared to the culture used on first enrichment broth it may prove useful in routine laboratory work because at least in pcr - negative cases it does not require microbiological tests . the samples examined , all being positive to pcr tests for listeria , confirm that these kinds of products are often contaminated with listeria , but that such condition rarely evolves into hazardous l. monocytogenes concentrations . in all the samples with unfavourable aw and ph values for l. monocytogenes growth , the pcr test for l. monocytogenes was negative , confirming that even if the product was contaminated in the production phase , l. monocytogenes would be overwhelmed by the product 's unfavourable environment . the m - pcr method adopted was rapid , reproducible , and cost - effective . its use however is not suggested to identify rare serotypes . for a serotype - specific analysis of isolated strains the traditional sero - agglutination method is recommended . the latter is no doubt less reproducible , more expensive , and requires specially trained staff , but it enables the detection of rare serotypes which in the years to come may be more frequently associated to cases of listeriosis . it is striking that 10 of the 11 l. monocytogenes strains isolated from salmon belonged to serotype 1/2a , as already confirmed by other scientific sources [ 3941 ] . the agglutination method to confirm the serotypes of the single serogroups detected with m - pcr rare serotypes have been linked to an epidemic outbreak , as confirmed in a finnish study by maijala et al . in 2000 . an epidemic case of listeriosis caused by the strain l. monocytogenes serotype 3a , isolated in packaged butter , was described therein . figure 2 shows the variability of strains found in the environment underscoring the need to develop methods that can assess the characterisation of pathogenicity , since same strains may correspond to different levels of pathogenicity . literature , in fact , documents l. monocytogenes and considers it a ubiquitous microorganism capable of adapting to different environmental conditions . however , despite the various reports on genotyping and characterization [ 43 , 44 ] , there is still very little empirical evidence on the correlation between its presence in food and the ensuing pathologies its consumption generates in humans . this ought to encourage a more ample use of molecular characterization to provide information , even at the epidemiological level , on the distribution of l. monocytogenes in the environment , which though it maintains phenotypic homogeneity , undergoes ongoing phenomena of clonal evolution with small , yet significant , genome variations . in addition , it is interesting to note that the 4 strains grouped in the upper part of the dendrogram identified as rg 1296 , rg 1369 , and rg 1533 were all isolated in samples analysed at time 0 . while ribogroups 564 , 568 , and 1340 were instead isolated in samples analysed both at the beginning as well as at the end of the shelf - life period , this suggests that the latter may be more resistant in time to different storage conditions . because of the relatively small number of tested samples , this work should be considered as a preliminary study . results should be confirmed throught further studies on different kind of products and increasing the number of samples . in fact , the refrigeration chain is not always respected especially in case of products with a few weeks of shelf - life , as the products in this report , which may be subjected to temperature changes which do not bring about any evident organoleptic alterations . production companies must therefore bear this in mind when certifying their products , using challenge tests to establish how safe a product is in terms of l. monocytogenes . they must also foresee temperature tests , in particular for products such as smoked salmon , traditionally from northern european countries having lower average temperatures than countries like italy also having less difficulty in managing the cold chain . products with aw and ph characteristics that favour l. monocytogenes growth were the only ones to result positive to microbiological and pcr tests in this work , in support of the potential hazard of these products . the highest percentage related to serotype 1/2a confirm similar investigations in seafood products and the genetic characterization using ribotyping demonstrates the genomic variability of l. monocytogenes strains also in this kind of food . this distinctive features could be interesting related to l. monocytogenes survival capacity under different storage conditions . nearly 30 years have gone by since canadians schelch demonstrated that l. monocytogenes can infect human via contaminated food . the significant number of studies conducted since then has provided much data on this pathogen . currently a lot is known about pathogenesis in humans and about its ability to resist and grow in different types of food . rightfully defined as an evolving pathogen by bryan , it changes its phenotypic and genotypic characteristics under outside - induced stress conditions , with acidic substances , new additives , and with the innovative food technologies applied by food industries . the latter , when producing all the latest food products , have to constantly monitor them for this versatile pathogen . in tandem education campaigns geared at the consumers who may be exposed to l. monocytogenes must provide general rules and guidelines for proper food storage and for food preparation in the domestic environment .

the risk of exposure to listeria monocytogenes ( l. monocytogenes ) when consuming ready - to - eat ( rte ) seafood was assessed in the veneto region ( italy ) . thirty - eight samples were analyzed , each sample consisted of three subunits belonging to the same batches . the first of the three units was examined immediately , the second was stored at + 4c ( for all of its shelf - life ) and the third at + 10c ( for the latter third of its shelf - life ) before the analysis . chemical - physical and microbiological parameters were tested simultaneously . culture results showed the presence of viable l. monocytogenes in 9 ( 23,68% ) of the 38 samples analysed , 3 ( 33,33% ) of which with a concentration > 100 cfu / g . pcr tests yielded 12 l. monocytogenes positive samples . semipreserves with aw ( water activity ) and ph values that favour l. monocytogenes growth were the only ones to result positive to microbiological and pcr tests . temperature proved to be an important factor as it limits the growth of l. monocytogenes , including products with potentially high competitive microbial charges . four different serotypes were recovered and ribotyping has helped to highlight the genomic variability of l. monocytogenes strains in food . this supports the hypothesis that l. monocytogenes continues to evolve genetically to the detriment of phenotypic conservation .

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

l. monocytogenes is an intracellular pathogen which , especially if foodborne , may induce what is known as listeriosis . once ingested , l. monocytogenes can penetrate the intestinal endothelial barrier , the placental or the hematoencephalic barrier [ 1 , 2 ] . the groups at higher risk of contracting the disease are young people , the old , ( over 65 ) , pregnant women , and immune compromised people , the yopi , an acronym coined by de cesare et al . , l. monocytogenes can lead to episodes of gastroenteritis and fever [ 5 , 6 ] . listeriosis is considered a rare disease , its incidence in humans ranges between 0.1 and 11.3 cases / million , with a high mortality rate , up to 30% in the categories most at risk ( yopi ) . europe has some well - documented episodes particularly in france , finland , switzerland , the uk , belgium , and ireland . distinct psychrotolerant characteristics allow l. monocytogenes to adapt to acidic conditions and to low water activity environments , making it an insidious threat to some kinds of food as ready - to - eat food ( rte ) that is characterized by mild treatments and a medium - to - long shelf - life a highly sought - after quality by today 's consumer [ 1522 ] . in europe , smoked salmon , more than all the other products , was reported to have surpassed the maximum threshold limits allowed for l. monocytogenes contamination . the risk of consuming rte seafood does not so much entail the contamination of the raw product , which will often have l. monocytogenes , but at low concentrations , as much as the product 's characteristics which in time encourage its growth [ 2628 ] . inadequate consumer knowledge on how to store rte food at home , at the right refrigerated temperature , has led to higher risks of l. monocytogenes growth [ 29 , 30 ] . the goal of this study is to determine the distribution of l. monocytogenes in rte fish semipreserves , as set out by ec regulations 2073/2005 related to this category which distinguishes the foods that favour l. monocytogenes growth from those that do not . packaged products marketed in the veneto region ( italy ) were sampled to determine the levels of l. monocytogenes . the products inspected included ones with intrinsically favourable characteristics , thus ideal for l. monocytogenes growth , and those with unfavourable characteristics ( ph 4.4 , aw 0.92 ; ph 5.0 and aw 0.94 ) . tests were carried on both these types of products stored , at 4c and at 10c , the latter being a more realistic simulation of household conditions which may experience thermal abuse . each sample unit , aside from examining l. monocytogenes ( qualitative and quantitative tests ) also analysed the following : total aerobic mesophilic count , total psychrophiles count , total psychrophiles h2s producers , moulds , yeasts , and lactic bacteria to establish whether correlation exists in relation to the presence of l. monocytogenes furthermore , enrichment broths were also tested for l. monocytogenes using pcr and serotyping and ribotyping of isolated strains and culture tests . the samples , each consisting of 3 sample units coming from a production batch , upon reaching the laboratory , were stored in a refrigerator in their original package at a temperature of 4c and 10c until their shelf - life expiry ( storage at 10c in the last third of their shelf - life expiry ) . product shelf - life ranged from 8 days ( raw salmon carpaccio ) to 70 days ( seafood salads ) . upon reaching the laboratory , one sample unit was examined immediately , the other two were on the last day of expiry . in total 38 samples were analysed , amounting to 114 sample units . l. monocytogenes count on aloa agar at 37c for 48 hours ( iso 11290 - 2:1998/amd 1 2004).detection of l. monocytogenes on aloa agar and palcam agar plates at 37c for 48 hours following selective enrichment in half fraser and fraser broth ( iso 11290 - 1:1996/amd 1 2004 ) . count of the moulds and yeasts on rose bengal chloramphenicol agar plates with incubation in aerobiosis at 25c for 5 days . detection of l. monocytogenes on aloa agar and palcam agar plates at 37c for 48 hours following selective enrichment in half fraser and fraser broth ( iso 11290 - 1:1996/amd 1 2004 ) . the quantitative and qualitative methods were carried out at the same time , the same day . ph measure using the mettler toledo mp 220 instrument , with temperature autocompensation.water activity ( aw ) using rotronic 29539 instrument ( iso 21807:2004 ) . detection of l. monocytogenes consisted in taking 1 ml of enrichment broth ( half fraser ) , after 24 hours of incubation , to extract dna and carry out subsequent pcr tests for listeriaspp and l. monocytogenes . dna extraction was carried out on pellet , obtained after centrifugation of the enrichment broth ( 2,000 g for 2 minutes , followed by 12,000 g for 5 minutes on the surnatant ) . nested pcr was the method used to target the codifying gene for 16s rrna in which the amplified product of the first reaction becomes the template for subsequent nested reactions . the primers used in the first reaction were the forward primer li1 5-ctc cat aaa ggt gac cct-3 and the reverse primer u1 5-cag cmg ccg cgg taa twc-3 . for amplification , the thermal cyler geneamp pcr system 9700 ( applied biosystem ) was used with temperatures set at initial denaturation at 95c for 3 min , followed by 25 cycles , each with a denaturation phase at 95c for 90 sec , and an annealing phase at 50c for 90 sec and an extension phase at 72c for 2 min , followed by a final extension phase at 72c for 10 min . the primers used for the nested reactions were forward primer ls1 5-acg acc gca adg ttg aaa ct-3 and reverse primer ls2 5-gac gtc atc ccc acc ttc ct-3 manufactured at the nucleic acids technology laboratory applied to foods at the istituto zooprofilattico sperimentale of brescia . analysis of foreseen amplification , of 301 bp , was carried out after electrophoresis in agarose gel at 2.5% stained with ethidium bromide ( final concentration on gel : 0.5 g / ml ) , by means of exposure to uv radiation . reaction was prepared using an end volume of 25 l with concentrations of 1x geneamp pcr buffer ii ( applied biosystems ) , 1.5 mm of mgcl2 , 0.2 mm for each dntp , 0.2 m of both primers , 0.75 u of ampli taq dna polymerase ( applied biosystems ) and 5 l of extracted dna . amplification was carried out in the thermal cycler geneamp pcr system 9700 ( applied biosystem ) with a temperature profile for initial denaturation set at 95c for 3 min , followed by 35 cycles , each comprising a denaturation phase at 95c for 30 sec , an annealing phase at 58c for 30 sec and an extension phase at 72c for 30 sec , followed by a final extension phase at 72c for 5 min . analysis of foreseen amplification , of 234 bp , was then conducted after electrophoresis in agarose gel at 2.5% stained with ethidium bromide ( final concentration on gel : 0,5 g / ml ) , by means of exposure to uv radiation . serotyping by m - pcr was performed using primers as described by doumith et al .. primers enable the identification of listeria and the subdivision of strains belonging only to the l. monocytogenes species into four distinct serogroups . the pcr mix included pcr master mix 1x ( qiagen , milan , italy ) , mix of primers ( lmo0737 , orf2819 , orf2110 , lmo1118 and prs ) , sterile distilled penta - h2o and the dna extracted . serotyping with antisera yielded 12 different serotypes of l. monocytogenes in terms of cellular surface , and the somatic seroagglutination was carried out to confirm the serotypes and serogroups obtained using the molecular method . subsequently , a batch containing eight containers to hold eight samples was inserted in the automated ribotyping instrument . finally , the instrument detects the signal emitted by a ccd camera and software converts the images in ribotyping patterns . ribotyping ( of the species ) is an automated process if more than 85% of the sample patterns resemble the reference patterns of the instrument 's database . , atcc , dsmz , or jmc ) and identified with a dup - id code ( dupont identification ) . each ribotyped strain was compared with all the patterns of the profiles contained in the database . in general , if there was a similarity 93% between the profile of the strain investigated and that of the databank , the strain was assigned to the corresponding ribogroup of that profile . the analyses were carried out on a range of 38 different commercial products 28 ( 73,68% ) of which had aw and ph values favourable for growth of l. monocytogenes , while 10 ( 26,32% ) had unfavourable values instead ( table 1 ) . of the 28 samples with favourable characteristics for growth of the pathogen conditions , nine were positive for l. monocytogenes ( 32,14% ) , according to the standard culture methods ( iso 11290 - 1:1996/amd 1 2004 ) ; of these , 3 ( 10,71% ) had values exceeding the limit ( 100 cfu / g ) established by ec reg . no sample with unfavourable characteristics was found positive for viable l. monocytogenes in microbiological testing . table 2 illustrates the corresponding values of l. monocytogenes qualitative test , quantitative test , and pcr results . listerial genus was present in all 38 samples analysed according to the amplification results of the genus specific pcr reaction . nonetheless , the species specific pcr demonstrated the presence of l. monocytogenes after enrichment in 12 samples only , of which 9 were also found to contain the pathogen by classical culture tests . in the 3 samples with a charge of > 100 cfu / g , l. monocytogenes grew despite the presence of competitive flora , both lactic and alternating . despite the increase of microbial values at the end of shelf - life , there were no significant changes in the product ph and aw values . in evidence that of three samples with a higher than 100 cfu / g value ( sample no . 2 , 8 and 10 ) , two were analysed after thermal abuse at 10c ( last third of its shelf - life ) , and one proved unfit at the time it was being taken . 2 , at the end of its shelf - life , had bulging but no unpleasant odours . serotyping of 15 isolated l. monocytogenes strains coming from rte fish semipreserves was carried out using multiplex pcr to separate the 4 main serotypes ( 1/2a , 1/2b , 1/2c and 4b ) into 4 distinct groups . the 15 strains of l. monocytogenes analysed came from 4 different serotypes : the highest percentage was related to serotype 1/2a ( 73,33% ) , followed by serotype 4b ( 13,33% ) , 1/2b ( 6,67% ) and 4d ( 6,67% ) . ribotyping of l.monocytogenes colonies in microbiological tests ( see table 4 ) has yielded the broad variation of isolated strains , identifying 15 particular strains on the basis of species . dup-20243 , dup-1043 , and -dup1052 were characterised in the strains examined , in 3 ( 20% ) , 2 ( 13.33% ) and 1 ( 6.66% ) sample , respectively . in terms of ribogroup distribution , rg-568 was the most diffuse , found in 9 of the 15 samples ( 60% ) , and the other ribogroups were each found in only one sample , except rg-1296 which was found in 2 samples . data from phylogenetic analysis on the restriction pattern ( figure 2 ) yielded a classification of three main strain groups identified with ribogroup 568 , being highly phylogenetically related , in turn distant , though still phylogenetically related to the 4 strains grouped in the upper part of the dendrogram . it was analysed after a shelf - life at a temperature of 4c being very different from rest of the l. monocytogenes that were characterized . the data demonstrate that there is a rather low probability of l.monocytogenes exceeding the 100 cfu / g limit in the rte seafood distributed in the veneto region , which would at any rate be associated with improper storage of the product or with thermal abuse . in fact , of the 3 positive samples out of the 38 , 2 had been stored at 10c in the last third period of their lifecycle and only one was defective at the time it was collected . analysis conducted at the end of shelf - life provides additional confirmation of the above - mentioned data . the total charge of psychrophiles and of lactic bacteria found in the three positive samples was high , differently from jameson 's theory which affirms that the lactic charge should develop a sort of microbial competition against l. monocytogenes . it is worth noting that 9 semi - preserves resulted positive at the qualitative microbiological test for l. monocytogenes ( found in 25 g ) . if at the end of shelf - life the above mentioned 9 samples had a < 5 cfu level , the producer would still have to demonstrate , with appropriate studies and challenge tests , that l. monocytogenes will not grow and multiply . in case of marinated products , just three samples had viable l. monocytogenes . at the end of shelf - life differently from salmon , these products have much lower aw and ph values , a factor that no doubt affects the growth of l. monocytogenes . moreover , smoked salmon production will have l. monocytogenes in the raw ingredient which can not be eliminated in the phases leading to final packaging but can only be contained . if there is any l. monocytogenes present in the end product , it implies that contamination occurred after the processing phases , after marinating . the higher number of positive samples in the molecular biology tests and the inability to isolate culture underscore the presence of viable not culturable ( vnc ) organisms or no longer viable cells . the pcr test compared to the culture used on first enrichment broth it may prove useful in routine laboratory work because at least in pcr - negative cases it does not require microbiological tests . the samples examined , all being positive to pcr tests for listeria , confirm that these kinds of products are often contaminated with listeria , but that such condition rarely evolves into hazardous l. monocytogenes concentrations . in all the samples with unfavourable aw and ph values for l. monocytogenes growth , the pcr test for l. monocytogenes was negative , confirming that even if the product was contaminated in the production phase , l. monocytogenes would be overwhelmed by the product 's unfavourable environment . the latter is no doubt less reproducible , more expensive , and requires specially trained staff , but it enables the detection of rare serotypes which in the years to come may be more frequently associated to cases of listeriosis . it is striking that 10 of the 11 l. monocytogenes strains isolated from salmon belonged to serotype 1/2a , as already confirmed by other scientific sources [ 3941 ] . the agglutination method to confirm the serotypes of the single serogroups detected with m - pcr rare serotypes have been linked to an epidemic outbreak , as confirmed in a finnish study by maijala et al . an epidemic case of listeriosis caused by the strain l. monocytogenes serotype 3a , isolated in packaged butter , was described therein . figure 2 shows the variability of strains found in the environment underscoring the need to develop methods that can assess the characterisation of pathogenicity , since same strains may correspond to different levels of pathogenicity . however , despite the various reports on genotyping and characterization [ 43 , 44 ] , there is still very little empirical evidence on the correlation between its presence in food and the ensuing pathologies its consumption generates in humans . this ought to encourage a more ample use of molecular characterization to provide information , even at the epidemiological level , on the distribution of l. monocytogenes in the environment , which though it maintains phenotypic homogeneity , undergoes ongoing phenomena of clonal evolution with small , yet significant , genome variations . in addition , it is interesting to note that the 4 strains grouped in the upper part of the dendrogram identified as rg 1296 , rg 1369 , and rg 1533 were all isolated in samples analysed at time 0 . while ribogroups 564 , 568 , and 1340 were instead isolated in samples analysed both at the beginning as well as at the end of the shelf - life period , this suggests that the latter may be more resistant in time to different storage conditions . because of the relatively small number of tested samples , this work should be considered as a preliminary study . in fact , the refrigeration chain is not always respected especially in case of products with a few weeks of shelf - life , as the products in this report , which may be subjected to temperature changes which do not bring about any evident organoleptic alterations . production companies must therefore bear this in mind when certifying their products , using challenge tests to establish how safe a product is in terms of l. monocytogenes . products with aw and ph characteristics that favour l. monocytogenes growth were the only ones to result positive to microbiological and pcr tests in this work , in support of the potential hazard of these products . the highest percentage related to serotype 1/2a confirm similar investigations in seafood products and the genetic characterization using ribotyping demonstrates the genomic variability of l. monocytogenes strains also in this kind of food . this distinctive features could be interesting related to l. monocytogenes survival capacity under different storage conditions . nearly 30 years have gone by since canadians schelch demonstrated that l. monocytogenes can infect human via contaminated food . in tandem education campaigns geared at the consumers who may be exposed to l. monocytogenes must provide general rules and guidelines for proper food storage and for food preparation in the domestic environment .

the plight of people with type 1 diabetes changed dramatically with the introduction of insulin therapy in 1922 ( 1 ) . type 1 diabetes was transformed from a uniformly fatal disease in the preinsulin era , with mortality occurring either acutely from diabetic ketoacidosis or subsequently from inanition owing to a chronic catabolic state , to a chronic degenerative disease . in the first 15 to 20 years of insulin therapy , a host of complications that had never been seen before was discovered in people with long - term diabetes ( 2 ) . these complications , affecting the eyes , kidneys , and peripheral nervous system , were collectively called microvascular complications , to distinguish them from the less diabetes - specific but highly prevalent macrovascular disease complications . microvascular disease and peripheral neuropathy resulted in blindness , kidney failure , and amputations ( 3 ) ; and macrovascular disease , exacerbated by renal dysfunction and autonomic neuropathy , increased the risk for myocardial infarctions and stroke to levels that were 10-fold or more than in the age - matched nondiabetic population ( 2,3 ) . the pathoetiology of the microvascular complications was vigorously debated during the mid-20th century ( 46 ) . some practitioners considered the complications a result of nonphysiologically controlled hyperglycemia ; others thought that they were a glycemia - independent feature of diabetes . perhaps the most sensible opinion regarding the role of glucose control , expressed by r.d . lawrence , the preeminent diabetologist of his time and who had type 1 diabetes himself , was as follows : the attempt to keep the blood sugar constantly normal may be ideal in theory , but in practice it is very difficult to achieve and makes the diabetic life unnecessarily hard without adequate benefit ( 7 ) . the devastating consequences of the long - term complications led in part to the formation of the national diabetes commission by an act of congress ( pl 93 - 354 ) . in 1975 , the commission issued the long - range plan to combat diabetes , which included the recommendation for the national institutes of health ( nih ) to initiate and support a 5-year clinical study to assess the effects of treatment of juvenile - onset diabetes on the development of microvascular and macrovascular complications . the advances necessary to perform a definitive clinical trial were finally in place by the early 1980s . these included : the ability to manage glucose levels in the near - normal range using multiple daily injection ( mdi ) therapy or continuous subcutaneous insulin infusion ( csii ) with external pumps , guided by self - monitoring of blood glucose ( smbg ) ; the means of measuring chronic glycemia objectively and accurately with the glycated hemoglobin ( hba1c ) assay ; and objective measures of long - term complications . with these tools available and with generous support from the national institute of arthritis , diabetes , and digestive and kidney diseases , which later became the national institute of diabetes and digestive and kidney diseases ( niddk ) , 21 centers were selected in 1982 to plan and conduct a study that would test what had become known as the glucose hypothesis . practically stated , the glucose hypothesis posited that achieving near - normal glucose would ameliorate the long - term complications of diabetes . over the course of more than a year , the investigators planned the diabetes control and complications trial ( dcct ) ( 8) . the two primary aims of the dcct consensus protocol were to determine whether , compared with conventional therapy ( con ) , an intensive treatment program designed to achieve glycemic control as close to the nondiabetic range as safely possible would prevent or delay the appearance of early background retinopathy ( primary prevention ) and would prevent the progression of early retinopathy to more advanced forms of retinopathy ( secondary intervention ) . after the successful completion of a 1-year feasibility phase , during which a substantial separation of hba1c levels between the intensive therapy ( int ) ( experimental ) and con ( standard ) groups was achieved ( 9 ) , an additional eight centers were added , and full - scale recruitment began . recruitment ended in 1989 , and the dcct was halted by its independent oversight committee in 1993 , approximately 1 year ahead of schedule , owing to the uniform and conclusive results achieved ( 10 ) . the original con group was taught int , and the entire cohort was invited to join a long - term observational study named the epidemiology of diabetes interventions and complications ( edic ) ( 11 ) . briefly , in order to address the primary prevention and secondary intervention questions , the following eligibility criteria were used : age 1339 years with type 1 diabetes diagnosed based on clinically accepted criteria and with fasting c - peptide concentrations < 0.2 nmol / l . the subjects had to be generally healthy with no history of cardiovascular disease ( cvd ) and without hypertension ( blood pressure < 140/90 mmhg ) or dyslipidemia ( 8) , and those with neuropathy requiring treatment were excluded . additionally , the primary prevention cohort had to have a duration of 15 years with no evidence of retinopathy on fundus photography and an albumin excretion rate ( aer ) < 40 mg per 24 h. the secondary intervention cohort could have a longer duration of diabetes ( 115 years ) and had to have at least one microaneurysm in either eye . this cohort could have an aer as high as 200 mg per 24 h. subjects in the primary prevention cohort and those in the secondary intervention cohort with < 5 years duration could have 2-h stimulated c - peptide levels as high as 0.5 nmol / l ; otherwise , it had to be 0.2 the clinical goals for both treatment groups included absence of frequent symptoms of hyperglycemia or frequent or severe hypoglycemia , defined as requiring assistance from another person . dcct int aimed to achieve hba1c levels that were < 2 sd above the mean value determined for similarly aged nondiabetic volunteers ( < 6.05% , 42.6 mmol / mol ) . hba1c was measured monthly to aid adjustment of int and quarterly as a process outcome in both therapy groups . int was adjusted based on four or more smbg tests with the following self - monitored glucose targets : premeals 70120 mg / dl ( 3.96.7 mmol / l ) and 2-h postmeals < 180 mg / dl ( 10 mmol / l ) . in addition , a weekly 3:00 a.m. blood glucose was to be > 65 mg / dl ( 3.6 mmol / l ) in order to protect against otherwise unappreciated nocturnal hypoglycemia . to achieve the glycemic goals , participants randomly assigned to int used at least three insulin injections per day ( mdi ) or csii . the insulins used were those that were available at the time : clear zinc ( regular ) insulin for premeal boluses and in the insulin pump and nph , lente , and beef ultralente insulin for basal delivery in mdi regimens . there was no single mdi or csii regimen , and clinic staff and participants worked together to individualize the regimens to match lifestyle factors and achieve the smbg and hba1c goals ( 12 ) . con was consistent with standard care in the 1980s and usually included one or two daily injections of insulin with daily urine or smbg . the only numeric glycemic target was if hba1c exceeded 13.5% , the mean + 2 sd of the cohort s baseline value , in which case treatment was intensified independent of whether the subject had symptoms . retinopathy , which was measured objectively with stereoscopic fundus photography and graded with standardized methods by a central reading center ( 13 ) , was the primary outcome used for power and sample - size calculations . similarly important outcomes were nephropathy and retinopathy . the measurements and their frequency and definitions of outcomes are included in table 1 . major outcome measurements in the wake of the successful completion of the dcct ( 10 ) , the dcct investigators and the niddk decided that longer - term follow - up would provide important information regarding the durability of the original dcct int effects and , in particular , the effects of int on the more - advanced stages of complications and cvd ( 11 ) . after the end of the dcct and before initiating the long - term follow - up called edic , all of the con participants were offered training in int . in addition , diabetes care was returned to the subjects own care provider , some of whom were dcct / edic investigators . whereas the dcct was a controlled clinical trial , edic was observational . the frequency of interactions with the subjects and of the outcome measurements decreased substantially ( table 1 ) ; however , the methods of measuring glycemia , other metabolic outcomes , and complications remained identical to those used during dcct . the clinical goals for both treatment groups included absence of frequent symptoms of hyperglycemia or frequent or severe hypoglycemia , defined as requiring assistance from another person . dcct int aimed to achieve hba1c levels that were < 2 sd above the mean value determined for similarly aged nondiabetic volunteers ( < 6.05% , 42.6 mmol / mol ) . hba1c was measured monthly to aid adjustment of int and quarterly as a process outcome in both therapy groups . int was adjusted based on four or more smbg tests with the following self - monitored glucose targets : premeals 70120 mg / dl ( 3.96.7 mmol / l ) and 2-h postmeals < 180 mg / dl ( 10 mmol / l ) . in addition , a weekly 3:00 a.m. blood glucose was to be > 65 mg / dl ( 3.6 mmol / l ) in order to protect against otherwise unappreciated nocturnal hypoglycemia . to achieve the glycemic goals , participants randomly assigned to int used at least three insulin injections per day ( mdi ) or csii . the insulins used were those that were available at the time : clear zinc ( regular ) insulin for premeal boluses and in the insulin pump and nph , lente , and beef ultralente insulin for basal delivery in mdi regimens . there was no single mdi or csii regimen , and clinic staff and participants worked together to individualize the regimens to match lifestyle factors and achieve the smbg and hba1c goals ( 12 ) . con was consistent with standard care in the 1980s and usually included one or two daily injections of insulin with daily urine or smbg . the only numeric glycemic target was if hba1c exceeded 13.5% , the mean + 2 sd of the cohort s baseline value , in which case treatment was intensified independent of whether the subject had symptoms . retinopathy , which was measured objectively with stereoscopic fundus photography and graded with standardized methods by a central reading center ( 13 ) , was the primary outcome used for power and sample - size calculations . similarly important outcomes were nephropathy and retinopathy . the measurements and their frequency and definitions of outcomes are included in table 1 . in the wake of the successful completion of the dcct ( 10 ) , the dcct investigators and the niddk decided that longer - term follow - up would provide important information regarding the durability of the original dcct int effects and , in particular , the effects of int on the more - advanced stages of complications and cvd ( 11 ) . after the end of the dcct and before initiating the long - term follow - up called edic , all of the con participants were offered training in int . in addition , diabetes care was returned to the subjects own care provider , some of whom were dcct / edic investigators . whereas the dcct was a controlled clinical trial , edic was observational . the frequency of interactions with the subjects and of the outcome measurements decreased substantially ( table 1 ) ; however , the methods of measuring glycemia , other metabolic outcomes , and complications remained identical to those used during dcct . the characteristics of the dcct cohort at baseline and at study end , which represents the edic baseline , and at the most recent edic annual examination in 2012 are shown in table 2 . the baseline characteristics were well matched between the int and con for the primary prevention and secondary intervention cohorts . although in most long - term studies loss to follow - up may compromise the integrity and interpretation of study results , the follow - up in dcct and subsequently in edic has been virtually complete . at the end of dcct , after an average of 6.5 years ( range 39 ) , more than 99% ( 1,422 of 1,441 ) completed the study . after another 20 years of follow - up in edic , 88% of the original cohort ( 95% of the survivors ) is being actively followed in dcct / edic clinical centers . clinical characteristics of dcct / edic participants at dcct baseline , dcct closeout , and edic year 18 the dcct int did not uniformly achieve the goal hba1c of < 6.05% ; however , 44% reached that level at least once during the trial ( 10 ) . the median of the quarterly measured hba1c levels in int was 7% compared with 9% in con ( fig . there was almost no crossover between int and con during the dcct , other than the protocol - dictated change to int for women assigned to con who were planning pregnancy and during pregnancy ( 14 ) . the adoption of int by the original con group and the transition for all subjects to their own health care providers resulted in a narrowing and then disappearance of the differences in hba1c maintained during dcct ( fig . median hba1c concentrations during dcct , the training period between dcct and edic , and edic . p < 0.001 for int vs. con during entire dcct and for the first 3 years during edic . reprinted and modified with permission from nathan et al . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . the two major adverse events experienced by int subjects were hypoglycemia and weight gain ( 1517 ) . the definition established for severe hypoglycemia , which has subsequently been adopted by many studies , was meant to be relatively inclusive but not to include episodes that were recognized and treated by the patients . to qualify as severe hypoglycemia , an episode had to require assistance from another and included coma or seizures or episodes requiring glucagon , iv dextrose , or oral carbohydrate administered by another person . although the intent was to limit bias of ascertainment by collecting the hypoglycemia events at quarterly visits for both int and con subjects , int subjects were seen and contacted more frequently than those in the con group , and some of the differences in hypoglycemia may be attributable to differences in the frequency of ascertainment . the frequency of severe hypoglycemia ( 62/100 patient - years ) and the subset of episodes involving coma or seizure ( 16/100 patient - years ) were both threefold higher than in the con group . despite the increased frequency of hypoglycemia , there were no adverse effects of int or of repeated severe episodes , on rigorously and repeatedly measured cognitive function in adults or adolescents , either during the dcct or after even longer - term follow - up ( 1820 ) . weight gain with int resulted in significantly more subjects becoming overweight or obese compared with con ( 17 ) . more detailed descriptions of the individual outcomes are presented in the subsequent articles in this series ( 2125 ) . in brief , dcct int reduced the early stages of microvascular complications by 3576% compared with con ( fig . 2 ) ( 10 ) . the magnitude and consistent direction of the effects on retinopathy , neuropathy , and nephropathy led to the termination of the study 1 year ahead of schedule by the independent oversight group . analyses of the relationship between metabolic control , measured by the updated mean hba1c and including approximately 18,000 hba1c measurements for each therapy group , revealed a strong association with each of the three complications ( 26,27 ) . the difference in updated mean hba1c levels between the therapy groups explained the vast majority ( > 98% ) of the difference in complications between the groups ( 26 ) , leaving little room for other established or putative risk factors to play a role in the trial s differential outcomes . summary of reduction in major complications with int compared with con during dcct , edic , and combined study periods . 3+step devel , prim : three - step or more development of retinopathy based on early treatment of diabetic retinopathy scale ( ref . microalb : microalbuminuria defined as albumin excretion 40 mg/24 h. macroalb : macroalbuminuria defined as albumin excretion > 300 mg/24 h. reduced gfr : estimated gfr < 60 ml / min/1.73 m. cvd events : cvd including myocardial infarctions , stroke , and cvd death . reprinted with permission from nathan et al . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . diabetes 2013;62:39763986 . considering the powerful effect that glycemic separation had on the outcomes during dcct , the subsequent narrowing and then disappearance of the difference in hba1c levels between the two original therapy groups during edic could logically have been expected to result in the subsequent parallel development of complications . however , the first 4 years of the edic follow - up demonstrated a further widening of the differences in outcomes , after adjusting for edic baseline outcomes ( 28 ) . this phenomenon of a durable effect on complications of prior metabolic control was named metabolic memory . studies during edic suggested that glycation of long - lived proteins , such as dermal collagen , might account for this persistent effect ( 33 ) . the long - term edic follow - up has allowed the study of the impact of int versus con on more advanced complications than were studied during the dcct . major beneficial effects of int on advanced complications ( 34 ) , including retinopathy ( 35 ) , nephropathy ( reduced glomerular filtration rate [ gfr ] ) ( 36 ) , and autonomic manifestations of neuropathy ( 37 ) , have been demonstrated ( fig . , measurements of atherosclerosis in several macrovascular beds , including carotid intima media thickness ( 38 ) and computed tomography measured coronary artery calcification ( 39 ) , have revealed less atherosclerosis in the int group . the clinical expression of these changes , fatal and nonfatal myocardial infarctions and stroke , were also reduced by int , with a 58% reduction in cvd events after a mean of 18 years of follow - up from the beginning of the dcct ( 40 ) . the characteristics of the dcct cohort at baseline and at study end , which represents the edic baseline , and at the most recent edic annual examination in 2012 are shown in table 2 . the baseline characteristics were well matched between the int and con for the primary prevention and secondary intervention cohorts . although in most long - term studies loss to follow - up may compromise the integrity and interpretation of study results , the follow - up in dcct and subsequently in edic has been virtually complete . at the end of dcct , after an average of 6.5 years ( range 39 ) , more than 99% ( 1,422 of 1,441 ) completed the study . after another 20 years of follow - up in edic , 88% of the original cohort ( 95% of the survivors ) is being actively followed in dcct / edic clinical centers . clinical characteristics of dcct / edic participants at dcct baseline , dcct closeout , and edic year 18 the dcct int did not uniformly achieve the goal hba1c of < 6.05% ; however , 44% reached that level at least once during the trial ( 10 ) . the median of the quarterly measured hba1c levels in int was 7% compared with 9% in con ( fig . there was almost no crossover between int and con during the dcct , other than the protocol - dictated change to int for women assigned to con who were planning pregnancy and during pregnancy ( 14 ) . the adoption of int by the original con group and the transition for all subjects to their own health care providers resulted in a narrowing and then disappearance of the differences in hba1c maintained during dcct ( fig . median hba1c concentrations during dcct , the training period between dcct and edic , and edic . p < 0.001 for int vs. con during entire dcct and for the first 3 years during edic . reprinted and modified with permission from nathan et al . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . the two major adverse events experienced by int subjects were hypoglycemia and weight gain ( 1517 ) . the definition established for severe hypoglycemia , which has subsequently been adopted by many studies , was meant to be relatively inclusive but not to include episodes that were recognized and treated by the patients . to qualify as severe hypoglycemia , an episode had to require assistance from another and included coma or seizures or episodes requiring glucagon , iv dextrose , or oral carbohydrate administered by another person . although the intent was to limit bias of ascertainment by collecting the hypoglycemia events at quarterly visits for both int and con subjects , int subjects were seen and contacted more frequently than those in the con group , and some of the differences in hypoglycemia may be attributable to differences in the frequency of ascertainment . the frequency of severe hypoglycemia ( 62/100 patient - years ) and the subset of episodes involving coma or seizure ( 16/100 patient - years ) were both threefold higher than in the con group . despite the increased frequency of hypoglycemia , there were no adverse effects of int or of repeated severe episodes , on rigorously and repeatedly measured cognitive function in adults or adolescents , either during the dcct or after even longer - term follow - up ( 1820 ) . weight gain with int resulted in significantly more subjects becoming overweight or obese compared with con ( 17 ) . the 4.6 kg difference in weight during the dcct largely dissipated during the edic . more detailed descriptions of the individual outcomes are presented in the subsequent articles in this series ( 2125 ) . in brief , dcct int reduced the early stages of microvascular complications by 3576% compared with con ( fig . 2 ) ( 10 ) . the magnitude and consistent direction of the effects on retinopathy , neuropathy , and nephropathy led to the termination of the study 1 year ahead of schedule by the independent oversight group . analyses of the relationship between metabolic control , measured by the updated mean hba1c and including approximately 18,000 hba1c measurements for each therapy group , revealed a strong association with each of the three complications ( 26,27 ) . the difference in updated mean hba1c levels between the therapy groups explained the vast majority ( > 98% ) of the difference in complications between the groups ( 26 ) , leaving little room for other established or putative risk factors to play a role in the trial s differential outcomes . summary of reduction in major complications with int compared with con during dcct , edic , and combined study periods . 3+step devel , prim : three - step or more development of retinopathy based on early treatment of diabetic retinopathy scale ( ref . microalb : microalbuminuria defined as albumin excretion 40 mg/24 h. macroalb : macroalbuminuria defined as albumin excretion > 300 mg/24 h. reduced gfr : estimated gfr < 60 ml / min/1.73 m. cvd events : cvd including myocardial infarctions , stroke , and cvd death . reprinted with permission from nathan et al . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . considering the powerful effect that glycemic separation had on the outcomes during dcct , the subsequent narrowing and then disappearance of the difference in hba1c levels between the two original therapy groups during edic could logically have been expected to result in the subsequent parallel development of complications . however , the first 4 years of the edic follow - up demonstrated a further widening of the differences in outcomes , after adjusting for edic baseline outcomes ( 28 ) . this phenomenon of a durable effect on complications of prior metabolic control was named metabolic memory . studies during edic suggested that glycation of long - lived proteins , such as dermal collagen , might account for this persistent effect ( 33 ) . the long - term edic follow - up has allowed the study of the impact of int versus con on more advanced complications than were studied during the dcct . major beneficial effects of int on advanced complications ( 34 ) , including retinopathy ( 35 ) , nephropathy ( reduced glomerular filtration rate [ gfr ] ) ( 36 ) , and autonomic manifestations of neuropathy ( 37 ) , have been demonstrated ( fig . , measurements of atherosclerosis in several macrovascular beds , including carotid intima media thickness ( 38 ) and computed tomography measured coronary artery calcification ( 39 ) , have revealed less atherosclerosis in the int group . the clinical expression of these changes , fatal and nonfatal myocardial infarctions and stroke , were also reduced by int , with a 58% reduction in cvd events after a mean of 18 years of follow - up from the beginning of the dcct ( 40 ) . the dcct and its observational edic follow - up were designed to determine whether the long - term complications that affect people with type 1 diabetes could be ameliorated by intensive glycemic therapy . the dcct / edic convincingly demonstrated that the glucose hypothesis was correct and that an intervention that aimed to achieve glycemia as close to the nondiabetic range as safely possible reduced all of the microvascular and cardiovascular complications of diabetes . translating the findings of the dcct / edic into clinical care

objectivethe diabetes control and complications trial ( dcct ) was designed to test the glucose hypothesis and determine whether the complications of type 1 diabetes ( t1 dm ) could be prevented or delayed . the epidemiology of diabetes interventions and complications ( edic ) observational follow - up determined the durability of the dcct effects on the more - advanced stages of diabetes complications including cardiovascular disease ( cvd).research design and methodsthe dcct ( 19821993 ) was a controlled clinical trial in 1,441 subjects with t1 dm comparing intensive therapy ( int ) , aimed at achieving levels of glycemia as close to the nondiabetic range as safely possible , with conventional therapy ( con ) , which aimed to maintain safe asymptomatic glucose control . int utilized three or more daily insulin injections or insulin pump therapy guided by self - monitored glucose . edic ( 1994present ) is an observational study of the dcct cohort.resultsthe dcct followed > 99% of the cohort for a mean of 6.5 years and demonstrated a 3576% reduction in the early stages of microvascular disease with int , with a median hba1c of 7% , compared with conv , with a median hba1c of 9% . the major adverse effect of int was a threefold increased risk of hypoglycemia , which was not associated with a decline in cognitive function or quality of life . edic showed a durable effect of initial assigned therapies despite a loss of the glycemic separation ( metabolic memory ) and demonstrated that the reduction in early - stage complications during the dcct translated into substantial reductions in severe complications and cvd.conclusionsdcct/edic has demonstrated the effectiveness of int in reducing the long - term complications of t1 dm and improving the prospects for a healthy life span .

Introduction Research Design and Methods DCCT Interventions and Metabolic Goals Outcomes EDIC Design and Outcomes Results Subjects Glycemia Adverse Effects Outcomes EDIC/Metabolic Memory Conclusions

the plight of people with type 1 diabetes changed dramatically with the introduction of insulin therapy in 1922 ( 1 ) . type 1 diabetes was transformed from a uniformly fatal disease in the preinsulin era , with mortality occurring either acutely from diabetic ketoacidosis or subsequently from inanition owing to a chronic catabolic state , to a chronic degenerative disease . in the first 15 to 20 years of insulin therapy , a host of complications that had never been seen before was discovered in people with long - term diabetes ( 2 ) . microvascular disease and peripheral neuropathy resulted in blindness , kidney failure , and amputations ( 3 ) ; and macrovascular disease , exacerbated by renal dysfunction and autonomic neuropathy , increased the risk for myocardial infarctions and stroke to levels that were 10-fold or more than in the age - matched nondiabetic population ( 2,3 ) . the pathoetiology of the microvascular complications was vigorously debated during the mid-20th century ( 46 ) . some practitioners considered the complications a result of nonphysiologically controlled hyperglycemia ; others thought that they were a glycemia - independent feature of diabetes . lawrence , the preeminent diabetologist of his time and who had type 1 diabetes himself , was as follows : the attempt to keep the blood sugar constantly normal may be ideal in theory , but in practice it is very difficult to achieve and makes the diabetic life unnecessarily hard without adequate benefit ( 7 ) . the devastating consequences of the long - term complications led in part to the formation of the national diabetes commission by an act of congress ( pl 93 - 354 ) . in 1975 , the commission issued the long - range plan to combat diabetes , which included the recommendation for the national institutes of health ( nih ) to initiate and support a 5-year clinical study to assess the effects of treatment of juvenile - onset diabetes on the development of microvascular and macrovascular complications . the advances necessary to perform a definitive clinical trial were finally in place by the early 1980s . these included : the ability to manage glucose levels in the near - normal range using multiple daily injection ( mdi ) therapy or continuous subcutaneous insulin infusion ( csii ) with external pumps , guided by self - monitoring of blood glucose ( smbg ) ; the means of measuring chronic glycemia objectively and accurately with the glycated hemoglobin ( hba1c ) assay ; and objective measures of long - term complications . with these tools available and with generous support from the national institute of arthritis , diabetes , and digestive and kidney diseases , which later became the national institute of diabetes and digestive and kidney diseases ( niddk ) , 21 centers were selected in 1982 to plan and conduct a study that would test what had become known as the glucose hypothesis . practically stated , the glucose hypothesis posited that achieving near - normal glucose would ameliorate the long - term complications of diabetes . over the course of more than a year , the investigators planned the diabetes control and complications trial ( dcct ) ( 8) . the two primary aims of the dcct consensus protocol were to determine whether , compared with conventional therapy ( con ) , an intensive treatment program designed to achieve glycemic control as close to the nondiabetic range as safely possible would prevent or delay the appearance of early background retinopathy ( primary prevention ) and would prevent the progression of early retinopathy to more advanced forms of retinopathy ( secondary intervention ) . after the successful completion of a 1-year feasibility phase , during which a substantial separation of hba1c levels between the intensive therapy ( int ) ( experimental ) and con ( standard ) groups was achieved ( 9 ) , an additional eight centers were added , and full - scale recruitment began . recruitment ended in 1989 , and the dcct was halted by its independent oversight committee in 1993 , approximately 1 year ahead of schedule , owing to the uniform and conclusive results achieved ( 10 ) . the original con group was taught int , and the entire cohort was invited to join a long - term observational study named the epidemiology of diabetes interventions and complications ( edic ) ( 11 ) . briefly , in order to address the primary prevention and secondary intervention questions , the following eligibility criteria were used : age 1339 years with type 1 diabetes diagnosed based on clinically accepted criteria and with fasting c - peptide concentrations < 0.2 nmol / l . the subjects had to be generally healthy with no history of cardiovascular disease ( cvd ) and without hypertension ( blood pressure < 140/90 mmhg ) or dyslipidemia ( 8) , and those with neuropathy requiring treatment were excluded . additionally , the primary prevention cohort had to have a duration of 15 years with no evidence of retinopathy on fundus photography and an albumin excretion rate ( aer ) < 40 mg per 24 h. the secondary intervention cohort could have a longer duration of diabetes ( 115 years ) and had to have at least one microaneurysm in either eye . this cohort could have an aer as high as 200 mg per 24 h. subjects in the primary prevention cohort and those in the secondary intervention cohort with < 5 years duration could have 2-h stimulated c - peptide levels as high as 0.5 nmol / l ; otherwise , it had to be 0.2 the clinical goals for both treatment groups included absence of frequent symptoms of hyperglycemia or frequent or severe hypoglycemia , defined as requiring assistance from another person . int was adjusted based on four or more smbg tests with the following self - monitored glucose targets : premeals 70120 mg / dl ( 3.96.7 mmol / l ) and 2-h postmeals < 180 mg / dl ( 10 mmol / l ) . to achieve the glycemic goals , participants randomly assigned to int used at least three insulin injections per day ( mdi ) or csii . the insulins used were those that were available at the time : clear zinc ( regular ) insulin for premeal boluses and in the insulin pump and nph , lente , and beef ultralente insulin for basal delivery in mdi regimens . the only numeric glycemic target was if hba1c exceeded 13.5% , the mean + 2 sd of the cohort s baseline value , in which case treatment was intensified independent of whether the subject had symptoms . retinopathy , which was measured objectively with stereoscopic fundus photography and graded with standardized methods by a central reading center ( 13 ) , was the primary outcome used for power and sample - size calculations . major outcome measurements in the wake of the successful completion of the dcct ( 10 ) , the dcct investigators and the niddk decided that longer - term follow - up would provide important information regarding the durability of the original dcct int effects and , in particular , the effects of int on the more - advanced stages of complications and cvd ( 11 ) . after the end of the dcct and before initiating the long - term follow - up called edic , all of the con participants were offered training in int . whereas the dcct was a controlled clinical trial , edic was observational . the frequency of interactions with the subjects and of the outcome measurements decreased substantially ( table 1 ) ; however , the methods of measuring glycemia , other metabolic outcomes , and complications remained identical to those used during dcct . int was adjusted based on four or more smbg tests with the following self - monitored glucose targets : premeals 70120 mg / dl ( 3.96.7 mmol / l ) and 2-h postmeals < 180 mg / dl ( 10 mmol / l ) . to achieve the glycemic goals , participants randomly assigned to int used at least three insulin injections per day ( mdi ) or csii . the insulins used were those that were available at the time : clear zinc ( regular ) insulin for premeal boluses and in the insulin pump and nph , lente , and beef ultralente insulin for basal delivery in mdi regimens . the only numeric glycemic target was if hba1c exceeded 13.5% , the mean + 2 sd of the cohort s baseline value , in which case treatment was intensified independent of whether the subject had symptoms . retinopathy , which was measured objectively with stereoscopic fundus photography and graded with standardized methods by a central reading center ( 13 ) , was the primary outcome used for power and sample - size calculations . in the wake of the successful completion of the dcct ( 10 ) , the dcct investigators and the niddk decided that longer - term follow - up would provide important information regarding the durability of the original dcct int effects and , in particular , the effects of int on the more - advanced stages of complications and cvd ( 11 ) . after the end of the dcct and before initiating the long - term follow - up called edic , all of the con participants were offered training in int . whereas the dcct was a controlled clinical trial , edic was observational . the frequency of interactions with the subjects and of the outcome measurements decreased substantially ( table 1 ) ; however , the methods of measuring glycemia , other metabolic outcomes , and complications remained identical to those used during dcct . the characteristics of the dcct cohort at baseline and at study end , which represents the edic baseline , and at the most recent edic annual examination in 2012 are shown in table 2 . although in most long - term studies loss to follow - up may compromise the integrity and interpretation of study results , the follow - up in dcct and subsequently in edic has been virtually complete . at the end of dcct , after an average of 6.5 years ( range 39 ) , more than 99% ( 1,422 of 1,441 ) completed the study . after another 20 years of follow - up in edic , 88% of the original cohort ( 95% of the survivors ) is being actively followed in dcct / edic clinical centers . clinical characteristics of dcct / edic participants at dcct baseline , dcct closeout , and edic year 18 the dcct int did not uniformly achieve the goal hba1c of < 6.05% ; however , 44% reached that level at least once during the trial ( 10 ) . the median of the quarterly measured hba1c levels in int was 7% compared with 9% in con ( fig . the adoption of int by the original con group and the transition for all subjects to their own health care providers resulted in a narrowing and then disappearance of the differences in hba1c maintained during dcct ( fig . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . although the intent was to limit bias of ascertainment by collecting the hypoglycemia events at quarterly visits for both int and con subjects , int subjects were seen and contacted more frequently than those in the con group , and some of the differences in hypoglycemia may be attributable to differences in the frequency of ascertainment . the frequency of severe hypoglycemia ( 62/100 patient - years ) and the subset of episodes involving coma or seizure ( 16/100 patient - years ) were both threefold higher than in the con group . despite the increased frequency of hypoglycemia , there were no adverse effects of int or of repeated severe episodes , on rigorously and repeatedly measured cognitive function in adults or adolescents , either during the dcct or after even longer - term follow - up ( 1820 ) . weight gain with int resulted in significantly more subjects becoming overweight or obese compared with con ( 17 ) . more detailed descriptions of the individual outcomes are presented in the subsequent articles in this series ( 2125 ) . in brief , dcct int reduced the early stages of microvascular complications by 3576% compared with con ( fig . the magnitude and consistent direction of the effects on retinopathy , neuropathy , and nephropathy led to the termination of the study 1 year ahead of schedule by the independent oversight group . the difference in updated mean hba1c levels between the therapy groups explained the vast majority ( > 98% ) of the difference in complications between the groups ( 26 ) , leaving little room for other established or putative risk factors to play a role in the trial s differential outcomes . summary of reduction in major complications with int compared with con during dcct , edic , and combined study periods . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . considering the powerful effect that glycemic separation had on the outcomes during dcct , the subsequent narrowing and then disappearance of the difference in hba1c levels between the two original therapy groups during edic could logically have been expected to result in the subsequent parallel development of complications . however , the first 4 years of the edic follow - up demonstrated a further widening of the differences in outcomes , after adjusting for edic baseline outcomes ( 28 ) . this phenomenon of a durable effect on complications of prior metabolic control was named metabolic memory . the long - term edic follow - up has allowed the study of the impact of int versus con on more advanced complications than were studied during the dcct . major beneficial effects of int on advanced complications ( 34 ) , including retinopathy ( 35 ) , nephropathy ( reduced glomerular filtration rate [ gfr ] ) ( 36 ) , and autonomic manifestations of neuropathy ( 37 ) , have been demonstrated ( fig . , measurements of atherosclerosis in several macrovascular beds , including carotid intima media thickness ( 38 ) and computed tomography measured coronary artery calcification ( 39 ) , have revealed less atherosclerosis in the int group . the clinical expression of these changes , fatal and nonfatal myocardial infarctions and stroke , were also reduced by int , with a 58% reduction in cvd events after a mean of 18 years of follow - up from the beginning of the dcct ( 40 ) . the characteristics of the dcct cohort at baseline and at study end , which represents the edic baseline , and at the most recent edic annual examination in 2012 are shown in table 2 . although in most long - term studies loss to follow - up may compromise the integrity and interpretation of study results , the follow - up in dcct and subsequently in edic has been virtually complete . at the end of dcct , after an average of 6.5 years ( range 39 ) , more than 99% ( 1,422 of 1,441 ) completed the study . after another 20 years of follow - up in edic , 88% of the original cohort ( 95% of the survivors ) is being actively followed in dcct / edic clinical centers . clinical characteristics of dcct / edic participants at dcct baseline , dcct closeout , and edic year 18 the dcct int did not uniformly achieve the goal hba1c of < 6.05% ; however , 44% reached that level at least once during the trial ( 10 ) . the median of the quarterly measured hba1c levels in int was 7% compared with 9% in con ( fig . there was almost no crossover between int and con during the dcct , other than the protocol - dictated change to int for women assigned to con who were planning pregnancy and during pregnancy ( 14 ) . the adoption of int by the original con group and the transition for all subjects to their own health care providers resulted in a narrowing and then disappearance of the differences in hba1c maintained during dcct ( fig . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . the definition established for severe hypoglycemia , which has subsequently been adopted by many studies , was meant to be relatively inclusive but not to include episodes that were recognized and treated by the patients . although the intent was to limit bias of ascertainment by collecting the hypoglycemia events at quarterly visits for both int and con subjects , int subjects were seen and contacted more frequently than those in the con group , and some of the differences in hypoglycemia may be attributable to differences in the frequency of ascertainment . the frequency of severe hypoglycemia ( 62/100 patient - years ) and the subset of episodes involving coma or seizure ( 16/100 patient - years ) were both threefold higher than in the con group . despite the increased frequency of hypoglycemia , there were no adverse effects of int or of repeated severe episodes , on rigorously and repeatedly measured cognitive function in adults or adolescents , either during the dcct or after even longer - term follow - up ( 1820 ) . weight gain with int resulted in significantly more subjects becoming overweight or obese compared with con ( 17 ) . the 4.6 kg difference in weight during the dcct largely dissipated during the edic . more detailed descriptions of the individual outcomes are presented in the subsequent articles in this series ( 2125 ) . in brief , dcct int reduced the early stages of microvascular complications by 3576% compared with con ( fig . the magnitude and consistent direction of the effects on retinopathy , neuropathy , and nephropathy led to the termination of the study 1 year ahead of schedule by the independent oversight group . analyses of the relationship between metabolic control , measured by the updated mean hba1c and including approximately 18,000 hba1c measurements for each therapy group , revealed a strong association with each of the three complications ( 26,27 ) . the difference in updated mean hba1c levels between the therapy groups explained the vast majority ( > 98% ) of the difference in complications between the groups ( 26 ) , leaving little room for other established or putative risk factors to play a role in the trial s differential outcomes . summary of reduction in major complications with int compared with con during dcct , edic , and combined study periods . diabetes control and complications trial / epidemiology of diabetes interventions and complications study at 30 years : advances and contributions . considering the powerful effect that glycemic separation had on the outcomes during dcct , the subsequent narrowing and then disappearance of the difference in hba1c levels between the two original therapy groups during edic could logically have been expected to result in the subsequent parallel development of complications . however , the first 4 years of the edic follow - up demonstrated a further widening of the differences in outcomes , after adjusting for edic baseline outcomes ( 28 ) . this phenomenon of a durable effect on complications of prior metabolic control was named metabolic memory . the long - term edic follow - up has allowed the study of the impact of int versus con on more advanced complications than were studied during the dcct . major beneficial effects of int on advanced complications ( 34 ) , including retinopathy ( 35 ) , nephropathy ( reduced glomerular filtration rate [ gfr ] ) ( 36 ) , and autonomic manifestations of neuropathy ( 37 ) , have been demonstrated ( fig . , measurements of atherosclerosis in several macrovascular beds , including carotid intima media thickness ( 38 ) and computed tomography measured coronary artery calcification ( 39 ) , have revealed less atherosclerosis in the int group . the clinical expression of these changes , fatal and nonfatal myocardial infarctions and stroke , were also reduced by int , with a 58% reduction in cvd events after a mean of 18 years of follow - up from the beginning of the dcct ( 40 ) . the dcct and its observational edic follow - up were designed to determine whether the long - term complications that affect people with type 1 diabetes could be ameliorated by intensive glycemic therapy . the dcct / edic convincingly demonstrated that the glucose hypothesis was correct and that an intervention that aimed to achieve glycemia as close to the nondiabetic range as safely possible reduced all of the microvascular and cardiovascular complications of diabetes . translating the findings of the dcct / edic into clinical care

since their discovery more than 70 years ago , wittig rearrangements have evolved into powerful tools for the isomerization of -metalated ethers into alkoxides . wittig rearrangements can proceed through a concerted , orbital - symmetry - allowed -sigmatropic shift ( scheme 1 , route a ) , or a stepwise -migration involving a radical / radical anion pair ( route b ) . arguably , the -wittig rearrangement pathway has enjoyed more attention from both mechanistic and synthetic perspectives , resulting in an impressive display of applications such as the stereoselective assembly of adjacent chiral centers , the transfer of chirality , and the formation of olefins with specific geometries . although some of these features are also characteristics of the -wittig rearrangement , a narrower range of substrates are capable of efficient -migration , perhaps a reflection of the requisite radical - stabilizing groups ( i.e. , r in scheme 1 ) for facile c o bond homolysis . another complication is the inherent problem of regioselectivity that arises in ( alkoxyallyl)metal species ( a in scheme 1 ) , where the -migration competes with the -shift , leading to mixtures of products . relative to the - and -shifts , -wittig rearrangements ( routes c and d ) are unique in their ability to generate stereodefined enolates ( rather than alkyl alkoxides ) . in addition , -wittig rearrangements have the potential to transfer chirality and stereoselectively form adjacent chiral centers . although there is some evidence supporting a stepwise mechanism for the -pathway ( route c ) , a concerted process is allowed by orbital symmetry ( route d ) and might be operative in some instances . as such , the underlying factors that govern regiocontrol in favor of either the - or -pathway remain unclear . in general , the -shift is favored at lower temperatures , while the nature of the base and base counterion can affect the product distribution . however , the : -selectivity seems to be mostly substrate - dependent , and few studies have attempted to uncover features within a substrate type that can drive the rearrangement down one path over the other . although the ring contractions of macrocyclic ethers by means of wittig rearrangements via - and -pathways have been documented in the work of marshall and takahashi , the behavior of smaller cyclic ethers is limited to only a few examples explored in the course of mechanistic studies . although certain dihydrothiopyrans have been shown to isomerize to the corresponding cyclopropyl thioenolates under basic conditions at low temperatures , we are aware of only two -wittig rearrangements of cyclic allylic ethers . reported by rautenstrauch in 1972 , these examples involve the isomerization of dihydropyran and nerol oxide to the corresponding -cyclopropylacetaldehydes ( scheme 2 ) . surprisingly , these rearrangements did not receive further attention despite being complementary to simmons smith - type reactions , transition - metal - catalyzed diazoalkyl decomposition / olefin insertions , and intramolecular cyclizations , cycloisomerizations , or stepwise cyclopropanation reactions . in this paper we demonstrate that the regioselection in favor of the - or -pathway within the context of the wittig rearrangements of diastereomeric 2-silyl-6-aryl-5,6-dihydro-(2h)-pyrans ( scheme 2 ) is dependent on both electronic and steric factors . thus , through electronic tuning at the aromatic appendage , judicious choice of the silyl group , or selecting certain olefin substitution patterns , allows one to maneuver these ring contractions toward -silylcyclopentenols ( via [ 1.2]-wittig ) or ( -cyclopropyl)acylsilanes ( via -wittig ) , with excellent diastereoselectivities and in a stereoconvergent fashion . benzylic trichloroacetimidates s1 were prepared by addition of homoallylic alcohols to trichloroacetonitrile under basic conditions ( scheme 3 ) . -hydroxysilanes s2 were prepared by retro - brook rearrangement of in situ generated o - silylated allylic alcohols . the preparation of o - trimethylsilyl -hydroxysilanes s2-f and s2-g involved trapping of the corresponding alkoxides with ( tms)cl prior to aqueous workup ( scheme 3 ) . lewis acid - catalyzed etherification of -hydroxysilanes s2 with benzylic trichloroacetimidates s1 provided diastereomeric dienes s3 that were submitted to ring - closing metathesis to afford cyclic ethers 1 and 2 ( scheme 4 ) . without exception , diastereomeric producs 1 ( trans ) and 2 ( cis ) were completely separated by column chromatography , although in some cases these could be prepared from diastereomerically pure precursors syn- or anti - s3 . compounds s3-j and s3-x were synthesized by a three - component condensation of allyltrimethylsilane , a benzaldehyde derivative and o - trimethylsilyl -hydroxysilane s2-f or s2-g , whereas dienes s3-k were prepared by suzuki cross - coupling of precursors s3-cc with phenylboronic acid ( scheme 5 ) . values in parentheses refer to the yield of a mixture of diastereomers 1 and 2 prepared from the corresponding mixture of syn / anti precursors s3 . our group recently reported the highly selective -wittig rearrangement of allyl benzyl ether bearing a trimethylsilyl group at the -allylic position . given the ability of the silyl group to ( 1 ) allow a selective deprotonation step and ( 2 ) suppress the competitive -pathway , we envisioned that diastereomeric cyclic ethers 1a/2a ( trans and cis , respectively ) would be suitable substrates for wittig rearrangements ( scheme 6 ) . indeed , under optimized conditions ( thf , 78 c ) , the trans diastereomer 1a underwent fast and selective allylic deprotonation by n - butyllithium and rearrangement within 5 min to give a mixture of the trans -wittig ( -cyclopropyl)acylsilanes 3a and cis -wittig -silylcyclopentenol 4a in good overall yield ( 82% ) , albeit with modest selectivity ( 2.4:1 ) in favor of the -product . on the other hand , cis diastereomer 2a was significantly less reactive and required excess s - butyllithium for complete conversion in 3 h. to our surprise , the same major diastereomers for both the corresponding -shift ( 3a ) and -shift ( 4a ) were obtained in virtually the same : regioisomeric ratio ( 2:1 ) observed for the trans isomer and in good overall yield . the full stereoconvergence in the - and -wittig rearrangements of trans ( 1a ) and cis ( 2a ) diastereomers is of significant importance because , in particular for the -wittig pathway , it challenges the known stereochemical outcome of this manifold . however , this apparent conflict can be rationalized by invoking the intermediacy of a common intermediate , and we have gathered supporting evidence of such species ( vide infra ) that accounts for the observed stereoconvergence and the similar product ratios obtained from the isomerization of each diastereomer . our study continued with the evaluation of the electronic effects on the aromatic appendage ( table 1 ) . such modifications to pyrans 1a and 2a were expected to directly impact the ability of the benzylic carbon to migrate through a - or -mode . nonetheless , the same reactivity trend was observed for these compounds : trans diastereomers 1b l ( entries 111 ) underwent complete rearrangement within 10 min , whereas cis diastereomers 2b l ( entries 1222 ) required at least 3 h for complete conversion . conditions : ( a ) n - buli ( 1.2 equiv ) , 10 min ; ( b ) s - buli ( 3 equiv ) , 3 h. determined by h nmr of isolated material a 1.1 equiv amount of n - buli . at 78 c , 6 h , then rt , 20 h. total of 58% recovered 2h and isomeric enol cyclic ether . a second trend was clearly observed in the trans diastereomers series : electron - donating groups located at the ortho and para positions increased the -selectivity ( entries 3 , 4 , and 6 ) , with the p - methoxy - substituted compound 1d giving exclusive -selectivity . o - methoxy substrate 1b ( entry 1 ) is an exception that might be attributed to coordination of oxygen lone pairs to the lithium cation during rearrangement , leading to a slight decrease in : -selectivity relative to that of the unsubstituted analogue 1a . the inductively electron - withdrawing methoxy group located at the meta position ( entry 2 ) led to an evident decrease in : -selectivity , providing the -wittig product in slight excess over the -product . the weakly electron - donating methyl group located at the meta position ( entry 5 ) led to a negligible effect in product distribution relative to that of the model substrate 1a . the fine balance between resonance and inductive effects was even more evident in halogenated compounds ( entries 7 and 8) : p - fluoro - substituted compound 1h afforded a 6:1 : -selectivity , whereas p - chloro - substituted compound 1i gave the reverse regioselection (: = 1:1.5 ) . on the opposite side of the spectrum , a p - trifluoromethyl group at the phenyl ring led to exclusive formation of the -wittig product ( entry 9 ) , whereas a p - biphenyl and 2-naphthyl groups directly attached to the migrating carbon also afforded high selectivity in favor of the -shift ( entries 10 and 11 ) . in all pertinent cases , the -wittig product was obtained as a single diastereomer , whereas the -product was formed with high diastereoselection ( > 15:1 ) . evaluation of cis diastereomers ( entries 1222 , table 1 ) confirmed the stereoconvergence of the - and -wittig rearrangements . both - and -pathways proceeded with diastereoselectivity similar to that of their trans counterparts , and the same electronic effects in product distribution were observed in most cases . the sluggishness of cis diastereomers to undergo allylic deprotonation had a detrimental effect on the overall yield due to competitive reactions such as ortho metalation ( entries 14 and 18 ) , lithium halogen exchange ( entry 19 ) , and presumably competitive benzylic deprotonation ( entry 20 ) . competitive ortho metalation , in particular , seems to retard rearrangement significantly , as suggested by deuterium quenching experiments . we conducted a hammett plot analysis for para - substituted compounds ( x = meo , me , f , cl , and cf3 ) in the trans series ( table 1 , entries 3 and 69 ) because these diastereomers underwent clean rearrangement relative to their cis counterparts . since the -wittig rearrangement is believed to follow a stepwise mechanism and assuming the -shift proceeds by an analogous homolytic process , both would be dependent on the extent of radical stabilization at the migrating center . therefore , we initially attempted to correlate scales vs log(kx / k0 ) ( where kx = : ratio derived from para - substituted compounds and k0 = : ratio from 1a ) ; however , severe deviations from linearity were obtained . on the other hand , good correlation of our data with and parameters was obtained ( r > 0.96 ) ( figure 1 ) . this suggests that spin delocalization of a presumed benzylic radical is not as important as the polar effects induced by the para substituents in the transition state of c o bond cleavage , which is likely the rate - determining step . a buildup of negative charge at the benzylic migrating carbon appears to favor the -migration , whereas any increasingly positive character of this position favors the -shift . the large values ( 3.10 and 4.37 ) indicate a high sensitivity to the nature of the substituent x , and a balance between resonance and inductive effects seems to play an important role in determining the : -selectivity . we cautiously interpret these observations as supporting evidence for a stepwise mechanism for both - and -pathways , in which a relatively slow c o bond homolysis is followed by rapid intramolecular recombination of the diradical anion , leading to the observed products . hammett plots of log(kx / k0 ) vs and . we were cognizant of the possibility that the -wittig alkoxide and -wittig enolate ( that is , the primary rearrangement products ) might equilibrate prior to workup , giving a false electronic effect . in fact , -cyclopropyl ketones bearing anion - stabilizing groups attached to the ring are known to undergo ring expansion to their cyclopentenol isomers under basic conditions , whereas some cyclopropyl thioenolates isomerize to the corresponding cyclopentenyl thiolates . however , independent subjection of selected products to the reaction conditions a or b ( table 1 ) did not lead to any /-interconversion ( or vice versa ) ( scheme 7 ) . in line with this observation , the diastereoselectivity in both - and -ring contractions is defined during rearrangement , as little or no change in diastereomeric ratio was observed in these control experiments . in fact , only upon warming the reaction mixture of certain -products ( alkoxides ) for prolonged periods was epimerization ever observed . thus , we have established that the observed product ratios are a true consequence of electronic effects and a secondary equilibration pathway is not operative at 78 c . we next looked to tackle the modest : -selectivity obtained in most cases , as well as the adverse -selectivity in electron - deficient substrates . a closer inspection of the -wittig product reveals two adjacent stereocenters in which the bulkier groups ( ph and sime3 ) are in a cis relationship . since the -wittig pathway proceeds via a radical / radical anion intermediate ( scheme 8) , we rationalized that increasing the steric demand of the silyl ( sir3 ) group would inhibit recombination via the -pathway , indirectly stimulating the -migration . gratifyingly , our hypothesis was right , and a gradual increase in the steric demand of the silyl group consistently led to greater : -product ratios ( table 2 ) . in the trans series , changing a sime3 group to a sime2ph group increased the selectivity from 2.4:1 to 10:1 ( entries 1 and 2 ) , and the even larger simeph2 group facilitated exclusive -wittig rearrangement in excellent yield ( entry 3 ) . we believe the improved regioselectivity is primarily dominated by the sterics of the silyl group with little electronic contribution . indeed , increasing the bulkiness of the silyl group only with alkyl groups ( siet3 , entry 4 ) also led to excellent : -selectivity . conditions : ( a ) n - buli ( 1.2 equiv ) , 10 min ; ( b ) s - buli ( 3 equiv ) , 3 h. determined by h nmr of isolated material . the corresponding cis diastereomers afforded virtually the same product ratios with excellent diastereoselectivities . however , the more sterically demanding silyl groups were deleterious for the reactivity of these isomers . given that the reactivity of trans isomers was minimally affected , the severe loss of reactivity in cis diastereomers suggests bulkier silyl groups shift the conformational equilibrium to the less reactive conformation . the relevant conformers for both trans and cis diastereomers involve half - chair arrangements and are depicted in scheme 9 . although the system in discussion involves a dihydropyran structure , we believe it is reasonable to use a values ( derived from the cyclohexane system ) to estimate conformational ratios since the overall trend should remain . accordingly , in the case of trans diastereomer 1 , conformers i and ii are expected to exist in approximately a 1:2.5 ratio , with a small predominance of conformer ii on the basis of the conformational a values for trimethylsilyl and phenyl groups in cyclohexane ( 2.5 and 2.9 , respectively ) . as we have previously suggested in the case of -silyl acyclic ethers , we believe the optimum conformation for allylic deprotonation requires an antiperiplanar arrangement of the allylic c h and benzylic c o bonds , which presumably heightens the acidity of the allylic c h bond . in addition , the resulting pseudoequatorial carbanion is expected to be more stable than the axial one , as suggested by theoretical and experimental studies . conformer ii , in which the aryl ring is orientated pseudoequatorially and the silyl group pseudoaxially , meets this requirement . although an increase in the steric demand of the silyl group is expected to shift the conformational equilibrium to the less reactive conformer i , the extent of this perturbation is expected to be modest given the larger si c bond length ( 1.89 vs 1.54 for c c bonds ) and the absence of severe steric interactions of the pseudoaxial silyl group in conformer ii . on the other hand , cis diastereomers 2 are intrinsically locked in conformation iv in which both aryl and silyl groups are oriented in pseudoequatorial positions . the required conformation for deprotonation ( conformation iii ) presents a severe steric clash between these bulky groups , which is expected to get worse as the silyl group becomes more sterically demanding . because the conformational equilibrium was also expected to be dependent on the steric demand of the aryl group , we reasoned that installing a large ortho substituent would lead to an increase in the population of conformers ii and iv ( for the trans-1 and cis-2 diastereomers , respectively ) , leading to a further increase of reactivity of the trans diastereomer ( 1 ) and a decrease in the reactivity of the cis diastereomer ( 2 ) . consistent with this hypothesis is the observation that placement of a propyl group at the ortho position of the phenyl ring did not influence the reactivity of 1s ( full conversion in < 10 min as observed with 1a ) , whereas 2s reacted more slowly than the unsubstituted 2a ( only 63% conversion in 3 h vs full conversion in the case of 2a ) ( scheme 10 ) . of interest is the observation that the effect of increasing the steric demand of the aryl group on product distribution is equivalent to what was observed by changing the size of the silyl group . in the trans series the : ratio gradually increased from 2.4:1 for the unsubstituted substrate 1a to 5.3:1 for o - methyl analogue 1e and to 11:1 for o - propyl analogue 1s . a similar increase in : -selectivity is observed in corresponding cis diasteromers 2a ( 2:1 ) , 2e ( 5.8:1 ) , and 2s ( 8.4:1 ) . this is supportive of our initial hypothesis and the determining role of sterics in the product distribution . we also evaluated the competition between opposite steric and electronic effects as per the above discussion . for this purpose , we chose substrates that were electronically biased toward the -wittig rearrangement and studied the effect of silyl groups larger than sime3 , which were expected to shift the selectivity in favor of the -pathway ( table 3 ) . trans and cis diastereomers 1p and 2p bearing an m - methoxy group at the phenyl ring and the bulky sime2ph underwent rearrangement with higher : -selectivity ( 3.9:1 and 3.5:1 , respectively , entries 2 and 3 ) relative to the sime3 counterpart 1c ( 1:1.3 , entry 1 ) . following this trend , in the trans - substituted ( p - chlorophenyl)dihydropyran series , the : -selectivity was gradually reversed from 1:2.3 with the sime3 analogue 1i ( entry 4 ) to 1:1 for the bulkier sime2ph group in 1q ( entry 5 ) and to 2.8:1 for the siet3 derivative 1r ( entry 6 ) . the trans-2-naphthyl - substituted dihydropyrans bearing a sime3 ( 1l ) and siet3 ( 1s ) afforded 1:32 and 1:5 : -selectivities ( entries 7 and 8) , respectively . analysis of the crystal structure of -wittig product 4s ( figure 2 ) bearing the 2-naphthyl and siet3 groups in a cis relationship suggests a significant steric clash between these groups exists and thus the : -selectivity is higher for 1s vs 1l . conditions : ( a ) n - buli ( 1.2 equiv ) , 10 min ; ( b ) s - buli ( 3 equiv ) , 3 h. see the experimental section for the dr of the products . the opposite diastereomer 2s afforded a 5:1 : -selectivity ( entry 9 ) . the opposite regioselectivity obtained from diastereomers 1s and 2s is a consequence of the higher temperature and prolonged reaction time for the rearrangement of 2s . one possible explanation is that under these reaction conditions the initially formed -wittig alkoxide isomerizes to the corresponding -enolate . however , we also speculate that competitive arene metalation leads to an electron - rich species that following allylic deprotonation rearranges preferentially via the -wittig pathway . although no evidence of the intermediacy of such species was gathered in this specific case , we have determined that doubly lithiated species ( heteroaryl / allylic dianion ) rearrange with remarkably different selectivies relative to the monolithiated species ( see below ) . alkyl or alkenyl substitution proximal to the silyl group veered the selectivity exclusively to the -wittig rearrangement . cyclopentenols were obtained in good yields and excellent diastereoselectivity ; however , a noticeable decrease in reactivity was observed in both trans and cis diastereomeric ethers , and longer reaction times were required for complete conversion ( table 4 ) . we attempted to shift the selectivity in favor of the -pathway by electronic modification of the aromatic ring ( introducing para electron - donating groups ) , but only the -products were obtained , and no cyclopropyl compounds were observed in these cases . conditions : ( c ) ( 1u x ) n - buli ( 1.2 equiv ) , 30 min ; ( d ) ( 2u x ) s - buli ( 3 equiv ) , 67 h. total of 13% recovered 1x . in contrast to the previous examples , alkyl olefin substitution distal to the silyl group provided low : -product selectivity , but with good overall yields ( table 5 ) . the diastereoselectivity of the -shift remained high as in previous examples , but that of the -product , bearing an all - carbon quaternary center , was modest . the lower : -selectivity with respect to that of unsubstituted analogues ( scheme 6 ) suggests the methyl substitution slightly hinders the -shift due to steric reasons . conditions ( c ) ( 1y , 1z ) n - buli ( 1.2 equiv ) , 15 min ; ( d ) ( 2y , 2z ) : s - buli ( 3 equiv ) , 6 h. the exclusive -selectivity observed in substrates having olefin substitution proximal to the silyl group ( but not when distal ) can be attributed to an unfavorable steric interaction between the olefin substituent and the silyl group . we conjecture that this leads to partial localization of the allyl anion ( prior to rearrangement ) or of the putative diradical anion intermediate in a stepwise scenario ( scheme 8) , leading to a structural distortion that completely inhibits the -wittig migration . we have found that the : -selectivity in certain heteroaryl - substituted substrates diverged markedly when proceeding through the usual allylic monoanion or through a dianionic species generated by initial deprotonation at the heteroaryl ring followed by allylic deprotonation . diastereomeric 2-thiophene - yl ( 1aa/2aa ) and 2-furyl ( 1bb/2bb ) cyclic ethers were subjected to our standard reaction conditions for cis and trans diastereomers ( scheme 11 ) . the trans isomers 1aa and 1bb underwent complete rearrangement within 10 min with significantly different : -selectivities , revealing a high electronic dependence . the trans-2-thiophene - yl - substituted compound 1aa afforded a 3:1 : -product ratio in 97% overall yield , whereas the analogous trans-2-furyl - substituted compound 1bb rearranged exclusively via the -pathway to give cyclopentenol 4bb in 81% yield . on the contrary , the corresponding cis diasteromers 2aa ( 2-thiophene - yl ) and 2bb ( 2-furyl ) underwent exclusive -wittig rearrangements to give ( cyclopropyl)acylsilanes 3aa and 3bb ( > 20:1 selectivity ) in 84% and 48% yields ( based on recovered starting material ( brsm ) ) , respectively , both with low diastereoselectivity . the unexpected exclusive -wittig selectivity in the rearrangement of both cis diastereomers 2aa and 2bb suggested that competitive deprotonation at the heteroaryl system was taking place . indeed , deuterium - trapping experiments demonstrated the intermediacy of a dianionic species formed by deprotonation at the 5-position of both thiophene - yl and furyl rings and at the allylic position . additional control experiments discarded the potential isomerization of the -alkoxide to the -enolate within the reaction time ( 3 h ) at 78 c . for instance , both -enolate and -alkoxide products were generated in 10 min from the rearrangement of trans-2-thiophene - yl - substituted isomer 1aa , and the reaction mixture was kept at 78 c for an additional 3 h. both - and -wittig products 3aa and 4aa were isolated in a combined 81% overall yield and in a 5:1 ratio . that is , the : -selectivity was not significantly modified , and /[1.2]-equilibration does not take place to a significant extent . although this product ratio is slightly higher than that observed when the reaction was stopped after 10 min , the lower yield of the reaction suggests some product decomposition took place during the extended time , thereby influencing the measured product distribution . to ascertain the origin of stereoconvergence , we first studied the rearrangement of enantiomerically enriched substrates ( )-1a and ( + ) -2a ( both in 76% ee ) ( scheme 12 ) . although the -wittig shift is known to occur with high retention of stereochemistry at the migrating carbon , in both acyclic and cyclic ethers , the stereochemical course of the competing -wittig pathway has only been studied in one acyclic instance . as expected , -wittig rearrangement of ( )-1a and ( + ) -2a proceeded with very high retention of stereochemistry at the benzylic carbon to give enantiomeric cyclopentenols ( + ) -4a and ( )-4a in 73% and 74% ee , respectively . importantly , the -wittig shift of ( )-1a and ( + ) -2a also occurred with retention of stereochemistry at the migrating center to give enantiomeric ( -cyclopropyl)acylsilanes ( )-3a and ( + ) -3a in 62% and 56% ee , respectively . in separate experiments , we attempted to trap allylic carbanions a and b generated by deprotonation of 1a and 2a , respectively ( scheme 13 ) . quenching the reaction of 1a with d2o immediately after n - buli addition ( t < 1s ) led to deuterium incorporation ( 20% ) at the allylic position without epimerization ( dr > 20:1 ) . on the other hand , attempts to trap the allylic anion b derived from 2a with d2o were unsuccessful , suggesting such species undergoes immediate rearrangement . on the basis of these results , we speculate the fleeting carbanion b is the actual species undergoing rearrangement , which implies the allylic carbanion a , generated from 1a ( which can be trapped with d2o ) , has to isomerize to species b. this is consistent with the fact that the chiral information at the silicon - bearing center ( the allylic stereocenter ) is destroyed during both - and -wittig rearrangements and the absolute configurations of both products are determined exclusively by the configurations of the migrating carbon . it is important to note that the rearrangement of species b is in accord with the normal stereochemical course at the lithium - bearing carbon in the -wittig pathway ( assuming a localized character of ( -silylallyl)lithium b ) involving inversion of stereochemistry . lastly , the observation that the stereochemical course of the -wittig rearrangement in these cyclic systems mirrors that of the competing -migration strongly suggests both pathways involve the same stepwise mechanism . in summary , we have developed a method to access ( -cyclopropyl)acylsilanes and -silylcyclopentenol structures with high diastereoselectivities and overall high efficiency via - and -wittig rearrangements of 2-silyl-6-aryldihydropyrans . the regioselective allylic deprotonations that trigger these rearrangements are made possible by the silyl appendage . trans / cis diastereomers show markedly different reactivities toward the deprotonation step , which we have rationalized on the basis of the expected acidities of equilibrating conformers . we have shown that the : -selectivity is governed by both electronic and steric characteristics of the reacting cyclic ethers . the -wittig pathway is favored by increasing the electron density at the migrating carbon and also by increasing the steric demand of the silyl group . on the other hand , electron - deficient migrating centers favor the -pathway , and olefin substitution at the olefin proximal to the silyl group leads to exclusive -migration . the role of the silyl group in determining the : -selectivity seems to be predominantly steric ; however , electronic modification at the silyl group substituents has not been explored in this study . stereochemical experiments demonstrate both - and -wittig rearrangements of dihydropyrans proceed with high retention of stereochemistry at the migrating center . deuterium - trapping experiments support the intermediacy of a common intermediate , which is responsible for the observed stereoconvergence of both isomerization pathways . taken together , our results make it reasonable to conclude that the primary mechanism of the -wittig migration in these cyclic ethers involves a stepwise process analogous to the -pathway . unless otherwise noted all reactions were run under a positive atmosphere of nitrogen in oven - dried or flame - dried round - bottom flasks or disposable drum vials capped with rubber septa . tetrahydrofuran and diethyl ether were distilled from sodium benzophenone ketyl ; dichloromethane , benzene , diisopropylamine , triethylamine , and trimethylsilyl chloride were distilled from calcium hydride . methyllithium ( 1.4 m in diethyl ether ) , n - butyllithum ( 1.6 m in hexanes ) , and sec - butyllithium ( 1.4 m in cyclohexane ) were titrated with diphenylacetic acid ( average of three runs ) . h nmr spectra were collected on 500 and 600 mhz instruments using cdcl3 as the solvent , which was referenced at 7.24 ppm ( residual chloroform proton ) , and c nmr spectra were collected in cdcl3 at 126 or 151 mhz and referenced at 77 ppm . high - resolution mass spectrometry ( hrms ) analysis was performed on tof instruments . optical rotations were measured at a wavelength of 589 nm ( sodium d line ) in chloroform . the crystal structures of 4s and 7 were deposited in the cambridge crystallographic data centre and allocated deposition numbers ccdc 1027999 and 1028000 . to a solution of the corresponding homoallylic alcohol ( 110 mmol ) in diethyl ether ( 12 ml ) was added slowly sodium hydride ( 0.15 equiv , dispersion in mineral oil , 60% ( w / w ) ) . the mixture was stirred vigorously for 5 min and then cooled in an ice bath . trichloroacetonitrile ( 1 equiv ) was then added dropwise , within 5 min approximately . the ice bath was removed after 15 min and the mixture stirred for about 1 h at room temperature and then concentrated by rotary evaporation . a solution of dry methanol ( 0.15 equiv ) in pentane ( 12 ml ) was added to precipitate salts . the filtrate was concentrated by rotary evaporation , and the crude product could be used without further purification in the next step . however , in all cases the crude product was partially purified by silica gel column chromatography ( typically 5% etoac in hexanes ) buffered with 1% triethylamine . to a solution of the corresponding homoallylic alcohol ( 16 mmol ) in dichloromethane ( 80 ml ) was added dbu ( 0.18 equiv ) . the solution was cooled at 0 c , and trichloroacetonitrile ( 1.4 equiv ) was added . the reaction was followed by tlc ( typically 5% etoac in hexanes ) using triethylamine - prewashed plates . after completion of the reaction ( typically 34 h ) , the reaction mixture was concentrated by rotary evaporation , and the residue was partially purified by silica gel column chromatography ( typically 5% etoac in hexanes ) buffered with 1% triethylamine . a solution of the corresponding allylic alcohol in thf was cooled at 78 c , and n - butyllithium ( 1.6 m in hexanes ) was added dropwise over 5 min . after 30 min after the resulting solution was stirred for 1 h , sec - butyllithium or tert - butyllithium ( see below for details ) was added dropwise over 4560 min , and then the reaction was kept at the indicated temperature . to a solution of -silyl allylic alcohol s2 ( 4 mmol , 1 equiv ) in hexane ( 22 ml ) was added the desired trichloroacetimidate s1 ( 1.51.9 equiv ) . the solution was cooled at 0 c and 0.1 equiv of ( tms)otf in hexane was added dropwise . the reaction was warmed at room temperature and monitored by tlc . typically , formation of a thick suspension indicated the end of the reaction . the solid was filtered through a plug of celite and rinsed with hexanes ( 50 ml ) . the filtrate was extracted with nahco3(satd ) ( 3 20 ml ) , h2o ( 2 20 ml ) , and brine ( 20 ml ) and dried over mgso4 . after filtration and concentration , the residue was purified by column chromatography . to a solution of o - trimethylsilyl -(trimethylsilyl)allylic alcohol ( 10 mmol ) in dichloromethane ( 50 ml ) were added allyltrimethylsilane ( 1.1 equiv ) and the desired benzaldehyde derivative ( 1.1 equiv ) . the solution was cooled at 78 c , and ( tms)otf ( 0.2 equiv ) was added dropwise . the reaction was followed by tlc and usually required between 1 and 4 h. the reaction was quenched by adding nahco3(satd ) ( 20 ml ) . combined organic extracts were washed with nahco3(satd ) ( 2 20 ml ) , h2o ( 20 ml ) , and brine ( 20 ml ) and dried over mgso4 . to a solution of bisallylic ether s3 ( 0.96 mmol ) in dichloromethane ( 10 ml ) was added second - generation grubbs catalyst , and the mixture was stirred at room temperature under nitrogen . after 3 h the solution was concentrated by rotary evaporation and the residue purified by column chromatography . a round - bottom flask was charged with bisallylic ether s3 ( 0.96 mmol ) , which was dissolved in benzene ( 0.050.07 m ) . second - generation grubbs catalyst was added , and a condenser was attached to the flask . the system was flushed with nitrogen and then heated in an oil bath at 80 c for 1 h. the reaction mixture was then cooled at room temperature and concentrated and the product purified by column chromatography . important note : all 2-silyl-6-aryl-5,6-dihydropyrans 1 and 2 are air sensitive and upon isolation undergo autoxidation ( observable by h nmr within 1 h after isolation ) , which is minimized when the compound is diluted ( < 0.05 m ) . for this reason , freshly purified dihydropyrans 1 and 2 were immediately submitted to the wittig rearrangement conditions ( general procedure h ) . freshly prepared and purified 2-silyl-6-aryldihydropyran 1 or 2 was dissolved in thf under nitrogen ( concentration 0.08 m , unless otherwise noted ) and the solution cooled at 78 c ( dry ice / acetone bath ) , n - butyllithium ( 1.2 equiv , 1.6 m in hexanes , conditions a and c ) or sec - butyllithium ( 3.0 equiv , 1.4 m in cyclohexane , conditions b and d ) was added dropwise ( 1 drop / s ) to give a colored solution . after the indicated time ( 1030 min for trans diastereomers 1 or 37 h for cis diastereomers 2 ) , the reaction was quenched by adding nh4cl(satd ) and diluted with h2o and diethyl ether . the solution was dried over magnesium sulfate , filtered , quickly concentrated in a rotovap at room temperature ( no effort was made to remove all thf to minimize the time the crude reaction was concentrated ) , and immediately loaded into a buffered column ( packed with 1% triethylamine ) . elution with 5% and 10% etoac in hexanes afforded the acylsilane and cyclopentenol products , respectively . applying general procedure a to 1-(2-methoxyphenyl)but-3-en-1-ol ( 13 g , 73.4 mmol , 1 equiv ) , sodium hydride ( 0.44 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 10.6 , 73.4 mmol , 1 equiv ) , and diethyl ether ( 24 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 18.4 g ( 78% ) of compound s1-b as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.25 ( s , 1 h ) , 7.42 ( dd , j = 1.2 , 7.8 hz , 1 h ) , 7.24 ( m , 1 h ) , 6.94 ( t , j = 7.8 hz , 1 h ) , 6.87 ( d , j = 7.8 hz , 1 h ) , 6.28 ( t , j = 6.6 hz , 1 h ) , 5.84 ( m , 1 h ) , 5.08 ( dd , j = 1.8 , 16.8 hz , 1 h ) , 5.03 ( d , j = 10.2 hz , 1 h ) ; c nmr ( 151 mhz , cdcl3 ) 161.5 , 155.9 , 133.6 , 128.7 , 128.4 , 125.9 , 120.6 , 117.6 , 110.4 , 91.8 , 75.0 , 55.5 , 39.6 ; ir ( film ) 3344 , 3070 , 2955 , 1664 , 1300 , 1076 , 794 cm . applying general procedure a to 1-(3-methoxyphenyl)but-3-en-1-ol ( 13 g , 72.9 mmol , 1 equiv ) , sodium hydride ( 0.29 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 10.5 g , 72.9 mmol , 1 equiv ) , and diethyl ether ( 24 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 19.88 g ( 85% ) of compound s1-c as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.30 ( s , 1 h ) , 7.27 ( t , j = 8.0 hz , 1 h ) , 6.97 ( m , 2 h ) , 6.83 ( dd , j = 2.5 , 8.0 hz , 1 h ) , 5.86 ( m , 1 h ) , 5.81 ( m , 1 h ) , 5.13 ( m , 1 h ) , 5.08 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.78 ( m , 1 h ) , 2.64 ( m , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.4 , 159.6 , 141.3 , 133.0 , 129.4 , 118.4 , 118.1 , 113.3 , 111.6 , 91.7 , 79.9 , 55.1 , 41.0 ; ir ( film ) 3341 , 3070 , 2936 , 1664 , 1290 , 1078 , 796 cm . applying general procedure a to 1-(4-methoxyphenyl)but-3-en-1-ol ( 10.7 g , 60 mmol , 1 equiv ) , sodium hydride ( 0.36 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 8.7 g , 60 mmol , 1 equiv ) , and diethyl ether ( 20 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 12.6 g ( 65% ) of s1-d as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.26 ( s , 1 h ) , 7.33 ( m , 2 h ) , 6.87 ( m , 2 h ) , 5.83 ( m , 1 h ) , 5.77 ( m , 1 h ) , 5.11 ( m , 1 h ) , 5.06 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.79 ( m , 1 h ) , 2.61 ( m , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.5 , 159.3 , 133.2 , 131.6 , 127.7 ( 2 c ) , 118.1 , 113.7 ( 2 c ) , 91.8 , 79.9 , 55.2 , 40.9 ; ir ( film ) 3340 , 3065 , 2930 , 1664 , 1295 , 1076 , 796 cm . applying general procedure a to 1-(2-methylphenyl)but-3-en-1-ol ( 4.5 g , 27.74 mmol , 1 equiv ) , sodium hydride ( 0.166 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 4 g , 27.74 mmol , 1 equiv ) , and diethyl ether ( 10 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 7.29 g ( 61% ) of compound s1-e as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.28 ( s , 1 h ) , 7.25 ( t , j = 8.0 hz , 1 h ) , 7.21 ( m , 2 h ) , 7.12 ( d , j = 8.0 hz , 1 h ) , 5.86 ( dd , j = 5.0 , 7.5 hz , 1 h ) , 5.83 ( m , 1 h ) , 5.13 ( dq , j = 1.5 , 17.0 hz , 1 h ) , 5.09 ( m , 1 h ) , 2.78 ( m , 1 h ) , 2.62 ( m , 1 h ) , 2.36 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.5 , 139.6 , 137.9 , 133.2 , 128.7 , 128.3 , 126.8 , 123.1 , 118.1 , 91.7 , 80.2 , 41.1 , 21.5 ; ir ( film ) 3418 , 1653 , 1305 , 1085 cm . applying general procedure a to 1-(3-methylphenyl)but-3-en-1-ol ( 4.22 g , 26 mmol , 1 equiv ) , sodium hydride ( 0.156 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 3.75 g , 26 mmol , 1 equiv ) , and diethyl ether ( 15 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 6.37 g ( 80% ) of compound s1-f as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.22 ( s , 1 h ) , 7.44 ( m , 1 h ) , 7.207.13 ( m , 3 h ) , 6.04 ( dd , j = 5.0 , 8.0 hz , 1 h ) , 5.82 ( ddt , j = 7.0 , 10.5 , 17.5 hz , 1 h ) , 5.12 ( dq , j = 1.5 , 17.0 hz , 1 h ) , 5.07 ( m , 1 h ) , 2.74 ( m , 1 h ) , 2.57 ( m , 1 h ) , 2.42 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.4 , 138.2 , 135.0 , 133.3 , 130.2 , 127.8 , 126.2 , 125.5 , 118.0 , 91.7 , 77.1 , 40.3 , 19.2 ; ir ( film ) 3344 , 3078 , 2980 , 1662 , 1311 , 1078 , 796 cm . applying general procedure a to 1-(4-methylphenyl)but-3-en-1-ol ( 6.5 g , 40.07 mmol , 1 equiv ) , sodium hydride ( 0.24 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 5.79 g , 40.1 mmol , 1 equiv ) , and diethyl ether ( 14 ml ) afforded after column chromatography ( 4% etoac in hexanes , column buffered with et3n ) 11.08 g ( 90% ) of compound s1-g as a semisolid : h nmr ( 500 mhz , cdcl3 ) 8.26 ( s , 1 h ) , 7.29 ( d , j = 8.0 hz , 2 h ) , 7.16 ( d , j = 8.0 hz , 2 h ) , 5.84 ( dd , j = 5.0 , 7.5 hz , 1 h ) , 5.80 ( ddt , j = 7.0 , 10.5 , 17.5 hz , 1 h ) , 5.11 ( dq , j = 1.5 , 17.5 hz , 1 h ) , 5.07 ( m , 1 h ) , 2.77 ( m , 1 h ) , 2.61 ( m , 1 h ) , 2.33 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.5 , 137.7 , 136.6 , 133.2 , 129.1 ( 2 c ) , 126.2 ( 2 c ) , 118.1 , 91.7 , 80.1 , 41.0 , 21.2 ; ir ( film ) 3335 , 3060 , 1662 , 1310 , 1060 cm . applying general procedure b to 1-(4-fluorophenyl)-3-en-1-ol ( 4.07 g , 24.49 mmol , 1 equiv ) , trichloroacetonitrile ( 5.3 g , 36.74 mmol , 1 equiv ) , and dbu ( 810 mg , 5.31 mmol , 0.18 equiv ) in ch2cl2 ( 150 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 6.25 g ( 82% ) of compound s1-h as a yellow oil : h nmr ( 600 mhz , cdcl3 ) 8.28 ( s , 1 h ) , 7.36 ( m , 2 h ) , 7.02 ( m , 2 h ) , 5.84 ( dd , j = 5.4 , 7.8 hz , 1 h ) , 5.76 ( ddt , j = 7.2 , 10.2 , 17.4 , 1 h ) , 5.115.06 ( m , 2 h ) , 2.76 ( m , 1 h ) , 2.60 ( m , 1 h ) ; c nmr ( 151 mhz , cdcl3 ) 162.4 ( j = 246.4 hz ) , 161.4 , 135.3 ( d , j = 3.2 hz ) , 132.7 , 128.1 ( d , j = 8.5 hz , 2 c ) , 118.4 ( d , j = 3.2 hz ) , 115.3 ( d , j = 21.1 hz , 2 c ) , 91.6 , 79.4 ( d , j = 1.7 hz ) , 40.9 ; ir ( film ) 3343 , 3083 , 2982 , 1664 , 1512 , 1230 , 1076 , 796 cm . applying general procedure a to 1-(4-chlorophenyl)but-3-en-1-ol ( 11 g , 60.22 mmol , 1 equiv ) , sodium hydride ( 0.36 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 8.7 g , 60.22 mmol , 1 equiv ) , and diethyl ether ( 21 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 14.97 g ( 76% ) of compound s1-i as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.28 ( s , 1 h ) , 7.32 ( s , 4 h ) , 5.83 ( dd , j = 5.5 , 7.5 hz , 1 h ) , 5.77 ( m , 1 h ) , 5.115.06 ( m , 2 h ) , 2.75 ( m , 1 h ) , 2.60 ( m , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.4 , 138.1 , 133.8 , 132.6 , 128.6 ( 2 c ) , 127.7 ( 2 c ) , 118.6 , 91.5 , 79.4 , 40.8 ; ir ( film ) 3343 , 3081 , 2928 , 1664 , 1294 , 1078 , 796 cm . applying general procedure b to 1-(naphthalen-2-yl)-3-en-1-ol ( 4.63 g , 23.3 mmol , 1 equiv ) , trichloroacetonitrile ( 5.05 g , 34.95 mmol , 1 equiv ) , and dbu ( 640 mg , 4.19 mmol , 0.18 equiv ) in ch2cl2 ( 350 ml ) afforded after column chromatography ( 8% etoac in hexanes , column buffered with et3n ) 7.62 g ( 95% ) of compound s1-l as a cream - colored solid : mp 4243 c ; h nmr ( 600 mhz , cdcl3 ) 8.30 ( s , 1 h ) , 7.83 ( m , 4 h ) , 7.53 ( dd , j = 1.8 , 9.0 hz , 1 h ) , 7.47 ( m , 2 h ) , 6.05 ( m , 1 h ) , 5.83 ( m , 1 h ) , 5.13 ( m , 1 h ) , 5.08 ( m , 1 h ) , 2.88 ( m , 1 h ) , 2.71 ( m , 1 h ) ; c nmr ( 151 mhz , cdcl3 ) 161.5 , 137.0 , 133.1 , 133.06 , 133.01 , 128.3 , 128.1 , 127.7 , 126.2 , 126.1 , 125.5 , 124.0 , 118.3 , 91.7 , 80.3 , 40.9 ; ir ( film ) 3341 , 3059 , 1664 , 1304 , 1076 , 794 cm . applying general procedure b to 1-(2-propylphenyl)-3-en-1-ol ( 1.5 g , 7.88 mmol , 1 equiv ) , trichloroacetonitrile ( 1.7 g , 11.82 mmol , 1 equiv ) , and dbu ( 240 mg , 1.58 mmol , 0.2 equiv ) in ch2cl2 ( 40 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 2.15 g ( 81% ) of compound s1-t as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.23 ( s , 1 h ) , 7.47 ( m , 1 h ) , 7.21 ( m , 2 h ) , 7.16 ( m , 1 h ) , 6.10 ( dd , j = 4.5 , 9.0 hz , 1 h ) , 5.87 ( ddt , j = 7.5 , 10.5 , 17.5 hz , 1 h ) , 5.14 ( dq , j = 1.5 , 17.0 hz , 1 h ) , 5.08 ( m , 1 h ) , 2.75 ( m , 2 h ) , 2.67 ( m , 1 h ) , 2.54 ( m , 1 h ) , 1.771.62 ( m , 2 h ) , 1.00 ( t , j = 7.5 hz , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.4 , 139.5 , 137.8 , 133.6 , 129.3 , 127.8 , 126.1 , 125.7 , 117.9 , 91.7 , 76.9 , 41.1 , 34.6 , 24.0 , 14.2 ; ir ( film ) 3346 , 3078 , 2961 , 1664 , 1309 , 1076 , 794 cm . applying general procedure a to 3-methyl-1-(p - tolyl)but-3-en-1-ol ( 4 g , 22.64 mmol , 1 equiv ) , sodium hydride ( 0.136 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 3.27 g , 42 mmol , 1 equiv ) , and diethyl ether 8.5 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 3.1 g ( 43% ) of compound s1-z as a white solid : mp 5960 c ; h nmr ( 500 mhz , cdcl3 ) 8.23 ( s , 1 h ) , 7.30 ( d , j = 8.0 hz , 2 h ) , 7.15 ( d , j = 8.0 hz , 2 h ) , 5.95 ( dd , j = 5.0 , 9.0 hz , 1 h ) , 4.81 ( m , 1 h ) , 4.76 ( m , 1 h ) , 2.77 ( dd , a of abx system , j = 9.0 , 14.5 hz , 1 h ) , 2.48 ( dd , b of abx system , j = 5.0 , 14.5 hz , 2 h ) , 2.33 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.6 , 140.9 , 137.7 , 137.1 , 129.1 ( 2 c ) , 126.2 ( 2 c ) , 113.7 , 91.7 , 79.2 , 45.1 , 22.8 , 21.2 ; ir ( film ) 3343 , 3070 , 2924 , 1660 , 1304 , 1080 , 794 cm . applying general procedure b to 1-(thiophene-2-yl)but-3-en-1-ol ( 2.5 g , 16.21 mmol , 1 equiv ) , trichloroacetonitrile ( 3.51 g , 24.31 mmol , 1 equiv ) , and dbu ( 0.44 g , 2.92 mmol , 0.18 equiv ) in ch2cl2 ( 100 ml ) afforded after column chromatography ( 7% etoac in hexanes , column buffered with et3n ) 4.1 g ( 95% ) of compound s1-aa as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.37 ( s , 1 h ) , 7.26 ( dd , j = 1.5 , 5.5 hz , 1 h ) , 7.10 ( m , 1 h ) , 6.96 ( dd , j = 3.5 , 5.0 hz , 1 h ) , 6.20 ( dd , j = 6.0 , 7.5 hz , 1 h ) , 5.81 ( ddt , j = 6.5 , 10.0 , 17.0 hz , 1 h ) , 5.16 ( dq , j = 1.5 , 17.5 hz , 1 h ) , 5.10 ( m , 1 h ) , 2.88 ( m , 1 h ) , 2.75 ( m , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.5 , 141.9 , 132.6 , 126.4 , 126.0 , 125.4 , 118.6 , 91.5 , 75.6 , 40.7 ; ir ( film ) 3341 , 3078 , 2943 , 1662 , 1290 , 1072 , 794 cm . applying general procedure b to 1-(furan-2-yl)but-3-en-1-ol ( 2.53 g , 18.31 mmol , 1 equiv ) , trichloroacetonitrile ( 3.97 g , 27.47 mmol , 1 equiv ) , and dbu ( 0.5 g , 3.3 mmol , 0.18 equiv ) in ch2cl2 ( 170 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 2.17 g ( 42% ) of compound s1-bb as a yellow oil : h nmr ( 500 mhz , cdcl3 ) 8.37 ( s , 1 h ) , 7.39 ( dd , j = 1.0 , 2.0 hz , 1 h ) , 6.39 ( d , j = 3.0 hz , 1 h ) , 6.33 ( dd , j = 2.0 , 3.0 hz , 1 h ) , 6.00 ( t , j = 6.5 hz , 1 h ) , 5.77 ( ddt , j = 7.0 , 10.5 , 17.0 hz , 1 h ) , 5.15 ( dq , j = 1.5 , 17.5 hz , 1 h ) , 5.08 ( m , 1 h ) , 2.902.78 ( m , 2 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.7 , 151.5 , 142.6 , 132.5 , 118.4 , 110.2 , 108.9 , 91.5 , 73.0 , 36.9 ; ir ( film ) 3343 , 3080 , 2926 , 1662 , 1300 , 1076 , 796 cm . applying general procedure a to 1-(4-bromophenyl)but-3-en-1-ol ( 9.54 g , 42 mmol , 1 equiv ) , sodium hydride ( 0.25 g , 60% ( w / w ) oil dispersion , 0.15 equiv ) , trichloroacetonitrile ( 6.06 g , 42 mmol , 1 equiv ) , and diethyl ether ( 14 ml ) afforded after column chromatography ( 5% etoac in hexanes , column buffered with et3n ) 13.4 g ( 86% ) of s1-cc as a yellow solid : mp 3738 c ; h nmr ( 500 mhz , cdcl3 ) 8.31 ( s , 1 h ) , 7.48 ( m , 2 h ) , 7.27 ( m , 2 h ) , 5.83 ( dd , j = 5.5 , 7.5 hz , 1 h ) , 5.77 ( ddt , j = 6.5 , 10.0 , 17.0 hz , 1 h ) , 5.135.07 ( m , 2 h ) , 2.76 ( m , 1 h ) , 2.61 ( m , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.3 , 138.6 , 132.5 , 131.5 ( 2 c ) , 128.0 ( 2 c ) , 121.9 , 118.6 , 91.5 , 79.3 , 40.7 ; ir ( film ) 3343 , 3081 , 2934 , 1664 , 1294 , 1072 , 794 cm . a solution of allyl alcohol ( 2.73 g , 47.06 mmol ) in thf ( 117 ml ) was cooled to 78 c . n - buli ( 1.61 m in hexanes , 31.57 ml , 50.83 mmol ) was added dropwise and the mixture stirred for 1 h. then freshly distilled ( tms)cl ( 5.97 ml , 47.06 mmol ) was added slowly from a syringe . after 1.5 h t - buli ( 1.66 m in pentane , 34.02 ml , 63.98 mmol ) was added dropwise and the reaction stirred for an additional 1.5 h. the reaction was quenched by the addition of aqueous nh4cl and diluted with et2o , and the mixture was warmed to room temperature . after the layers were separated , the aqueous phase was washed with et2o ( 3 50 ml ) . then all the organic phases were combined , washed with h2o ( 4 25 ml ) and brine ( 3 17 ml ) , and dried over na2so4 overnight . filtration and concentration furnished the crude product s2-a ( 65% ) as a yellow thf solution ( 90.1% pure ) , which was used in the next step without further purification : h nmr ( 500 mhz , cdcl3 ) 6.73 ( ddd , j = 5.5 , 11.0 , 17.0 hz , 1 h ) , 5.77 ( dt , j = 2.0 , 17.0 hz , 1 h ) , 5.69 ( dt , j = 2.0 , 11.0 hz , 1 h ) , 4.72 ( m , 1 h ) 0.76 ( s , 9 h ) ; c nmr ( 62.8 mhz , cdcl3 ) 140.1 , 109.6 , 69.3 , 4.05 ; ir ( neat ) 3430 , 2959 , 1634 , 1250 cm . s2-a is a known compound and has spectral data in accord with the reported data . applying general procedure c to allyl alcohol ( 3 g , 51.65 mmol , 1 equiv ) in thf ( 130 ml ) at 78 c , n - buli ( 1.6 m in hexanes , 35 ml , 55.78 mmol , 1.08 equiv ) , phenyldimethylsilyl chloride ( 9.52 g , 55.78 mmol , 1.08 equiv ) , and t - buli ( 1.7 m in pentane , 36.5 ml , 62 mmol , 1.2 equiv ) afforded 7.15 g ( 72% ) of s2-b as a colorless oil after column chromatography : h nmr ( 500 mhz , cdcl3 ) 7.55 ( m , 2 h ) , 7.36 ( m , 3 h ) , 5.98 ( ddd , j = 5.5 , 11.0 , 17.5 hz , 1 h ) , 5.06 ( dt , j = 1.5 , 17.0 hz , 1 h ) , 4.98 ( dt , j = 1.5 , 11.0 hz , 1 h ) , 4.20 ( m , 1 h ) , 0.33 ( s , 3 h ) , 0.32 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 139.3 , 136.0 , 134.2 ( 2 c ) , 129.5 , 127.9 ( 2 c ) , 110.1 , 68.5 , 5.8 , 6.1 ; ir ( film ) 3426 , 3071 , 2959 , 1427 , 1250 , 1115 , 835 cm . s2-b is a known compound and has spectral data in accord with the reported data . applying general procedure c to allyl alcohol ( 2 g , 34.48 mmol , 1 equiv ) in thf ( 85 ml ) , n - buli ( 22 ml , 34.48 mmol , 1.0 equiv ) , methyldiphenylsilyl chloride ( 8.03 g , 34.48 mmol , 1.0 equiv ) , and t - buli ( 24 ml , 41.4 mmol , 1.2 equiv ) afforded 5.77 g ( 66% ) of s2-c as colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.62 ( m , 4 h ) , 7.437.35 ( m , 6 h ) , 6.04 ( ddd , j = 5.5 , 11.0 , 17.5 hz , 1 h ) , 5.12 ( dt , j = 2.0 , 17.0 hz , 1 h ) , 5.02 ( dt , j = 2.0 , 11.0 hz , 1 h ) , 4.59 ( m , 1 h ) , 1.43 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.0 , 135.1 ( 2 c ) , 135.0 ( 2 c ) , 134.4 , 134.1 , 129.7 , 129.68 , 127.93 ( 2 c ) , 129.1 ( 2 c ) , 110.8 , 67.6 , 7.1 ; ir ( film ) 3431 , 3071 , 3041 , 3964 , 1427 , 1115 , 904 , 790 cm . applying general procedure c to allyl alcohol ( 2 g , 34.5 mmol , 1 equiv ) in thf ( 70 ml ) , n - buli ( 23.7 ml , 37.9 mmol , 1.1 equiv ) , triethylsilyl chloride ( 5.7 g , 37.9 mmol , 1.1 equiv ) , and s - buli ( 30 ml , 41.4 mmol , 1.2 equiv ) afforded 5.75 g ( 97% ) of s2-d as colorless oil : h nmr ( 500 mhz , cdcl3 ) 6.05 ( ddd , j = 5.0 , 10.5 , 16.0 hz , 1 h ) , 5.07 ( dd , j = 1.5 , 17.0 hz , 1 h ) , 4.96 ( dd , j = 1.5 , 10.5 hz , 1 h ) , 4.16 ( m , 1 h ) , 0.97 ( t , j = 8.0 hz , 9 h ) , 0.60 ( q , j = 8.0 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.4 , 109.0 , 67.4 , 7.4 , 1.6 ; ir ( film ) 3402 , 2955 , 1458 , 1097 cm . s2-d is a known compound and has spectral data in accord with the reported data . compound s2-e was prepared by a slight modification of general procedure c according to the literature . to a solution of methallyl alcohol ( 4 g , 55.5 mmol , 1 equiv ) in thf ( 100 ml ) at 78 c was added dropwise n - buli ( 38 ml , 61 mmol , 1.1 equiv ) . after 1 h trimethylsilyl chloride ( 7 ml , 55.5 mmol , 1 equiv ) was added and the mixture stirred for an additional 1 h at 78 c . then t - buli ( 42 ml , 72 mmol , 1.3 equiv ) was added dropwise over a period of 45 min and the temperature raised to 35 c for 6 h. the reaction was quenched by adding a solution of acetic acid ( 4.1 ml , 72 mmol , 1.3 equiv ) in thf ( 10 ml ) and removal of the cold bath . after 15 min the reaction was diluted with nahco3(satd ) and pentane ( 300 ml ) . the organic phase was washed with h2o ( 3 50 ml ) and brine ( 50 ml ) and dried over na2so4 . after filtration the residue was carefully concentrated to give 79% s2-e as a thf solution ( ca . 76% , w / w ) : h nmr ( 600 mhz , cdcl3 ) 4.77 ( m , 1 h ) , 4.74 ( m , 1 h ) , 3.85 ( s , 1 h ) , 1.68 ( m , 3 h ) , 1.37 ( s , 1 h ) , 0.05 ( s , 9 h ) . compound s2-f was prepared by a slight modification of general procedure c prior to workup . following application of general procedure c to allyl alcohol ( 1 g , 17.24 mmol , 1 equiv ) in thf ( 45 ml ) at 78 c , n - buli ( 1.6 m in hexanes , 11.6 ml , 18.62 mmol , 1.08 equiv ) , trimethylsilyl chloride ( 2.2 ml , 17.24 mmol , 1 equiv ) , and t - buli ( 1.7 m in pentane , 12.2 ml , 20.69 mmol , 1.2 equiv ) ( added dropwise over 50 min and stirred at 78 c for 2.5 h ) , the reaction was slowly treated ( 5 min ) with trimethylsilyl chloride ( 5.47 ml , 43.1 mmol , 2.5 equiv ) and the mixture stirred at 78 c for 1 h and at room temperature for 1 h. the reaction was cooled at 78 c and quenched with nahco3(satd ) ( 50 ml ) and the mixture diluted immediately with et2o ( 100 ml ) . filtration and evaporation of solvent gave the crude product ( 80% ) , which was almost pure and was used in the next step without further purification . analytically pure ( colorless oil ) s2-f was obtained by column chromatography on buffered silica gel : h nmr ( 300 mhz , cdcl3 ) ( 5.87 ( ddd , j = 5.0 , 10.5 , 17.0 hz , 1 h ) , 5.01 ( dd , j = 2.0 , 17.0 hz , 1 h ) , 4.88 ( dd , j = 2.0 , 10.5 hz , 1 h ) , 3.93 ( dd j = 2.0 , 5.0 hz , 1 h ) , 0.067 ( s , 9 h ) , 0.027 ( s , 9 h ) ; c nmr ( 62.8 mhz , cdcl3 ) 139.4 , 109.5 , 69.2 , 0.08 , 4.1 ; ir ( neat ) 2959 , 2901 , 2818 , 1250 , 1020 , 841 cm . compound s2-g was prepared by a slight modification of general procedure c prior to workup . following general procedure c , to 2-butyn-1-ol ( 3 g , 42.8 mmol , 1 equiv ) in thf ( 150 ml ) at 78 c was slowly added n - buli ( 1.6 m in hexanes , 31 ml , 46.2 mmol , 1.08 equiv ) . after 30 min trimethylsilyl chloride ( 5 g , 46.2 mmol , 1.08 equiv ) was added and the mixture stirred at the same temperature for 1 h. then t - buli ( 1.7 m in pentane , 31 ml , 51.3 mmol , 1.2 equiv ) was added dropwise over 1 h , and the yellow mixture was stirred at 78 c for 3 h. trimethylsilyl chloride ( 6.93 g , 64.2 mmol , 1.5 equiv ) was added slowly ( 5 min ) and the mixture stirred at 78 c for 1 h and at room temperature for 1 h. the reaction was cooled at 78 c and quenched with nahco3(satd ) ( 50 ml ) and the mixture immediately diluted with et2o ( 100 ml ) . column chromatography ( 2% etoac in hexanes ) afforded 6.4 g ( 70% ) of s2-g as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 3.94 ( q , j = 2.5 hz , 1 h ) , 1.83 ( d , j = 2.5 hz , 3 h ) , 0.11 ( s , 9 h ) , 0.05 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 82.4 , 79.4 , 56.5 , 3.9 , 0.0 , 4.2 . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 2.78 g , 54.7% ( w / w ) in thf , 1 mmol ) , trichloroacetimidate s1-b ( 6.7 g , 20.73 mmol , 1.8 equiv ) , and ( tms)otf ( trace ) in cyclohexane ( 64 ml ) afforded after column chromatography ( 10% ch2cl2 in hexanes ) 2.58 g ( 77% ) of syn / anti - s3-b ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : anti - s3-b = 1:1 ) ; h nmr ( 500 mhz , cdcl3 ) 7.45 ( dd , j = 1.5 , 8.0 hz , 1 h ) , 7.34 ( dd , j = 1.5 , 7.5 hz , 1 h ) , 7.18 ( m , 2 h ) , 6.94 ( t , j = 7.5 hz , 1 h ) , 6.92 ( t , j = 7.5 hz , 1 h ) , 6.82 ( dd , j = 1.0 , 8.5 hz , 1 h ) , 6.79 ( dd , j = 1.0 , 8.0 hz , 1 h ) , 5.87 ( ddt , j = 7.0 , 10.5 , 17.5 hz , 1 h ) , 5.77 ( m , 2 h ) , 5.65 ( ddd , j = 7.0 , 10.5 , 17.5 hz , 1 h ) , 5.014.87 ( m , 8 h ) , 4.80 ( m , 2 h ) , 3.79 ( dt , j = 1.5 , 7.5 hz , 1 h ) , 3.78 ( s , 3 h ) , 3.76 ( s , 3 h ) , 3.44 ( dt , j = 1.0 , 7.5 hz , 1 h ) , 2.472.35 ( m , 4 h ) , 0.04 ( s , 9 h ) , 0.01 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 157.4 , 155.7 , 138.02 , 137.98 , 135.9 , 135.4 , 132.3 , 131.0 , 127.8 , 127.5 , 127.4 , 127.3 , 120.4 , 120.2 , 116.2 , 115.9 , 112.7 , 111.7 , 110.3 , 109.9 , 75.6 , 74.4 , 73.2 , 72.5 , 55.4 , 55.3 , 41.9 , 39.9 , 3.7 , 3.9 ; ir ( film ) 3076 , 2957 , 2835 , 1489 , 1244 , 841 cm ; hrms ( ei ) m / z 290.1700 [ ( m ) , calcd for c17h26o2si , 290.1702 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.83 g , 54.7% ( w / w ) in thf , 7.68 mmol ) , trichloroacetimidate s1-c ( 4.46 g , 13.8 mmol , 1.8 equiv ) , and ( tms)otf ( 97 l , 0.538 mmol , 0.07 equiv ) in cyclohexane ( 43 ml ) afforded after column chromatography ( 15% ch2cl2 in hexanes ) 1.34 g ( 60% ) of syn / anti - s3-c ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : anti - s3-c = 1:0.4 ) ; h nmr ( 500 mhz , cdcl3 ) 7.21 ( t , j = 8.0 hz , 0.4 h ) , 7.19 ( t , j = 8.0 hz , 1 h ) , 6.88 ( m , 1 h ) , 6.85 ( m , 1 h ) , 6.80 ( m , 1.2 h ) , 6.75 ( ddd , j = 1.0 , 2.5 , 8.0 hz , 1 h ) , 5.80 ( m , 0.4 h ) , 5.71 ( m , 1.4 h ) , 5.67 ( ddd , j = 2.0 , 10.5 , 17.0 hz , 1 h ) , 5.034.95 ( m , 3.6 h ) , 4.92 ( dt , j = 1.5 , 17.0 hz , 1 h ) , 4.83 ( dt , j = 1.5 , 11.0 hz , 1 h ) , 4.40 ( dd , j = 6.0 , 8.0 hz , 0.4 h ) , 4.34 ( t , j = 6.0 hz , 1 h ) , 3.79 ( s , 1.2 h ) , 3.78 ( s , 3 h ) , 3.77 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 3.44 ( dt , j = 1.5 , 7.5 hz , 0.4 h ) , 2.48 ( m , 1.4 h ) , 2.41 ( m , 1 h ) , 2.32 ( m , 0.4 h ) , 0.04 ( s , 9 h ) , 0.02 ( s , 3.6 h ) ; c nmr ( 126 mhz , cdcl3 ) 159.6 , 159.3 , 145.3 , 144.3 , 137.8 , 137.5 , 135.4 , 134.8 , 129.0 , 128.8 , 119.8 , 119.0 , 116.8 , 116.4 , 113.0 , 112.9 , 112.3 ( 2 c ) , 112.1 , 111.9 , 80.8 , 79.1 , 75.7 , 72.9 , 55.14 , 55.12 , 43.0 , 41.6 , 3.7 , 3.9 ; ir ( film ) 3076 , 2957 , 1248 , 1047 , 841 cm ; hrms ( ei ) m / z 290.1685 [ ( m ) , calcd for c17h26o2si , 290.1702 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 2 g , 15.35 mmol ) , trichloroacetimidate s1-d ( 9.9 g , 30.7 mmol , 2 equiv ) , and ( tms)otf ( 194 l , 1.07 mmol , 0.07 equiv ) in cyclohexane ( 85 ml ) afforded after column chromatography ( 15% ch2cl2 in hexanes ) 4.35 g ( 60% ) of syn / anti - s3-d ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-d : h nmr ( 500 mhz , cdcl3 ) 7.20 ( d , j = 8.5 hz , 2 h ) , 6.82 ( d , j = 8.5 hz , 2 h ) , 5.745.62 ( m , 2 h ) , 4.96 ( m , 2 h ) , 4.92 ( dt , j = 2.0 , 17.0 hz , 1 h ) , 4.82 ( dt , j = 1.5 , 10.0 hz , 1 h ) , 4.30 ( t , j = 6.0 hz , 1 h ) , 3.78 ( s , 3 h ) , 3.76 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.49 ( m , 1 h ) , 2.39 ( m , 1 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 158.5 , 138.0 , 135.7 , 135.0 , 127.7 ( 2 c ) , 116.7 , 113 ( 2 c ) , 111.6 , 80.7 , 75.5 , 55.2 , 41.5 , 3.7 ; ir ( film ) 3076 , 2957 , 1514 , 1248 , 1039 , 841 cm ; hrms ( ei ) m / z 290.1688 [ ( m ) , calcd for c17h26o2si , 290.1702 ] . spectroscopic data for anti - s3-d : h nmr ( 500 mhz , cdcl3 ) 7.14 ( d , j = 8.5 hz , 2 h ) , 6.84 ( d , j = 8.0 hz , 2 h ) , 5.825.68 ( m , 2 h ) , 4.97 ( m , 4 h ) , 4.36 ( t , j = 7.0 hz , 1 h ) , 3.79 ( s , 3 h ) , 3.39 ( dt , j = 1.0 , 7.0 hz , 1 h ) , 2.51 ( m , 1 h ) , 2.31 ( m , 1 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 158.9 , 137.7 , 135.6 , 134.5 , 128.5 , 116.2 , 113.5 , 112.7 , 78.7 , 72.5 , 55.2 , 43.0 , 4.0 ; ir ( film ) 3076 , 2957 , 1514 , 1258 , 1039 , 841 cm ; hrms ( ei ) m / z 290.1695 [ ( m ) , calcd for c17h26o2si , 290.1702 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.17 g , 85.5% ( w / w ) in et2o , 8.98 mmol ) , trichloroacetimidate s1-e ( 3.85 g , 12.57 mmol , 1.4 equiv ) , and ( tms)otf ( 40 l , 0.225 mmol , 0.025 equiv ) in cyclohexane ( 45 ml ) afforded after column chromatography ( hexanes ) 1.81 g ( 73% ) of syn / anti - s3-e ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : antis3-e = 1.0:0.5 ) ; h nmr ( 500 mhz , cdcl3 ) 7.45 ( dd , j = 1.0 , 7.5 hz , 1 h ) , 7.38 ( dd , j = 1.5 , 7.5 hz , 0.5 h ) , 7.23 ( m , 4.5 h ) , 5.86 ( ddt , j = 7.0 , 10.0 , 17.0 hz , 0.5 h ) , 5.79 ( m , 1.5 h ) , 5.63 ( ddd , j = 7.5 , 10.5 , 18.0 hz , 1 h ) , 5.064.96 ( m , 4 h ) , 4.91 ( ddd , j = 1.5 , 2.0 , 17.5 hz , 1 h ) , 4.81 ( ddd , j = 1.5 , 2.0 , 10.0 hz , 1 h ) , 4.77 ( dd , j = 5.0 , 8.0 hz , 0.5 h ) , 4.56 ( dd , j = 5.5 , 6.5 hz , 1 h ) , 3.79 ( dt , j = 1.0 , 7.5 hz , 1 h ) , 3.39 ( dt , j = 1.5 , 8.0 hz , 0.5 h ) , 2.522.44 ( m , 1.5 h ) , 2.412.29 ( m , 1.5 h ) , 2.28 ( s , 4.5 h ) , 0.07 ( s , 9 h ) , 0.01 ( s , 4.5 h ) ; c nmr ( 151 mhz , cdcl3 ) ( syn - s3-e , major ) 142.1 , 138.06 , 135.1 , 134.0 , 129.8 , 126.7 , 126.57 , 125.7 , 116.7 , 111.8 , 78.2 , 75.0 , 41.1 , 19.3 , 3.7 ; ( antis3-e , minor ) 140.7 , 138.1 , 135.9 , 135.6 , 129.9 , 126.8 , 126.60 , 125.9 , 116.3 , 113.1 , 76.6 , 73.0 , 42.3 , 19.1 , 3.9 ; ir ( film ) 3077 , 2957 , 1247 , 1060 , 842 cm ; hrms ( ei ) m / z 274.1753 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.17 g , 85.5% ( w / w ) in et2o , 8.98 mmol ) , trichloroacetimidate s1-f ( 3.85 g , 12.57 mmol , 1.4 equiv ) , and ( tms)otf ( 162 l , 0.898 mmol , 0.1 equiv ) in cyclohexane ( 45 ml ) afforded after column chromatography ( hexanes ) 1.38 g ( 56% ) of syn / anti - s3-f ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-f : h nmr ( 500 mhz , cdcl3 ) 7.17 ( t , j = 8.0 hz , 1 h ) , 7.08 ( m , 2 h ) , 7.02 ( d , j = 7.5 hz , 1 h ) , 5.72 ( ddt , j = 7.0 , 10.0 , 17.5 hz , 1 h ) , 5.67 ( ddd , j = 7.0 , 10.5 , 17.5 hz , 1 h ) , 5.014.93 ( m , 2 h ) , 4.91 ( dt , j = 2.0 , 17 hz , 1 h ) , 4.83 ( ddd , j = 1.5 , 2.0 , 10.5 hz , 1 h ) , 4.33 ( t , j = 6.0 hz , 1 h ) , 3.79 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.50 ( m , 1 h ) , 2.40 ( m , 1 h ) , 2.33 ( s , 3 h ) , 0.05 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.5 , 137.9 , 137.3 , 135.0 , 127.7 , 127.6 , 127.3 , 123.7 , 116.7 , 111.8 , 104.7 , 81.0 , 75.6 , 41.6 , 21.5 , 3.7 ; ir ( film ) 3079 , 2958 , 1247 , 910 , 845 cm ; hrms ( ei ) m / z 274.1750 [ ( m ) , calcd for c17h26osi , 274.1753 ] . spectroscopic data for anti - s3-f : h nmr ( 500 mhz , cdcl3 ) 7.19 ( t , j = 7.5 hz , 1 h ) , 7.06 ( m , 2 h ) , 7.02 ( d , j = 8.0 hz , 1 h ) , 5.81 ( ddt j = 7.0 , 10.0 , 17.0 hz , 1 h ) , 5.73 ( ddd , j = 7 , 10.5 , 17.0 hz , 1 h ) , 5.01 ( m , 1 h ) , 4.97 ( m , 1 h ) , 4.39 ( dd , j = 5.5 , 8.0 hz , 1 h ) , 3.42 ( dt , j = 1.0 , 7.9 hz , 1 h ) , 2.51 ( m , 1h ) , 2.34 ( s , 3 h ) , 2.31 ( m 1 h ) , 0.02 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 142.5 , 137.63 , 137.62 , 135.6 , 128.0 , 127.96 , 127.95 , 124.4 , 116.2 , 112.7 , 79.2 , 72.8 , 43.1 , 21.4 , 3.9 ; ir ( film ) 3079 , 2959 , 1247 , 911 , 842 cm ; hrms ( ei ) m / z 274.1741 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 2.78 g , 54% ( w / w ) in et2o , 11.52 mmol , 1 equiv ) , trichloroacetimidate s1-g ( 6 g , 19.58 mmol , 1.7 equiv ) , and ( tms)otf ( trace , < 0.02 equiv ) in hexane ( 64 ml ) afforded after column chromatography ( hexanes ) 2.38 g ( 75% ) of syn / anti - s3-g ( 1:1 ) as a colorless oil . spectroscopic data for syns3-g : h nmr ( 500 mhz , cdcl3 ) 7.17 ( d , j = 8.5 hz , 2 h ) , 7.09 ( d , j = 8.5 hz , 2 h ) , 5.72 ( m , 1 h ) , 5.67 ( m , 1 h ) , 5.014.90 ( m , 3 h ) , 4.83 ( m , 1 h ) , 4.34 ( t , j = 6.0 hz , 1 h ) , 3.78 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.51 ( m , 1 h ) , 2.41 ( m , 1 h ) , 2.32 ( s , 3 h ) , 0.05 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.5 , 137.9 , 136.4 , 135.0 , 128.6 ( 2c ) , 126.5 ( 2c ) , 116.7 , 111.7 , 80.8 , 75.5 , 41.5 , 21.1 , 3.7 ; ir ( film ) 3070 , 2959 , 1514 , 1248 , 1062 , 910 , 841 cm ; hrms ( ei ) m / z 274.1749 [ ( m ) , calcd for c17h26osi , 274.1753 ] . spectroscopic data for anti - s3-g : h nmr ( 500 mhz , cdcl3 ) 7.13 ( s , 4 h ) , 5.81 ( m , 1 h ) , 5.74 ( m , 1 h ) , 5.044.95 ( m , 4 h ) , 4.40 ( dd , j = 6.0 , 7.5 hz , 1 h ) , 3.43 ( dt , j = 1.0 , 7.5 hz , 1 h ) , 2.53 ( m , 1 h ) , 2.34 ( s , 3 h ) , 2.33 ( m , 1 h ) , 0.01 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.4 , 137.7 , 136.9 , 135.6 , 128.8 ( 2 c ) , 127.3 ( 2 c ) , 116.2 , 112.8 , 79.0 , 72.7 , 43.1 , 21.2 , 4.0 ; ir ( film ) 3076 , 2957 , 1514 , 1248 , 1057 , 910 , 841 cm ; hrms ( ei ) m / z 274.1753 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.26 g , 69.9% ( w / w ) in thf , 6.73 mmol , 1 equiv ) , trichloroacetimidate s1-h ( 2.9 g , 9.43 mmol , 1.4 equiv ) , and ( tms)otf ( 121 l , 0.673 mmol , 0.1 equiv ) in hexane ( 37 ml ) afforded after column chromatography ( hexanes ) a total of 724 mg ( 39% ) of syn / anti - s3-h ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-h : h nmr ( 500 mhz , cdcl3 ) 7.23 ( dd , j = 6.0 , 9.0 hz , 2 h ) , 6.96 ( t , j = 9.0 hz , 2 h ) , 5.65 ( m , 2 h ) , 4.96 ( m , 2 h ) , 4.87 ( dt , j = 1.5 , 17.0 hz , 1 h ) , 4.83 ( dt , j = 1.5 , 10.5 hz , 1 h ) , 4.33 ( t , j = 6.0 hz , 1 h ) , 3.77 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.48 ( m , 1 h ) , 2.38 ( m , 1 h ) , 0.03 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.8 ( d , j = 245.1 hz ) , 139.2 ( d , j = 3.2 hz ) , 137.7 , 134.5 , 128.1 ( d , j = 7.9 hz , 2 c ) , 117.1 , 114.6 ( d , j = 21.7 hz , 2 c ) , 111.9 , 80.4 , 75.8 , 41.4 , 3.8 ; ir ( film ) 3078 , 2959 , 1518 , 1224 , 839 cm ; hrms ( ei ) m / z 278.1505 [ ( m ) , calcd for c16h23osif , 278.1502 ] . spectroscopic data for anti - s3-h : h nmr ( 500 mhz , cdcl3 ) 7.18 ( dd , j = 5.5 , 8.0 hz , 2 h ) , 6.99 ( t , j = 8.5 hz , 2 h ) , 5.73 ( m , 2 h ) , 5.024.94 ( m , 4 h ) , 4.39 ( t , j = 6.5 hz , 1 h ) , 3.36 ( d , j = 7.5 hz , 1 h ) , 2.50 ( m , 1 h ) , 2.31 ( m , 1 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 162.2 ( d , j = 245.3 hz ) , 138.1 ( d , j = 3.2 hz ) , 137.4 , 135.1 , 128.8 ( d , j = 7.9 hz , 2 c ) , 116.6 , 114.9 ( d , j = 21.3 hz , 2 c ) , 113.0 , 78.6 , 73.0 , 43.0 , 4.0 ; ir ( film ) 3078 , 2959 , 1518 , 1224 , 835 cm ; hrms ( ei ) m / z 278.1492 [ ( m ) , calcd for c16h23osif , 278.1502 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 5.5 g , solution 36.2% ( w / w ) in thf , 15.35 mmol , 1 equiv ) , trichloroacetimidate s1-i ( 10 g , 30.7 mmol , 2 equiv ) , and ( tms)otf ( 190 l , 1.07 mmol , 0.07 equiv ) in cyclohexane ( 85 ml ) afforded after column chromatography ( hexanes ) 1.425 g ( 54% ) of syn / anti - s3-i ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : antis3-i = 0.7:1.0 ) ; h nmr ( 500 mhz , cdcl3 ) 7.27 ( m , 3.4 h ) , 7.24 ( m , 2 h ) , 7.18 ( m , 1.4 h ) , 5.825.62 ( m , 3.4 h ) , 5.04 ( m , 0.7 h ) , 5.024.96 ( m , 4.1 h ) , 4.90 ( dt , j = 1.5 , 17.0 hz , 1 h ) , 4.85 ( dt , j = 1.5 , 11.0 hz , 1 h ) , 4.42 ( dd , j = 6.0 , 7.0 hz , 0.7 h ) , 4.36 ( t , j = 6.0 hz , 1 h ) , 3.80 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 3.39 ( m , 0.7 h ) , 2.552.30 ( m , 3.4 h ) , 0.06 ( s , 9 h ) , 0.01 ( s , 7.3 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.1 , 140.9 , 137.7 , 137.3 , 134.9 , 134.7 , 134.3 , 133.0 , 132.5 , 128.7 ( 2 c ) , 128.3 ( 2 c ) , 128.2 , 128.0 ( 2 c ) , 127.9 ( 2 c ) , 127.4 , 117.2 , 116.8 , 113.1 , 112.1 , 80.3 , 78.6 , 75.9 , 73.1 , 42.9 , 41.3 , 3.8 , 4.0 ; ir ( film ) 3078 , 2957 , 1491 , 1248 , 1089 , 841 cm ; hrms ( ei ) m / z 294.1218 [ ( m ) , calcd for c16h23osicl , 294.1207 ] . applying general procedure e to alcohol s2-f ( 1 g , 4.95 mmol , 1 equiv ) , 4-(trifluoromethyl)benzaldehyde ( 862 mg , 4.95 mmol , 1 equiv ) , allyltrimethylsilane ( 625 mg , 4.95 mmol , 1 equiv ) , and ( tms)otf ( 180 l , 0.989 mmol , 0.2 equiv ) in ch2cl2 ( 50 ml ) for 30 min at 78 c afforded after workup and column chromatography ( 10% ch2cl2 in hexanes ) 765 mg of a mixture of anti / syn - s3-j ( 4:1 ) as a colorless oil : mixture of diastereomers ( anti : syn - s3-j = 0.8:0.2 ) ; h nmr ( 600 mhz , cdcl3 ) 7.59 ( d , j = 7.8 hz , 1.6 h ) , 7.56 ( d , j = 7.8 hz , 0.4 h ) , 7.42 ( d , j = 8.4 hz , 0.4 h ) , 7.37 ( d , j = 7.8 hz , 1.6 h ) , 5.835.73 ( m , 1.6 h ) , 5.715.64 ( m , 0.4 h ) , 5.07 ( m , 0.8 h ) , 5.034.98 ( m , 2.8 h ) , 4.92 ( dt , j = 1.8 , 17.4 hz , 0.2 h ) , 4.86 ( dt , j = 1.8 , 10.8 hz , 0.2 h ) , 4.53 ( dd , j = 6.6 , 7.8 hz , 0.8 h ) , 4.47 ( t , j = 6.0 hz , 0.2 h ) , 3.84 ( dt , j = 1.8 , 7.2 hz , 0.2 h ) , 3.40 ( d , j = 7.8 hz , 0.8 h ) , 2.53 ( m , 1 h ) , 2.46 ( m , 0.2 h ) , 2.36 ( m , 0.8 h ) , 0.09 ( s , 1.8 h ) , 0.02 ( s , 7.2 h ) ; c nmr ( 151 mhz , cdcl3 ) ( anti ) 146.7 , 137.2 , 134.6 , 129.6 ( q , j = 32.3 hz ) , 127.6 ( 2 c ) , 125.1 ( q , j = 3.78 hz , 2 c ) , 124.3 ( q , j = 272.0 hz ) , 117.0 , 113.4 , 78.7 , 73.5 , 42.9 , 4.0 ; ( syn ) 147.7 , 137.5 , 134.0 , 129.1 ( q , j = 32.1 hz ) , 126.8 ( 2 c ) , 124.9 ( q , j = 3.6 hz , 2 c ) , cf3 carbon could not be located , 117.5 , 112.4 , 80.3 , 76.2 , 41.3 , 3.8 ; ir ( film ) 3080 , 2959 , 1325 , 1128 , 841 cm ; hrms ( ei ) m / z 328.1457 [ ( m ) , calcd for c17h23osif3 , 328.1470 ] . to a solution of syn / anti - s3-z ( 1:1 , 342 mg , 1.01 mmol , 1 equiv ) in thf ( 2.2 ml ) was added s - phos ( 4.2 mg , 0.02 mmol , 0.02 equiv ) , phenylboronic acid ( 184 mg , 1.512 mmol , 1.5 equiv ) , and k3po42h2o ( 500 mg , 2.016 mmol , 2 equiv ) . the mixture was degassed with three freeze pump thaw cycles , and then pd(oac)2 ( 2.3 mg , 0.01 mmol , 0.01 equiv ) was added at room temperature . the mixture was stirred at room temperature under a nitrogen atmosphere and the reaction monitored by tlc ( hexanes ) . after 9 h the reaction was concentrated and the residue subjected to column chromatography ( 10% ch2cl2 in hexanes ) to afford a total of 327 mg ( 53% ) of syn / anti - s3-k ( 1:1 ) . spectroscopic data for syn - s3-k : h nmr ( 600 mhz , cdcl3 ) 7.60 ( m , 2 h ) , 7.54 ( d , j = 8.4 hz , 2 h ) , 7.43 ( t , j = 7.2 hz , 2 h ) , 7.37 ( d , j = 8.4 hz , 2 h ) , 7.33 ( tt , j = 1.2 , 7.2 hz , 1 h ) , 5.77 ( ddt , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 5.72 ( ddd , j = 7.2 . 10.8 , 18.0 hz , 1 h ) , 5.044.98 ( m , 2 h ) , 4.96 ( dt , j = 1.8 , 16.8 hz , 1 h ) , 4.87 ( dt , j = 1.8 , 10.8 hz , 1 h ) , 4.45 ( t , j = 6.0 hz , 1 h ) , 3.85 ( dt , j = 1.2 , 7.2 hz , 1 h ) , 2.57 ( m , 1 h ) , 2.49 ( m , 1 h ) , 0.09 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 142.7 , 141.1 , 139.7 , 137.9 , 134.8 , 128.7 ( 2 c ) , 127.04 , 127.02 ( 2 c ) , 126.97 ( 2 c ) , 126.6 ( 2 c ) , 116.9 , 112.0 , 80.6 , 75.7 , 41.4 , 3.7 ; ir ( film ) 3077 , 2957 , 1250 , 840 cm ; hrms ( ei ) m / z 336.1923 [ ( m ) , calcd for c22h28osi , 336.1909 ] . spectroscopic data for anti - s3-k : h nmr ( 600 mhz , cdcl3 ) 7.61 ( d , j = 7.8 hz , 2 h ) , 7.56 ( d , j = 7.8 hz , 2 h ) , 7.44 ( t , j = 7.8 hz , 2 h ) , 7.33 ( m , 3 h ) , 5.85 ( ddt , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 5.77 ( ddd , j = 7.8 , 10.8 , 18.0 hz , 1 h ) , 5.075.00 ( m , 4 h ) , 4.50 ( dd , j = 5.4 , 7.8 hz , 1 h ) , 3.49 ( d , j = 7.2 hz , 1 h ) , 2.58 ( m , 1 h ) , 2.40 ( m , 1 h ) , 0.01 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 141.6 , 140.9 , 140.2 , 137.6 , 135.4 , 128.7 ( 2 c ) , 127.7 ( 2 c ) , 127.2 , 127.0 ( 2 c ) , 126.8 ( 2 c ) , 116.5 , 112.9 , 78.9 , 72.9 , 43.1 , 3.9 ; ir ( film ) 3077 , 2957 , 1247 , 840 cm ; hrms ( ei ) m / z 336.1920 [ ( m ) , calcd for c22h28osi , 336.1909 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.07 g , 44% ( w / w ) in thf , 5.72 mmol , 1 equiv ) , trichloroacetimidate s1-l ( 2.74 g , 8 mmol , 1.4 equiv ) , and ( tms)otf ( 47 l , 0.259 mmol , 0.05 equiv ) in hexane ( 32 ml ) afforded after column chromatography ( 10% ch2cl2 in hexanes ) a total of 852 mg ( 48% ) of syn / anti - s3-l ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-l : h nmr ( 500 mhz , cdcl3 ) 7.81 ( m , 2 h ) , 7.78 ( dd , j = 1.5 , 8.5 hz , 1 h ) , 7.72 ( s , 1 h ) , 7.45 ( m , 3 h ) , 5.805.63 ( m , 2 h ) , 5.024.96 ( m , 2 h ) , 4.92 ( dq , j = 2.0 , 15.0 hz , 1 h ) , 4.81 ( dq , j = 1.5 , 10.5 hz , 1 h ) , 4.53 ( m , 1 h ) , 3.59 ( dq , j = 1.5 , 7.0 hz , 1 h ) , 2.61 ( m , 1 h ) , 2.51 ( m , 1 h ) , 0.08 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.1 , 137.9 , 134.8 , 133.1 , 132.8 , 127.9 , 127.6 , 127.5 , 125.8 , 125.4 , 125.3 , 125.0 , 116.9 , 111.9 , 81.3 , 75.9 , 41.5 , 3.7 ; ir ( film ) 3060 , 2959 , 2825 , 1241 , 860 , 841 cm ; hrms ( ei ) m / z 310.1753 [ ( m ) , calcd for c20h26osi , 310.1753 ] . spectroscopic data for anti - s3-l : h nmr ( 500 mhz , cdcl3 ) 7.81 ( m , 3 h ) , 7.65 ( s , 1 h ) , 7.44 ( m , 3 h ) , 5.865.72 ( m , 2 h ) , 5.064.96 ( m , 4 h ) , 4.59 ( dd , j = 6.0 , 7.5 hz , 1 h ) , 3.44 ( m , 1 h ) , 2.62 ( m , 1 h ) , 2.43 ( m , 1 h ) , 0.02 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.8 , 137.5 , 135.3 , 133.1 , 133.0 , 128.0 , 127.8 , 127.7 , 126.5 , 125.9 , 125.6 , 125.1 , 116.5 , 112.9 , 79.3 , 72.9 , 42.8 , 4.0 ; ir ( film ) 3057 , 2959 , 2831 , 1246 , 859 , 841 cm ; hrms ( ei ) m / z 310.1745 [ ( m ) , calcd for c20h26osi , 310.1753 ] . applying general procedure d to s2-b ( 256 mg , 1.331 mmol , 1 equiv ) , the trichloroacetimidate of 1-phenylbut-3-en-1-ol ( 662 mg , 2.26 mmol , 1.7 equiv ) , and ( tms)otf ( 24 l , 0.133 mmol , 0.1 equiv ) in hexane ( 7 ml ) afforded after column chromatography ( hexanes ) a total of 250 mg ( 58% ) of syn / anti - s3-m ( 1:1 ) as a colorless oil . compounds syn- and anti - s3-m were separable by column chromatography . spectroscopic data for syn - s3-m : h nmr ( 600 mhz , cdcl3 ) 7.58 ( m , 2 h ) , 7.35 ( m , 3 h ) , 7.26 ( m , 2 h ) , 7.21 ( m , 3 h ) , 5.655.55 ( m , 2 h ) , 4.914.87 ( m , 3 h ) , 4.81 ( m , 1 h ) , 4.28 ( t , j = 6.0 hz , 1 h ) , 3.98 ( dt , j = 1.8 , 7.2 hz , 1 h ) 2.45 ( m , 1 h ) , 2.35 ( m , 1 h ) , 0.36 ( s , 3 h ) , 0.31 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 143.3 , 137.4 , 137.0 , 134.7 , 134.3 ( 2 c ) , 129.2 , 127.8 ( 2 c ) , 127.6 ( 2 c ) , 126.9 , 126.6 ( 2 c ) , 81.1 , 75.2 , 41.5 , 5.2 , 5.5 ; ir ( film ) 3071 , 2961 , 1427 , 1248 , 1115 , 837 cm ; hrms ( ei ) m / z 322.1751 [ ( m ) , calcd for c21h26osi , 322.1753 ] . spectroscopic data for anti - s3-m : h nmr ( 600 mhz , cdcl3 ) 7.50 ( m , 2 h ) , 7.36 ( m , 1 h ) , 7.32 ( m , 2 h ) , 7.21 ( m , 3 h ) , 7.06 ( m , 2 h ) , 5.78 ( ddt , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 5.69 ( ddd , j = 7.2 , 10.8 , 17.4 hz , 1 h ) , 5.024.93 ( m , 4 h ) , 4.43 ( dd , j = 5.4 , 7.8 hz , 1 h ) , 3.60 ( d , j = 7.8 hz , 1 h ) , 2.50 ( quintet , a of abx system , j = 7.2 hz , 1 h ) , 2.32 ( quintet , b of abx system , j = 7.2 hz , 1 h ) , 0.28 ( s , 3 h ) , 0.25 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 142.1 , 137.1 , 136.8 , 135.4 , 134.4 ( 2 c ) , 129.0 , 128.0 ( 2 c ) , 127.4 ( 2 c ) , 127.30 ( 2 c ) , 127.26 , 79.2 , 72.5 , 43.0 , 5.3 , 6.0 ; ir ( film ) 3071 , 2961 , 1427 , 1248 , 1115 , 837 cm ; hrms ( ei ) m / z 322.1753 [ ( m ) , calcd for c21h26osi , 322.1753 ] . applying general procedure d to s2-c ( 2.17 g , 8.53 mmol , 1 equiv ) , the trichloroacetimidate of 1-phenylbut-3-en-1-ol ( 5 g , 17.07 mmol , 2 equiv ) , and ( tms)otf ( 230 l , 1.28 mmol , 0.15 equiv ) in cyclohexane ( 41 ml ) afforded after column chromatography ( 10% ch2cl2 in hexanes ) 2.7 g ( 83% ) of syn / anti - s3-n ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-n : h nmr ( 500 mhz , cdcl3 ) 7.68 ( m , 2 h ) , 7.59 ( m , 2 h ) , 7.397.33 ( m , 5 h ) , 7.27 ( m , 3 h ) , 7.22 ( m , 3 h ) , 5.67 ( ddd , j = 7.0 , 10.5 , 17.5 hz , 1 h ) , 5.47 ( m , 1 h ) , 4.93 ( dt , j = 2.0 , 17.5 hz , 1 h ) , 4.854.81 ( m , 3 h ) , 4.31 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 4.27 ( t , j = 7.0 hz , 1 h ) , 2.44 ( m , 1 h ) , 2.31 ( m , 1 h ) , 0.59 ( m , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.1 , 137.1 , 135.4 ( 2 c ) , 135.2 ( 2 c ) , 134.8 , 134.6 , 129.4 , 129.3 , 127.8 ( 2 c ) , 127.7 ( 2 c ) , 127.6 ( 2 c ) , 127.0 , 126.7 , 81.4 , 74.7 , 41.5 , 6.5 ; ir ( film ) 3071 , 2975 , 1429 , 1115 , 734 cm ; hrms ( ei ) m / z 384.1901 [ ( m ) , calcd for c26h28osi , 384.1909 ] . spectroscopic data for anti - s3-n : h nmr ( 500 mhz , cdcl3 ) 7.63 ( m , 2 h ) , 7.49 ( m , 2 h ) , 7.40 ( m , 1 h ) , 7.377.29 ( m , 5 h ) , 7.19 ( m , 3 h ) , 6.97 ( m , 2 h ) , 5.845.73 ( m , 2 h ) , 5.054.97 ( m , 4 h ) , 4.50 ( dd , j = 5.5 , 7.5 hz , 1 h ) , 3.93 ( dt , j = 1.5 , 8.0 hz , 1 h ) , 2.53 ( m , 1 h ) , 2.35 ( m , 1 h ) , 0.53 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.6 , 136.7 , 135.5 ( 2 c ) , 135.4 , 135.3 ( 2 c ) , 135.2 , 129.4 , 129.2 , 128.0 ( 2 c ) , 127.54 ( 2 c ) , 127.52 ( 2 c ) , 127.46 ( 2 c ) , 127.3 , 116.5 , 114.8 , 79.2 , 72.1 , 42.9 , 6.6 ; ir ( film ) 3071 , 3976 , 1429 , 1115 , 724 cm ; hrms ( ei ) m / z 384.1889 [ ( m ) , calcd for c26h28osi , 384.1909 ] . applying general procedure d to s2-d ( 583 mg , 3.38 mmol , 1 equiv ) , the trichloroacetimidate of 1-phenylbut-3-en-1-ol ( 1.48 g , 5.07 mmol , 1.5 equiv ) , and ( tms)otf ( 31 l , 0.169 mmol , 0.05 equiv ) in hexane ( 19 ml ) afforded after column chromatography ( hexanes ) a total of 720 mg ( 70% ) of syn / anti - s3-o ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-o : h nmr ( 500 mhz , cdcl3 ) 7.26 ( m , 4 h ) , 7.20 ( m , 1 h ) , 5.68 ( m , 2 h ) , 4.984.88 ( m , 3 h ) , 4.80 ( dd , j = 1.0 , 10.0 hz , 1 h ) , 4.35 ( t , j = 6.0 hz , 1 h ) , 3.98 ( dd , j = 1.5 , 7.5 hz , 1 h ) , 2.52 ( m , 1 h ) , 2.42 ( m , 1 h ) , 0.98 ( t , j = 8.0 hz , 9 h ) , 0.62 ( dq , j = 1.5 , 7.5 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.7 , 138.3 , 134.8 , 127.8 ( 2 c ) , 126.8 , 126.6 ( 2 c ) , 116.8 , 111.8 , 80.9 , 74.3 , 41.3 , 7.5 , 1.8 ; ir ( film ) 3078 , 2953 , 1454 , 1014 , 910 cm ; hrms ( ei ) m / z 302.2063 [ ( m ) , calcd for c19h30osi , 302.2066 ] . spectroscopic data for anti - s3-o : h nmr ( 600 mhz , cdcl3 ) 7.30 ( m , 2 h ) , 7.24 ( m , 3 h ) , 5.78 ( m , 2 h ) , 5.024.95 ( m , 4 h ) , 4.41 ( dd , j = 5.4 , 7.8 hz , 1 h ) , 3.55 ( dt , j = 1.2 , 7.8 hz , 1 h ) , 2.52 ( m , 1 h ) , 2.34 ( m , 1 h ) , 0.88 ( t , j = 7.8 hz , 9 h ) , 0.54 ( dq , j = 2.4 , 7.8 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.2 , 138.0 , 135.5 , 128.1 ( 2 c ) , 127.43 ( 2 c ) , 127.37 , 116.3 , 112.8 , 79.0 , 71.5 , 42.9 , 7.3 , 1.6 ; ir ( film ) 3064 , 2953 , 1450 , 1011 , 910 cm ; hrms ( ei ) m / z 302.2065 [ ( m ) , calcd for c19h30osi , 302.2066 ] . applying general procedure d to s2-b ( 1 g , 5.2 mmol , 1 equiv ) , trichloroacetimidate s1-c ( 2.68 g , 8.32 mmol , 1.6 equiv ) , and ( tms)otf ( 94 l , 0.52 mmol , 0.1 equiv ) in hexane ( 29 ml ) afforded after column chromatography ( 15% ch2cl2 in hexanes ) a total of 1.298 g ( 71% ) of syn / anti - s3-p ( 1:1 ) . diastereomers were partially separated and obtained as colorless oils . spectroscopic data for syn - s3-p : h nmr ( 600 mhz , cdcl3 ) 7.61 ( m , 2 h ) , 7.36 ( m , 3 h ) , 7.19 ( t , j = 7.8 hz , 1 h ) , 6.85 ( d , j = 0.6 hz , 1 h ) , 6.81 ( dd , j = 0.6 , 7.2 hz , 1 h ) , 6.76 ( ddd , j = 0.6 , 2.4 , 7.8 hz , 1 h ) , 5.705.59 ( m , 2 h ) , 4.94 ( m , 3 h ) , 4.86 ( dt , j = 10.8 hz , 1 h ) , 4.29 ( t , j = 6.0 hz , 1 h ) , 4.00 ( dt , j = 1.2 , 7.8 hz , 1 h ) , 3.78 ( s , 3 h ) , 2.46 ( m , 1 h ) , 2.37 ( m , 1 h ) , 0.39 ( s , 3 h ) , 0.35 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 159.3 , 145.0 , 137.4 , 136.9 , 134.7 , 134.3 ( 2 c ) , 129.2 , 128.8 , 127.6 ( 2 c ) , 119.0 , 116.8 , 112.5 , 112.4 , 111.9 , 81.0 , 75.2 , 55.1 , 41.6 , 5.2 , 5.6 ; ir ( film ) 3071 , 2958 , 1254 , 1046 , 837 cm ; hrms ( ei ) m / z 352.1855 [ ( m ) , calcd for c22h28o2si , 352.1859 ] . spectroscopic data for anti - s3-p : h nmr ( 600 mhz , cdcl3 ) 7.52 ( m , 2 h ) , 7.33 ( m , 3 h ) , 7.14 ( t , j = 7.8 hz , 1 h ) , 6.76 ( ddd , j = 0.6 , 2.4 , 7.8 hz , 1 h ) , 6.68 ( m , 2 h ) , 5.80 ( ddt , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 5.69 ( ddd , j = 7.2 , 10.2 , 18.0 hz , 1 h ) , 5.034.94 ( m , 4 h ) , 4.43 ( dd , j = 5.4 , 7.8 hz , 1 h ) , 3.69 ( s , 3 h ) , 3.66 ( dt , j = 1.2 , 7.2 hz , 1 h ) , 2.51 ( m , 1 h ) , 2.33 ( m , 1 h ) , 0.29 ( s , 3 h ) , 0.27 ( s , 3 h ) . ) . c nmr ( 151 mhz , cdcl3 ) 159.5 , 143.8 , 137.1 , 136.8 , 135.4 , 134.4 ( 2 c ) , 129.04 , 129.03 , 127.4 ( 2 c ) , 119.8 , 116.4 , 113.7 , 113.2 , 112.2 , 79.1 , 72.5 , 43.0 , 5.3 , 5.8 ; ir ( film ) 3071 , 2958 , 1254 , 1046 , 837 cm ; hrms ( ei ) m / z 352.1859 [ ( m ) , calcd for c22h28o2si , 352.1859 ] . applying general procedure d to s2-b ( 1 g , 5.2 mmol , 1 equiv ) , trichloroacetimidate s1-i ( 2.72 g , 8.32 mmol , 1.6 equiv ) , and ( tms)otf ( 94 l , 0.52 mmol , 0.1 equiv ) in hexane ( 29 ml ) afforded after column chromatography ( hexanes and 10% ch2cl2 in hexanes ) a total of 1.686 g ( 91% ) of syn / anti - s3-q ( 1:1 ) . spectroscopic data for syn - s3-q : h nmr ( 600 mhz , cdcl3 ) 7.61 ( m , 2 h ) , 7.407.36 ( m , 3 h ) , 7.25 ( m , 2 h ) , 7.18 ( m , 2 h ) , 5.675.55 ( m , 2 h ) , 4.954.85 ( m , 4 h ) , 4.29 ( t , j = 6.0 hz , 1 h ) , 4.00 ( dt , j = 1.2 , 5.4 hz , 1 h ) , 2.45 ( m , 1 h ) , 2.35 ( m , 1 h ) , 0.39 ( s , 3 h ) , 0.35 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 141.8 , 137.2 , 136.7 , 134.3 ( 2 c ) , 134.2 , 132.5 , 129.2 , 128.0 ( 2 c ) , 127.9 ( 2 c ) , 127.6 ( 2 c ) , 117.2 , 112.6 , 80.4 , 75.4 , 41.3 , 5.3 , 5.7 ; ir ( film ) 3072 , 2961 , 1490 , 1114 , 913 , 836 cm ; hrms ( ei ) m / z 356.1352 [ ( m ) , calcd for c21h25osicl , 356.1363 ] . spectroscopic data for anti - s3-q : h nmr ( 600 mhz , cdcl3 ) 7.52 ( m , 2 h ) , 7.39 ( tt , j = 1.8 , 7.8 hz , 1 h ) , 7.34 ( t , j = 7.2 hz , 2 h ) , 7.17 ( m , 2 h ) , 6.95 ( m , 2 h ) , 5.76 ( m , 1 h ) , 5.71 ( ddd , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 5.03 ( dt , j = 1.8 , 10.8 hz , 1 h ) , 5.014.96 ( m , 3 h ) , 4.42 ( dd , j = 6.0 , 7.8 hz , 1 h ) , 3.56 ( d , j = 7.8 hz , 1 h ) , 2.48 ( m , 1 h ) , 2.30 ( m , 1 h ) , 0.31 ( s , 3 h ) , 0.26 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 140.5 , 136.8 , 136.6 , 134.8 , 134.4 ( 2 c ) , 132.9 , 129.1 , 128.6 ( 2 c ) , 128.2 ( 2 c ) , 127.5 ( 2 c ) , 116.8 , 113.7 , 78.5 , 72.7 , 42.9 , 31.6 , 22.7 , 14.1 , 5.3 , 6.3 ; ir ( film ) 3076 , 2961 , 1489 , 1093 , 911 , 830 cm ; hrms ( ei ) m / z 356.1355 [ ( m ) , calcd for c21h25osicl , 356.1363 ] . applying general procedure d to s2-d ( 0.73 g , 4.23 mmol , 1 equiv ) , trichloroacetimidate s1-i ( 1.8 g , 5.5 mmol , 1.3 equiv ) , and ( tms)otf ( 76 l , 0.24 mmol , 0.1 equiv ) in hexane ( 24 ml ) afforded after column chromatography ( hexanes and 10% ch2cl2 in hexanes ) a total of 0.96 g ( ca . only compound syn - s3-q was purified by subsequent column chromatography : h nmr ( 600 mhz , cdcl3 ) 7.24 ( m , 2 h ) , 7.19 ( m , 2 h ) , 5.64 ( m , 2 h ) , 4.964.93 ( m , 2 h ) , 4.88 ( dt , j = 1.8 , 17.4 hz , 1 h ) , 4.81 ( dt , j = 1.8 , 10.8 , 1 h ) , 4.32 ( t , j = 6.0 hz , 1 h ) , 3.96 ( dt , j = 1.2 , 7.8 hz , 1 h ) , 2.48 ( m , 1 h ) , 2.37 ( m , 1 h ) , 0.97 ( t , j = 7.8 hz , 9 h ) , 0.61 ( dq , j = 3.0 , 7.8 hz , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 142.2 , 138.1 , 134.2 , 132.4 , 127.9 ( 2 c ) , 117.2 , 112.0 , 80.3 , 74.6 , 41.2 , 7.4 , 1.7 ; ir ( film ) 3033 , 2957 , 1491 , 833 cm ; hrms ( ei ) m / z 336.1663 [ ( m ) , calcd for c19h29osicl , 336.1676 ] . applying general procedure d to s2-d ( 860 mg , 5 mmol , 1 equiv ) , trichloroacetimidate s1-l ( 2.4 g , 7 mmol , 1.4 equiv ) , and ( tms)otf ( 22.5 l , 0.125 mmol , 0.025 equiv ) in hexane ( 28 ml ) afforded after column chromatography ( hexanes and 10% ch2cl2 in hexanes ) a total of 793 mg ( 45% ) of syn / anti - s3-s ( 1:1 ) . spectroscopic data for syn - s3-s : h nmr ( 500 mhz , cdcl3 ) 7.84 ( m , 2 h ) , 7.81 ( d , j = 8.5 hz , 1 h ) , 7.74 ( s , 1 h ) , 7.47 ( m , 3 h ) , 5.74 ( m , 2 h ) , 5.044.95 ( m , 3 h ) , 4.82 ( m , 1 h ) , 4.55 ( d , j = 6.0 hz , 1 h ) , 4.08 ( dt , j = 1.0 , 7.0 hz , 1 h ) , 2.65 ( m , 1 h ) , 2.54 ( m , 1 h ) , 1.05 ( t , j = 8.0 hz , 9 h ) , 0.69 ( dq , j = 2.0 , 8.0 hz , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 141.2 , 138.3 , 134.7 , 133.1 , 132.7 , 127.9 , 127.7 , 127.5 , 125.7 , 125.4 , 125.3 , 125.0 , 116.9 , 111.9 , 81.2 , 74.6 , 41.4 , 7.5 , 1.8 ; ir ( film ) 3057 , 2953 , 2878 , 1414 , 1018 , 910 , 817 cm ; hrms ( ei ) m / z 352.2210 [ ( m ) , calcd for c23h32osi , 352.2222 ] . spectroscopic data for anti - s3-s : h nmr ( 500 mhz , cdcl3 ) 7.80 ( m , 3 h ) , 7.63 ( s , 1 h ) , 7.467.41 ( m , 3 h ) , 5.79 ( m , 2 h ) , 5.044.94 ( m , 4 h ) , 4.59 ( dd , j = 6.0 , 8.0 hz , 1 h ) , 3.58 ( d , j = 8.0 hz , 1 h ) , 2.60 ( m , 1 h ) , 2.41 ( m , 1 h ) , 0.88 ( t , j = 8.0 hz , 9 h ) , 0.55 ( dq , j = 4.0 , 8.0 hz , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 139.7 , 138.0 , 135.4 , 133.1 , 133.09 , 128.0 , 127.8 , 127.7 , 126.6 , 125.9 , 125.6 , 125.1 , 116.5 , 112.9 , 79.1 , 71.6 , 42.9 , 7.4 , 1.6 ; ir ( film ) 3059 , 2953 , 2876 , 1458 , 1020 , 910 cm ; hrms ( ei ) m / z 352.2222 [ ( m ) , calcd for c23h32osi , 352.2222 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 380 mg , 69.6% ( w / w ) in thf , 2.03 mmol , 1 equiv ) , trichloroacetimidate s1-t ( 679 mg , 2.03 mmol , 1 equiv ) , and ( tms)otf ( 37 l , 0.2 mmol , 0.1 equiv ) in hexane ( 11 ml ) afforded after column chromatography ( hexanes ) a total of 144.5 mg ( 24% ) of syn / anti - s3-t ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-t : h nmr ( 500 mhz , cdcl3 ) 7.45 ( m , 1 h ) , 7.15 ( m , 2 h ) , 7.07 ( m , 1 h ) , 5.80 ( ddt , j = 7.0 , 10.0 , 17.0 hz , 1 h ) , 5.61 ( ddd , j = 7.5 , 10.5 , 17.5 hz , 1 h ) , 5.034.97 ( m , 2 h ) , 4.87 ( dt , j = 2.0 , 17.5 hz , 1 h ) , 4.79 ( ddd , j = 1.5 , 2.0 , 10.5 hz , 1 h ) , 4.58 ( dd , j = 5.0 , 7.5 hz , 1 h ) , 3.78 ( dt , j = 1.5 , 7.5 hz , 1 h ) , 2.53 ( m , 2 h ) , 2.46 ( m , 1 h ) , 2.36 ( m , 1 h ) , 1.59 ( m , 2 h ) , 0.96 ( t , j = 7.5 hz , 3 h ) , 0.05 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.6 , 138.5 , 138.1 , 135.4 , 128.8 , 126.9 , 126.6 , 125.6 , 116.6 , 111.8 , 77.9 , 76.6 , 42.1 , 34.5 , 24.2 , 14.2 , 3.8 ; ir ( film ) 3074 , 2959 , 1248 , 841 cm ; hrms ( ei ) m / z 302.2078 [ ( m ) , calcd for c19h30osi , 302.2066 ] . spectroscopic data for anti - s3-t : h nmr ( 500 mhz , cdcl3 ) 7.41 ( dd , j = 1.5 , 7.5 hz , 1 h ) , 7.18 ( m , 2 h ) , 7.10 ( m , 1 h ) , 5.92 ( m , 1 h ) , 5.76 ( ddd , j = 7.5 , 11.0 , 17.5 hz , 1 h ) , 5.064.96 ( m , 4 h ) , 4.78 ( dd , j = 4.5 , 9.0 hz , 1 h ) , 3.39 ( dt , j = 1.0 , 7.0 hz , 1 h ) , 2.53 ( m , 2 h ) , 2.47 ( m , 1 h ) , 2.28 ( m , 1 h ) , 1.55 ( m , 2 h ) , 0.95 ( t , j = 7.0 hz , 3 h ) , 0.00 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.6 , 140.4 , 138.1 , 135.9 , 129.1 , 126.8 , 126.7 , 126.0 , 116.1 , 112.8 , 74.4 72.8 , 43.2 , 34.5 , 24.6 , 14.2 , 4.0 ; ir ( film ) 3076 , 2959 , 1248 , 841 cm ; hrms ( ei ) m / z 302.2080 [ ( m ) , calcd for c19h30osi , 302.2066 ] . applying general procedure d to s2-e ( 2.6 g , 76.9% ( w / w ) in thf , 13.86 mmol , 1 equiv ) , the trichloroacetimidate of 1-phenylbut-3-en-1-ol ( 7.3 g , 24.95 mmol , 1.8 equiv ) , and ( tms)otf ( 0.25 ml , 1.386 mmol , 0.1 equiv ) in hexane ( 70 ml ) afforded after column chromatography ( hexanes ) a total of 1.67 g ( 44% ) of syn / anti - s3-u ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : anti - s3-u = 1:1 ) ; h nmr ( 500 mhz , cdcl3 ) 7.377.19 ( m , 10 h ) , 5.81 ( ddt , j = 7.0 , 10.5 , 17.0 hz , 1 h ) , 5.67 ( ddt , j = 7.0 , 10.5 , 17.0 hz , 1 h ) , 5.004.93 ( m , 3 h ) , 4.80 ( m , 1 h ) , 4.66 ( m , 2 h ) , 4.32 ( t , j = 6.0 hz , 1 h ) , 4.28 ( dd , j = 6.0 , 8.0 hz , 1 h ) , 3.77 ( s , 1 h ) , 3.31 ( s , 1 h ) , 2.52 ( m , 2 h ) , 2.48 ( m , 1 h ) , 2.34 ( m , 1 h ) , 1.63 ( m , 3 h ) , 1.51 ( m , 3 h ) , 0.07 ( s , 9 h ) , 0.02 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 145.0 , 144.4 , 143.4 , 142.3 , 135.5 , 134.6 , 128.1 ( 2 c ) , 127.8 ( 2 c ) , 127.5 ( 2 c ) , 127.4 , 126.8 , 126.6 ( 2 c ) , 116.9 , 116.4 , 109.9 , 109.5 , 80.0 , 79.0 , 77.8 , 75.4 , 43.0 , 40.5 , 20.4 , 20.3 , 3.0 , 3.2 ; ir ( film ) 3072 , 2959 , 1248 , 1060 , 839 cm ; hrms ( ei ) m / z 274.1753 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure d to s2-e ( 660 mg , 79% ( w / w ) in thf , 3.6 mmol , 1 equiv ) , trichloroacetimidate s1-d ( 1.86 g , 5.77 mmol , 1.6 equiv ) , and ( tms)otf ( 0.32 l , 0.18 mmol , 0.05 equiv ) in hexane ( 20 ml ) afforded after column chromatography ( 15% and 20% ch2cl2 in hexanes ) a total of 918 mg ( 83% ) of syn / anti - s3-v ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-v : h nmr ( 500 mhz , cdcl3 ) 7.20 ( m , 2 h ) , 6.81 ( m , 2 h ) , 5.66 ( ddt , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 4.984.93 ( m , 2 h ) , 4.67 ( m , 1 h ) , 4.65 ( m , 1 h ) , 4.27 ( t , j = 6.6 hz , 1 h ) , 3.78 ( s , 3 h ) , 3.75 ( s , 1 h ) , 2.52 ( m , 1 h ) , 2.43 ( m , 1 h ) , 0.06 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 158.4 , 145.1 , 135.5 , 134.8 , 127.7 ( 2 c ) , 116.8 , 113.1 ( 2 c ) , 109.3 , 79.8 , 77.7 , 55.1 , 40.5 , 20.3 , 2.9 ; ir ( film ) 3033 , 2950 , 1238 , 840 cm ; hrms ( ei ) m / z 304.1853 [ ( m ) , calcd for c18h28o2si , 304.1859 ] . spectroscopic data for anti - s3-v : h nmr ( 600 mhz , cdcl3 ) 7.15 ( d , j = 8.4 hz , 2 h ) , 6.85 ( d , j = 8.4 hz , 2 h ) , 5.78 ( ddt , j = 6.6 , 9.6 , 16.8 hz , 1 h ) , 4.994.94 ( m , 2 h ) , 4.80 ( s , 1 h ) , 4.66 ( s , 1 h ) , 4.23 ( t , j = 7.2 hz , 1 h ) , 3.80 ( s , 3 h ) , 3.30 ( s , 1 h ) , 2.53 ( m , 1 h ) , 2.33 ( m , 1 h ) , 0.02 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 158.9 , 144.5 , 135.6 , 134.3 , 128.7 ( 2 c ) , 116.3 , 113.4 ( 2 c ) , 109.8 , 78.5 , 75.0 , 55.1 , 43.0 , 20.4 , 3.2 ; ir ( film ) 3074 , 2955 , 1247 , 824 cm ; hrms ( ei ) m / z 304.1859 [ ( m ) , calcd for c18h28o2si , 304.1859 ] . applying general procedure d to s2-e ( 380 mg , 79% ( w / w ) in thf , 2.083 mmol , 1 equiv ) , trichloroacetimidate s1-g ( 1.02 g , 3.33 mmol , 1.6 equiv ) , and ( tms)otf ( 19 l , 0.104 mmol , 0.05 equiv ) in hexane ( 12 ml ) afforded after column chromatography ( 2% ch2cl2 in hexanes ) a total of 390 mg ( 61% ) of syn / anti - s3-w ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : anti - s3-w = 1.4:1.0 ) ; h nmr ( 600 mhz , cdcl3 ) 7.18 ( d , j = 8.4 hz , 2.8 h ) , 7.13 ( s , 4 h ) , 7.09 ( d , j = 7.8 hz , 2.8 h ) , 5.81 ( ddt , j = 7.2 , 10.2 , 17.4 hz , 1 h ) , 5.68 ( ddt , j = 6.6 , 9.6 , 16.8 hz , 1.4 h ) , 5.014.94 ( m , 4.8 h ) , 4.81 ( m , 1 h ) , 4.69 ( m , 1 h ) , 4.67 ( m , 2.8 h ) , 4.31 ( t , j = 5.4 hz , 1.4 h ) , 4.27 ( t , j = 6.6 hz , 1 h ) , 3.78 ( s , 1.4 h ) , 3.33 ( s , 1 h ) , 2.53 ( m , 2.4 h ) , 2.46 ( m , 1.4 h ) , 2.35 ( s , 3 h ) , 2.34 ( heavily overlapped , m , 1 h ) , 2.33 ( s , 4.2 h ) , 1.64 ( d , j = 0.6 hz , 3 h ) , 1.53 ( d , j = 0.6 hz , 4.2 h ) , 0.07 ( s , 12.6 h ) , 0.08 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) ( syn - s3-v , major ) 145.0 , 140.4 , 136.2 , 134.8 , 128.5 ( 2c ) , 126.5 ( 2c ) , 116.8 , 109.4 , 79.8 , 77.6 , 40.5 , 21.1 , 20.3 , 3.0 ; ( anti - s3-w , minor ) 144.4 , 139.3 , 136.9 , 135.7 , 128.8 ( 2 c ) , 127.5 ( 2 c ) , 116.3 , 109.8 , 78.8 , 75.2 , 43.1 , 21.2 , 20.4 , 3.2 ; ir ( film ) 3075 , 2957 , 1247 , 840 cm ; hrms ( ei ) m / z 288.1895 [ ( m ) , calcd for c18h28osi , 288.1909 ] . applying general procedure e to compound s2-g ( 1 g , 4.66 mmol , 1 equiv ) , benzaldehyde ( 544 mg , 5.13 mmol , 1.1 equiv ) , allyltrimethylsilane ( 586 mg , 5.13 mmol , 1.1 equiv ) , and ( tms)otf ( 170 l , 0.932 mmol , 0.2 equiv ) in ch2cl2 ( 47 ml ) for 1 h at 78 c afforded after workup and column chromatography ( 1% etoac in hexanes ) 1.08 g ( 90% ) of a mixture of anti / syn - s3-x ( 1.6:1 ) as a yellow oil . spectroscopic data for syn - s3-x : h nmr ( 500 mhz , cdcl3 ) 7.357.28 ( m , 4 h ) , 7.21 ( tt , j = 1.5 , 7 hz , 1 h ) , 5.72 ( dddd , j = 7 , 10 , 14 , 17 hz , 1 h ) , 4.98 ( m , 2 h ) , 4.56 ( t , j = 6 hz , 1 h ) , 3.86 ( q , j = 2.5 hz , 1 h ) , 2.572.44 ( m , 2 h ) , 1.70 ( d , j = 3 hz , 3 h ) , 0.12 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.1 , 134.6 , 127.8 , 126.9 , 126.7 , 116.9 , 83.4 , 81.1 , 77.6 , 62.3 , 40.7 , 3.7 , 3.7 ; ir ( neat ) 3072 , 3030 , 2959 , 2363 , 2335 , 1641 , 1452 , 1248 , 1057 , 843 cm ; hrms ( ei ) m / z 272.1594 [ ( m ) , calcd for c17h24osi , 272.1596 ] . spectroscopic data for anti - s3-x : h nmr ( 500 mhz , cdcl3 ) 7.30 ( m , 2 h ) , 7.25 ( m , 3 h ) , 5.75 ( dddd , j = 7 , 10.5 , 14 , 17.5 hz , 1 h ) , 5.014.93 ( m , 2 h ) , 4.67 ( t , j = 7 hz , 1 h ) , 3.45 ( q , j = 2.5 hz , 1 h ) , 2.54 ( m , 1 h ) , 2 . 36 ( m , 1 h ) , 1.87 ( d , j = 2.5 hz , 3 h ) , 0.05 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.8 , 135.2 , 128.1 , 127.4 , 127.3 , 116.2 , 82.8 , 79.8 , 77.3 , 60.3 , 42.6 , 3.9 , 4 ; ir ( neat ) 3076 , 2959 , 2361 , 2336 , 1653 , 1539 , 1456 , 1248 , 844 cm ; hrms ( ei ) m / z 272.1590 [ ( m ) , calcd for c17h24osi , 272.1596 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.5 g , 44% ( w / w ) in thf , 5.07 mmol , 1 equiv ) , the trichloroacetimidate of 3-methyl-1-phenylbut-3-en-1-ol ( 2.49 g , 8.11 mmol , 1.6 equiv ) , and ( tms)otf ( 47 l , 0.5 mmol , 0.1 equiv ) in hexane ( 28 ml ) afforded after column chromatography ( 2% ch2cl2 in hexanes ) a total of 847 mg ( 61% ) of syn / anti - s3-y ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-y : h nmr ( 500 mhz , cdcl3 ) 7.26 ( m , 4 h ) , 7.19 ( m , 1 h ) , 5.62 ( ddd , j = 7.0 , 10.5 , 17.0 hz , 1 h ) , 4.88 ( m , 1 h ) , 4.79 ( m , 1 h ) , 4.70 ( m , 1 h ) , 4.60 ( m , 1 h ) , 4.41 ( t , j = 6.5 hz , 1 h ) , 3.76 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.52 ( dd , a of abx system , j = 7.0 , 14.0 hz , 1 h ) , 2.26 ( dd , b of abx system , j = 6.5 , 14.0 hz , 1 h ) , 1.67 ( s , 3 h ) , 0.02 ( s , 9 h ) ; ir ( film ) 3065 , 2957 , 1245 , 841 cm ; hrms ( ei ) m / z 274.1741 [ ( m ) , calcd for c17h26osi , 274.1753 ] . spectroscopic data for anti - s3-y : h nmr ( 500 mhz , cdcl3 ) 7.34 ( m , 2 h ) , 7.28 ( m , 3 h ) , 5.76 ( ddd , j = 7.5 , 10.5 , 17.5 hz , 1 h ) , 5.06 ( dq , j = 1.0 , 10.0 hz , 1 h ) , 4.99 ( dt , j = 1.5 , 17.5 hz , 1 h ) , 4.75 ( m , 1 h ) , 4.67 ( m , 1 h ) , 4.55 ( dd , j = 6.0 , 8.0 hz , 1 h ) , 3.42 ( dt , j = 1.0 , 7.5 hz , 1 h ) , 2.52 ( dd , a of abx system , j = 8.0 , 13.5 hz , 1 h ) , 2.29 ( dd , b of abx system , j = 5.0 , 13.5 hz , 1 h ) , 1.76 ( s , 3 h ) , 0.00 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.0 , 142.8 , 137.5 , 128.0 ( 2 c ) , 127.3 ( 2 c ) , 127.27 , 113.0 , 112.6 , 78.7 , 72.8 , 47.0 , 23.3 , 4.0 ; ir ( film ) 3076 , 2959 , 1247 , 840 cm ; hrms ( ei ) m / z 274.1745 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 0.97 g , 85.5% ( w / w ) in thf , 6.35 mmol , 1 equiv ) , trichloroacetimidate s1-z ( 2.85 g , 8.89 mmol , 1.4 equiv ) , and ( tms)otf ( 57 l , 0.317 mmol , 0.05 equiv ) in hexane ( 35 ml ) afforded after column chromatography ( 5% and 30% ch2cl2 in hexanes ) a total of 1.2 g ( 65% ) of syn / anti - s3-z ( 1:1 ) as a colorless oil . spectroscopic data for syn - s3-z : h nmr ( 500 mhz , cdcl3 ) 7.16 ( d , j = 7.0 hz , 2 h ) , 7.07 ( d , j = 7.5 hz , 2 h ) , 5.64 ( dddd , j = 1.0 , 7.0 , 10.5 , 17.0 hz , 1 h ) , 4.90 ( dq , j = 1.5 , 17.0 hz , 1 h ) , 4 . 80 ( m , 1 h ) , 4.70 ( m , 1 h ) , 4.61 ( m , 1 h ) , 4.39 ( t , j = 7.0 hz , 1 h ) , 3.75 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.51 ( dd , a of abx system , j = 7.0 , 14.0 hz , 1 h ) , 2.31 ( s , 3 h ) , 2.25 ( dd , b of abx system , j = 6.5 , 13.5 hz , 1 h ) , 1.67 ( d , j = 1.0 hz , 3 h ) , 0.02 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.6 , 141.0 , 138.2 , 136.4 , 128.5 ( 2 c ) , 126.6 ( 2 c ) , 113.0 , 111.6 , 80.5 , 76.0 , 46.1 , 23.1 , 21.1 , 3.7 ; ir ( film ) 3079 , 2961 , 1248 , 1060 , 841 cm ; hrms ( ei ) m / z 288.1900 [ ( m ) , calcd for c18h28osi , 288.1909 ] . spectroscopic data for anti - s3-z : h nmr ( 500 mhz , cdcl3 ) 7.11 ( m , 4 h ) , 5.72 ( ddd , j = 8.0 , 11.0 , 17.5 hz , 1 h ) , 5.00 ( ddd , j = 1.0 , 2.0 , 10.5 hz , 1 h ) , 4.95 ( ddd , j = 1.0 , 2.0 , 17.0 hz , 1 h ) , 4.71 ( m , 1 h ) , 4.64 ( m , 1 h ) , 4.49 ( dd , j = 5.0 , 8.0 hz , 1 h ) , 3.39 ( dt , j = 1.0 , 7.5 hz , 1 h ) , 2.46 ( dd , a of abx system , j = 9.0 , 13.5 hz , 1 h ) , 2.33 ( s , 3 h ) , 2.23 ( dd , b of abx system , j = 5.5 , 14.0 hz , 1 h ) , 1.72 ( m , 3 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.2 , 139.7 , 137.6 , 136.8 , 128.8 ( 2 c ) , 127.2 ( 2 c ) , 112.9 , 112.5 , 78.4 , 72.6 , 47.0 , 23.3 , 21.2 , 4.0 ; ir ( film ) 3076 , 2961 , 1248 , 1053 , 841 cm ; hrms ( ei ) m / z 288.1909 [ ( m ) , calcd for c18h28osi , 288.1909 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 1.03 g , 77.4% ( w / w ) in thf , 6.14 mmol , 1 equiv ) , trichloroacetimidate s1-aa ( 2.75 g , 9.21 mmol , 1.5 equiv ) , and ( tms)otf ( 55.5 l , 0.307 mmol , 0.05 equiv ) in hexane ( 34 ml ) afforded after column chromatography ( hexanes ) a total of 982 mg ( 60% ) of syn / anti - s3-aa ( 1:1 ) as a colorless oil . compounds syn- and anti - s3-aa were separable by column chromatography . spectroscopic data for syn - s3-aa : h nmr ( 500 mhz , cdcl3 ) 7.18 ( dd , j = 1.0 , 5.0 hz , 1 h ) , 6.91 ( dd , j = 3.0 , 5.0 hz , 1 h ) , 6.86 ( m , 1 h ) , 5.805.70 ( m , 2 h ) , 5.074.96 ( m , 3 h ) , 4.89 ( dt , j = 1.5 , 10.5 hz , 1 h ) , 4.64 ( t , j = 6.0 hz , 1 h ) , 3.83 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.632.50 ( m , 2 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 147.6 , 137.5 , 134.3 , 126.1 , 124.0 , 123.6 , 117.3 , 112.3 , 76.5 , 75.7 , 41.4 , 3.86 ; ir ( neat ) 3076 , 2957 , 1248 , 1062 , 912 , 841 cm ; hrms ( ei ) m / z 266.1148 [ ( m ) , calcd for c14h22osis , 266.1161 ] . spectroscopic data for anti - s3-aa : h nmr ( 500 mhz , cdcl3 ) 7.23 ( m , 1 h ) , 6.92 ( dd , j = 3.5 , 5.0 hz , 1 h ) , 6.87 ( m , 1 h ) , 5.79 ( dddd , j = 7.0 , 10.0 , 14.0 , 17.0 hz , 1 h ) , 5.71 ( ddd , j = 7.5 , 10.5 , 17.5 hz , 1 h ) , 5.04 ( m , 1 h ) , 5.01 ( m 2 h ) , 4.98 ( m , 1 h ) , 4.69 ( t , j = 7.0 hz , 1 h ) , 3.56 ( dt , j = 1.5 , 7.5 hz , 1 h ) , 2.61 ( m , 1 h ) , 2.45 ( m , 1 h ) 0.03 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 146.5 , 137.4 , 134.9 , 126.0 , 125.5 , 124.8 , 116.8 , 113.1 , 74.7 , 72.8 , 43.4 , 4.0 ; ir ( neat ) 3076 , 2957 , 1248 , 914 , 843 cm ; hrms ( ei ) m / z 266.1153 [ ( m ) , calcd for c14h22osis , 266.1161 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 990 mg , 77.4% ( w / w ) in thf , 5.9 mmol , 1 equiv ) , trichloroacetimidate s1-bb ( 2.17 g , 7.67 mmol , 1.3 equiv ) , and ( tms)otf ( 27 l , 0.147 mmol , 0.025 equiv ) in hexane ( 33 ml ) afforded after column chromatography ( hexanes ) a total of 720 mg ( 49% ) of syn / anti - s3-bb ( 1:1 ) as a colorless oil . compounds syn- and anti - s3-bb were separable by column chromatography . spectroscopic data for syn - s3-bb : h nmr ( 500 mhz , cdcl3 ) 7.32 ( m , 1h ) , 6.27 ( ddd , j = 0.5 , 2.0 , 3.0 hz , 1 h ) , 6.19 ( m , 1 h ) , 5.75 ( dddd , j = 7.0 , 10.5 , 14.0 , 17.5 hz , 1 h ) , 5.69 ( ddd , j = 7.0 , 10.5 , 17.0 hz , 1 h ) , 5.04 ( ddt , j = 1.5 , 2.0 , 17.0 hz , 1 h ) , 4.99 ( m , 1 h ) , 4.92 ( ddd , j = 1.5 , 2.0 , 17.5 hz , 1 h ) , 4.83 ( ddd , j = 1.0 , 2.0 , 11.0 hz , 1 h ) , 4.33 ( t , j = 6.5 hz , 1 h ) , 3.73 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.642.52 ( m , 2 h ) , 0.01 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 155.5 , 141.4 , 137.6 , 134.5 , 117.0 , 111.4 , 109.9 , 107.2 , 76.0 , 75.1 , 38.0 , 3.89 ; ir ( neat ) 3078 , 2959 , 1248 , 841 cm ; hrms ( ei ) m / z 250.1381 [ ( m ) , calcd for c14h22o2si , 250.1389 ] . spectroscopic data for anti - s3-bb : h nmr ( 500 mhz , cdcl3 ) 7.35 ( m , 1 h ) , 6.29 ( dd , j = 2.0 , 3.0 hz , 1 h ) , 6.17 ( dd , j = 1.0 , 3.5 hz , 1 h ) , 5.75 ( m , 2 h ) , 5.054.95 ( m , 4 h ) , 4.40 ( t , j = 7.0 hz , 1 h ) , 3.51 ( dt , j = 1.5 , 7.0 hz , 1 h ) , 2.60 ( m , 1 h ) , 2.52 ( m , 1 h ) , 0.07 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 154.8 , 141.9 , 137.3 , 134.7 , 116.7 , 112.4 , 109.7 , 108.0 , 73.1 , 72.7 , 39.4 , 4.2 ; ir ( neat ) 3078 , 2957 , 1248 , 841 cm ; hrms ( ei ) m / z 250.1379 [ ( m ) , calcd for c14h22o2si , 250.1389 ] . applying general procedure d to -(trimethylsilyl)allyl alcohol ( 2.3 g , solution 44% ( w / w ) in thf , 11.51 mmol , 1 equiv ) , trichloroacetimidate s1-cc ( 6.8 g , 18.4 mmol , 1.6 equiv ) , and ( tms)otf ( 208 l , 1.151 mmol , 0.1 equiv ) in hexane ( 64 ml ) afforded after column chromatography ( hexanes ) 1.425 g ( 37% ) of syn / anti - s3-cc ( 1:1 ) as a colorless oil : mixture of diastereomers ( syn : anti - s3-cc = 1:1 ) ; h nmr ( 600 mhz , cdcl3 ) 7.42 ( m , 2 h ) , 7.39 ( m , 2 h ) , 7.14 ( m , 2 h ) , 7.10 ( m , 2 h ) , 5.765.59 ( m , 4 h ) , 5.014.93 ( m , 6 h ) , 4.87 ( dt , j = 1.8 , 16.8 hz , 1 h ) , 4.83 ( dt , j = 1.8 , 10.8 hz , 1 h ) , 4.38 ( dd , j = 6.0 , 7.8 hz , 1 h ) , 4.32 ( t , j = 6.0 hz , 1 h ) , 3.77 ( dt , j = 1.8 , 7.2 hz , 1 h ) , 3.35 ( dt , j = 1.1 , 7.8 hz , 1 h ) , 2.49 ( m , 2 h ) , 2.40 ( m , 1 h ) , 2.35 ( m , 1 h ) , 0.03 ( s , 9 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.6 , 141.5 , 137.6 , 137.3 , 134.8 , 134.2 , 131.3 ( 2 c ) , 131.0 ( 2 c ) , 129.1 ( 2 c ) , 128.3 ( 2 c ) , 121.1 , 120.6 , 117.3 , 116.8 , 113.1 , 112.1 , 80.3 , 78.6 , 76.0 , 73.1 , 42.8 , 41.3 , 3.8 , 4.0 ; ir ( film ) 3078 , 2957 , 1487 , 1246 , 1070 , 1010 , 841 cm ; hrms ( ei ) m / z 338.0712 [ ( m ) , calcd for c16h23osibr , 338.0702 ] . applying general procedure f to syn - s3-a ( 184 mg , 0.707 mmol ) and grubbs second - generation catalyst ( 24 mg , 0.028 mmol , 0.04 equiv ) in ch2cl2 for 3 h followed by column chromatography ( 30% ch2cl2 in hexanes ) afforded 151 mg ( 92% ) of 1a as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.387.24 ( m , 5 h ) , 5.835.76 ( m , 2 h ) , 4.72 ( t , j = 5.5 hz , 1 h ) , 4.01 ( m , 1 h ) , 2.412.38 ( m , 2 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.3 , 128.5 ( 2c ) , 128.4 , 127.5 , 126.9 ( 2c ) , 120.3 , 72.6 , 70.4 , 30.4 , 2.7 ; hrms ( ci ) m / z 261.1664 [ ( m + h ) , calcd for c16h24osi , 261.1675 ] . the relative stereochemistry of 1a was confirmed on the basis of negative noesy signals between protons at 4.72 and 4.01 ppm . following general procedure f , to anti - s3-a ( 167 mg , 0.641 mmol ) in ch2cl2 ( 10 ml , 0.7 m ) was added grubbs second - generation catalyst ( 21.4 mg , 0.025 mmol , 0.04 equiv ) , and the solution was stirred under nitrogen at room temperature for 3 h. the reaction mixture was concentrated and purified by column chromatography ( 10% ch2cl2 in hexanes ) to afford 144 mg ( 97% ) of 2a as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.347.22 ( m , 5 h ) , 5.825.78 ( m , 2 h ) , 4.38 ( dd , j = 3.5 , 10 hz , 1 h ) , 4.174.15 ( m , 1 h ) , 2.262.12 ( m , 2 h ) , 0.08 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 144.0 , 128.10 ( 2c ) , 128.06 , 126.9 , 125.6 ( 2c ) , 121.1 , 75.3 , 71.6 , 34.2 , 4.0 ; hrms ( ci ) m / z 261.1681 [ ( m + h ) , calcd for c16h24osi , 261.1675 ] . the relative stereochemistry of 2a was assigned on the basis of positive noesy signals between protons at 4.15 and 2.262.12 ppm . applying general procedure f to syn / anti - s3-b ( 2:1 ratio , 280 mg , 0.964 mmol , 1 equiv ) and second - generation grubbs catalyst ( 33 mg , 0.039 mmol , 0.04 equiv ) in ch2cl2 ( 10 ml ) for 3 h afforded after column chromatography ( 25% ch2cl2 in hexanes and 7% etoac in hexanes ) 153 mg ( 62% ) of 1b and 82 mg ( 31% ) of 2b as colorless oils . spectroscopic data for 1b : h nmr ( 600 mhz , cdcl3 ) 7.42 ( dd , j = 1.8 , 7.8 hz , 1 h ) , 7.23 ( t , j = 8.4 hz , 1 h ) , 6.95 ( tt , j = 0.6 , 7.2 hz , 1 h ) , 6.85 , ( d , j = 8.4 hz , 1 h ) , 5.82 ( m , 2 h ) , 5.02 ( dd , j = 4.2 , 8.4 hz , 1 h ) , 4.14 ( m , 1 h ) , 3.81 ( s , 3 h ) , 2.29 ( m , 2 h ) , 0.09 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 156.5 , 131.0 , 128.2 , 127.8 , 127.0 , 120.9 , 120.6 , 110.3 , 72.3 , 67.0 , 55.3 , 30.7 , 2.7 ; ir ( film ) 1493 , 1248 , 1049 , 839 cm ; hrms ( ei ) m / z 262.1388 [ ( m ) , calcd for c15h22o2si , 262.1389 ] . spectroscopic data for 2b : h nmr ( 600 mhz , cdcl3 ) 7.47 ( dd , j = 1.8 , 7.8 hz , 1 h ) , 7.21 ( dt , j = 1.8 , 7.8 hz , 1 h ) , 6.98 ( dt , j = 1.2 , 7.8 hz , 1 h ) , 6.83 ( d , j = 1.2 , 7.8 hz , 1 h ) , 5.80 ( m , 2 h ) , 4.72 ( dd , j = 2.4 , 10.2 hz , 1 h ) , 4.16 ( m , 1 h ) , 3.80 ( s , 3 h ) , 2.35 ( m , 1 h ) , 1.98 ( m , 1 h ) , 0.10 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 155.6 , 132.7 , 127.7 , 127.6 , 126.1 , 121.7 , 120.8 , 109.9 , 71.4 , 70.0 , 55.3 , 32.9 , 3.9 ; ir ( film ) 1493 , 1248 , 1049 , 841 cm ; hrms ( ei ) m / z 262.1382 [ ( m ) , calcd for c15h22o2si , 262.1389 ] . applying general procedure f to syn - s3-c ( 95.9 mg , 0.33 mmol , 1 equiv ) and second - generation grubbs catalyst ( 9.8 mg , 0.012 mmol , 0.035 equiv ) in ch2cl2 ( 3.5 ml ) for 3 h afforded after column chromatography ( 35% ch2cl2 in hexanes ) 74 mg ( 86% ) of 1c as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.23 ( m , 1 h ) , 6.94 ( m , 2 h ) , 6.79 ( m , 1 h ) , 5.79 ( m , 2 h ) , 4.69 ( t , j = 5.5 hz , 1 h ) , 4.03 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.39 ( m , 2 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 159.6 , 143.9 , 129.2 , 128.1 , 120.0 , 118.9 , 112.6 , 112.3 , 72.3 , 70.2 , 55.2 , 30.3 , 2.9 ; ir ( film ) 1248 , 1051 , 841 cm ; hrms ( ei ) m / z 262.1400 [ ( m ) , calcd for c15h22o2si , 262.1389 ] . applying general procedure f to anti - s3-c ( 109 mg , 0.375 mmol , 1 equiv ) and second - generation grubbs catalyst ( 17.7 mg , 0.021 mmol , 0.04 equiv ) in ch2cl2 ( 5.5 ml ) for 3 h afforded after column chromatography ( 30% ch2cl2 in hexanes ) 91 mg ( 92% ) of 2c as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.23 ( t , j = 8.0 hz , 1 h ) , 6.92 ( m , 2 h ) , 6.78 ( ddd , j = 0.5 , 2.5 , 8.0 hz , 1 h ) , 5.79 ( m , 2 h ) , 4.37 ( dd , j = 3.0 , 10.0 hz , 1 h ) , 4.15 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.23 ( m , 1 h ) , 2.15 ( m , 1 h ) , 0.08 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 159.5 , 145.8 , 129.1 , 128.0 , 121.1 , 118.0 , 112.2 , 111.4 , 75.2 , 71.6 , 55.1 , 34.1 , 4.0 ; ir ( film ) 1248 , 1049 , 841 cm ; hrms ( ei ) m / z 262.1394 [ ( m ) , calcd for c15h22o2si , 262.1389 ] . applying general procedure f to syn - s3-d ( 155 mg , 0.534 mmol , 1 equiv ) and second - generation grubbs catalyst ( 14 mg , 0.016 mmol , 0.03 equiv ) in ch2cl2 ( 6 ml ) for 3 h afforded after column chromatography ( 35% ch2cl2 in hexanes ) 126 mg ( 90% ) of 1d as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.30 ( d , j = 9.0 hz , 2 h ) , 6.87 ( d , j = 9.0 hz , 1 h ) , 5.80 ( m , 2 h ) , 4.70 ( t , j = 5.0 hz , 1 h ) , 3.98 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.40 ( m , 2 h ) , 0.10 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 158.8 , 134.2 , 128.1 , 127.9 , 120.0 , 113.6 , 71.9 , 69.6 , 55.2 , 30.0 , 3.0 ; ir ( film ) 1513 , 1248 , 1038 , 840 cm ; hrms ( ei ) m / z 262.1403 [ ( m ) , calcd for c15h22o2si , 262.1389 ] . applying general procedure f to anti - s3-d ( 149 mg , 0.513 mmol , 1 equiv ) and second - generation grubbs catalyst ( 17.4 mg , 0.021 mmol , 0.04 equiv ) in ch2cl2 ( 6 ml ) for 3 h afforded after column chromatography ( 25% ch2cl2 in hexanes ) 122 mg ( 91% ) of 2d as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.27 ( m , 2 h ) , 6.87 ( m , 2 h ) , 5.79 ( m , 2 h ) , 4.34 ( dd , j = 4.0 , 9.0 hz , 1 h ) , 4.16 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.18 ( m , 2 h ) , 0.08 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 158.7 , 136.3 , 128.1 , 126.9 , 121.2 , 113.5 , 75.0 , 71.7 , 55.2 , 34.1 , 4.0 ; ir ( film ) 1248 , 1072 , 1039 , 841 cm ; hrms ( ei ) m / z 262.1390 [ ( m ) , calcd for c15h22o2si , 262.1389 ] . applying general procedure f to syn / anti - s3-e ( 2:1 ratio , 234 mg , 0.8526 mmol , 1 equiv ) and second - generation grubbs catalyst ( 29 mg , 0.034 mmol , 0.04 equiv ) in ch2cl2 ( 9 ml ) for 3 h afforded after column chromatography ( 15% ch2cl2 in hexanes and 5% etoac in hexanes ) 114 mg ( 62% ) of 1e and 79 mg ( 30% ) of 2e as colorless oils . spectroscopic data for 1e : h nmr ( 500 mhz , cdcl3 ) 7.41 ( m , 1 h ) , 7.19 ( m , 3 h ) , 5.90 ( m , 1 h ) , 5.82 ( dq , j = 2.0 , 10.0 hz , 1 h ) , 4.99 ( t , j = 5.0 hz , 1 h ) , 3.82 ( quintet , j = 3.0 hz , 1 h ) , 2.542.46 ( m , 1 h ) , 2.42 ( s , 3 h ) , 2.402.34 ( m , 2 h ) , 0.11 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.4 , 136.3 , 130.3 , 128.2 , 127.2 , 126.6 , 125.5 , 120.4 , 69.7 ( d , j = 5.8 hz ) , 68.5 ( d , j = 3.2 hz ) , 29.5 , 19.4 ( d , j = 1.4 hz , ) , 3.3 ; ir ( film ) 3028 , 2955 , 1247 , 1052 , 840 cm ; hrms ( ei ) m / z 246.1444 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 2e : h nmr ( 500 mhz , cdcl3 ) 7.45 ( d , j = 7.5 hz , 1 h ) , 7.23 ( t , j = 7.5 hz , 1 h ) , 7.17 ( dt , j = 1.5 , 7.5 hz , 1 h ) , 7.13 ( d , j = 7.0 hz , 1 h ) , 5.84 ( m , 2 h ) , 4.56 ( dd , j = 4.5 , 8.5 hz , 1 h ) , 4.20 ( m , 1 h ) , 2.35 ( s , 3 h ) , 2.22 ( m , 2 h ) , 0.12 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.8 , 134.6 , 130.0 , 128.0 , 126.8 , 126.1 , 125.6 , 121.5 , 73.1 ( d , j = 3.9 hz , 1 h ) , 71.8 , 32.4 , 19.2 , 3.9 ; ir ( film ) 3027 , 2957 , 1247 , 1072 , 843 cm ; hrms ( ei ) m / z 246.1436 [ ( m ) , calcd for c15h22osi , 246.1440 ] . applying general procedure f to syn / anti - s3-f ( 2:1 ratio , 228 mg , 0.8307 mmol , 1 equiv ) and second - generation grubbs catalyst ( 24.8 mg , 0.029 mmol , 0.035 equiv ) in ch2cl2 ( 9 ml ) for 3 h afforded after column chromatography ( 15% ch2cl2 in hexanes and 5% etoac in hexanes ) 120 mg ( 59% ) of 1f and 60 mg ( 29% ) of 2f as colorless oils . spectroscopic data for 1f : h nmr ( 600 mhz , cdcl3 ) 7.23 ( t , j = 7.8 hz , 1 h ) , 7.19 ( m , 2 h ) , 7.09 ( d , j = 7.8 hz , 1 h ) , 5.81 ( m , 2 h ) , 4.69 ( t , j = 6.0 hz , 1 h ) , 4.07 ( m , 1 h ) , 2.39 ( m , 2 h ) , 2.36 ( s , 3 h ) , 0.12 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 142.2 , 137.8 , 128.09 , 128.06 , 128.0 , 127.2 , 123.6 , 120.1 , 72.5 , 70.4 , 30.5 , 21.5 , 2.9 ; ir ( film ) 3028 , 2917 , 1247 , 1055 , 840 cm ; hrms ( ei ) m / z 246.1440 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 2f : h nmr ( 600 mhz , cdcl3 ) 7.24 ( t , j = 7.8 hz , 1 h ) , 7.18 ( m , 2 h ) , 7.06 ( d , j = 7.8 hz , 1 h ) , 5.83 ( m , 2 h ) , 4.38 ( dd , j = 3.0 , 9.6 hz , 1 h ) , 4.19 ( m , 1 h ) , 2.37 ( s , 3 h ) , 2.272.17 ( m , 2 h ) , 0.12 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 144.0 , 128.1 ( 2 c ) , 127.7 , 126.4 , 122.7 , 121.2 , 75.5 , 71.6 , 34.1 , 21.5 , 3.9 ; ir ( film ) 3028 , 2917 , 1247 , 1074 , 873 cm ; hrms ( ei ) m / z 246.1436 [ ( m ) , calcd for c15h22osi , 246.1440 ] . applying general procedure f to syn / anti - s3-g ( 1.15:1 ratio , 228.8 mg , 0.834 mmol , 1 equiv ) and second - generation grubbs catalyst ( 28.3 mg , 0.033 mmol , 0.04 equiv ) in ch2cl2 ( 9 ml ) for 3 h afforded after column chromatography ( 10% ch2cl2 in hexanes and 6% etoac in hexanes ) 108 mg ( 53% ) of 1 g and 88 mg ( 43% ) of 2 g as colorless oils . spectroscopic data for 1 g : h nmr ( 500 mhz , cdcl3 ) 7.26 ( d , j = 7.5 hz , 2 h ) , 7.14 ( d , j = 8 hz , 2 h ) , 5.835.76 ( m , 2 h ) , 4.71 ( t , j = 5.5 hz , 1 h ) , 4.00 ( m , 1 h ) , 2.39 ( m , 2 h ) , 2.33 ( s , 3 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.1 , 136.8 , 128.9 ( 2 c ) , 128.1 , 126.6 ( 2 c ) , 120.1 , 72.2 , 69.8 , 30.1 , 21.1 , 2.9 ; ir ( film ) 3028 , 2955 , 1248 , 1053 , 841 cm ; hrms ( ei ) m / z 246.1452 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 2 g : h nmr ( 500 mhz , cdcl3 ) 7.23 ( d , j = 8.0 hz , 2 h ) , 7.13 ( d , j = 8.0 hz , 2 h ) , 5.825.77 ( m , 2 h ) , 4.36 ( dd , j = 4.0 , 9.5 hz , 1 h ) , 4.15 ( m , 1 h ) , 2.33 ( s , 3 h ) , 2.242.13 ( m , 2 h ) , 0.08 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.1 , 136.5 , 128.8 ( 2 c ) , 128.1 , 125.6 ( 2 c ) , 121.2 , 75.3 , 71.6 , 34.1 , 21.1 , 4.0 ; ir ( film ) 3028 , 2957 , 1248 , 1072 , 858 , 841 cm ; hrms ( ei ) m / z 246.1440 [ ( m ) , calcd for c15h22osi , 246.1440 ] . applying general procedure f to syn - s3-h ( 95 mg , 0.341 mmol , 1 equiv ) and second - generation grubbs catalyst ( 11.6 mg , 0.014 mmol , 0.04 equiv ) in ch2cl2 ( 4 ml ) for 3 h afforded after column chromatography ( 30% ch2cl2 in hexanes ) 75.2 mg ( 88% ) of 1h as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.34 ( dd , j = 5.5 , 9.0 hz , 2 h ) , 7.01 ( t , j = 9.0 hz , 2 h ) , 5.79 ( m , 2 h ) , 4.70 ( t , j = 5.5 hz , 1 h ) , 3.98 ( m , 1 h ) , 2.442.32 ( m , 2 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 162.0 ( d , j = 247.1 hz ) , 137.7 ( d , j = 3.0 hz ) , 128.3 ( d , j = 8.0 hz , 2 c ) , 128.2 , 119.8 , 114.9 ( d , j = 21.2 hz , 2 c ) , 71.6 , 69.8 , 30.1 , 3.0 ; ir ( film ) 3030 , 2957 , 2899 , 1510 , 1248 , 1055 , 841 cm ; hrms ( ei ) m / z 250.1177 [ ( m ) , calcd for c14h19osif , 250.1189 ] . applying general procedure g to anti - s3-h ( 93 mg , 0.334 mmol , 1 equiv ) and second - generation grubbs catalyst ( 11.3 mg , 0.013 mmol , 0.04 equiv ) in benzene ( 4.2 ml ) for 1 h at 80 c afforded after column chromatography ( 10% ch2cl2 in hexanes ) 77.5 mg ( 93% ) of 2h as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.30 ( m , 2 h ) , 7.00 ( m , 2 h ) , 5.80 ( m , 2 h ) , 4.37 ( dd , j = 3.5,10.0 hz , 1 h ) , 4.17 ( m , 1 h ) , 2.21 ( m , 1 h ) , 2.13 ( m , 1 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.9 ( d , j = 244.9 hz ) , 139.8 ( d , j = 3.5 hz ) , 128.1 , 127.2 ( d , j = 7.9 hz , 2 c ) , 121.0 , 114.9 ( d , j = 21.2 hz , 2 c ) , 74.8 , 71.7 , 34.2 , 4.0 ; ir ( film ) 3030 , 2959 , 2775 , 1512 , 1248 , 839 cm ; hrms ( ei ) m / z 250.1183 [ ( m ) , calcd for c14h19osif , 250.1189 ] . applying general procedure f to syn / anti - s3-i ( 1:1.4 ratio , 210 mg , 0.746 mmol , 1 equiv ) and second - generation grubbs catalyst ( 25.3 mg , 0.03 mmol , 0.04 equiv ) in ch2cl2 ( 8 ml ) for 3 h afforded after column chromatography ( 10% and 25% ch2cl2 in hexanes ) 75.5 mg ( 38% ) of 1i and 105.6 mg ( 53% ) of 2i as colorless oils . spectroscopic data for 1i : h nmr ( 500 mhz , cdcl3 ) 7.30 ( m , 4 h ) , 5.78 ( m , 2 h ) , 4.71 ( dd , j = 5.0 , 10.0 hz , 1 h ) , 3.97 ( m , 1 h ) , 2.42 ( m , 1 h ) , 2.34 ( m , 1 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.6 , 132.9 , 128.3 ( 2 c ) , 128.2 , 128.0 ( 2 c ) , 119.7 , 71.6 , 69.7 , 30.0 , 3.0 ; ir ( film ) 3030 , 2956 , 1492 , 1248 , 1090 , 1055 , 1015 , 841 cm ; hrms ( ei ) m / z 266.0890 [ ( m ) , calcd for c14h19osicl , 266.0894 ] . spectroscopic data for 2i : h nmr ( 500 mhz , cdcl3 ) 7.29 ( m , 4 h ) , 5.81 ( m , 2 h ) , 4.37 ( dd , j = 3.5 , 10.0 hz , 1 h ) , 4.17 ( m , 1 h ) , 2.22 ( m , 1 h ) , 2.12 ( m , 1 h ) , 0.10 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.5 , 132.6 , 128.2 ( 2 c ) , 128.1 , 127.0 ( 2 c ) , 120.9 , 74.7 , 71.7 , 34.0 , 4.0 ; ir ( film ) 3030 , 2957 , 2896 , 1490 , 1248 , 1088 , 1073 , 1014 , 841 cm ; hrms ( ei ) m / z 266.0883 [ ( m ) , calcd for c14h19osicl , 266.0894 ] . applying general procedure f to syn / anti - s3-j ( 1:4.1 ratio , 296 mg , 0.904 mmol , 1 equiv ) and second - generation grubbs catalyst ( 31 mg , 0.036 mmol , 0.04 equiv ) in ch2cl2 ( 9.5 ml ) for 3 h afforded after column chromatography ( 10% and 25% ch2cl2 in hexanes ) 50 mg ( 18% ) of 1j and 201 mg ( 74% ) of 2j as colorless oils . spectroscopic data for 1j : h nmr ( 500 mhz , cdcl3 ) 7.58 ( d , j = 8.0 hz , 2 h ) , 7.48 ( d , j = 8.0 hz , 2 h ) , 5.80 ( m , 2 h ) , 4.77 ( t , j = 5.0 hz , 1 h ) , 4.01 ( m , 1 h ) , 2.46 ( m , 1 h ) , 2.36 ( m , 1 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 146.2 , 129.4 ( q , j = 32.1 hz ) , 128.3 , 126.8 ( 2 c ) , 125.1 ( q , j = 3.9 hz , 2 c ) , 124.3 ( q , j = 270.8 hz ) , 119.6 , 71.7 , 70.0 , 30.1 , 3.1 ; ir ( neat ) 1325 , 1250 , 1126 , 1068 , 839 cm ; hrms ( ei ) m / z 300.1151 [ ( m ) , calcd for c15h19osif3 , 300.1157 ] . spectroscopic data for 2j : h nmr ( 500 mhz , cdcl3 ) 7.59 ( d , j = 8.0 hz , 2 h ) , 7.47 ( d , j = 8.0 hz , 1 h ) , 5.875.79 ( m , 2 h ) , 4.46 ( dd , j = 3.0 , 10.0 hz , 1 h ) , 4.19 ( m , 1 h ) , 2.27 ( m , 1 h ) , 2.14 ( m , 1 h ) , 0.12 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 147.9 , 129.2 ( q , j = 38.4 hz ) , 128.1 , 125.9 ( 2 c ) , 125.1 ( q , j = 4.7 hz , 2 c ) , 124.3 ( q , j = 324.3 hz ) , 120.7 , 74.9 , 71.7 , 34.0 , 4.1 ; ir ( neat ) 1325 , 1250 , 1165 , 1126 , 1068 , 841 cm ; hrms ( ei ) m / z 300.1157 [ ( m ) , calcd for c15h19osif3 , 300.1157 ] . applying general procedure f to syn / anti - s3-k ( 2:1 ratio , 161 mg , 0.478 mmol , 1 equiv ) and second - generation grubbs catalyst ( 16.2 mg , 0.0191 mmol , 0.04 equiv ) in ch2cl2 ( 5 ml ) for 3 h afforded after column chromatography ( 15% and 35% ch2cl2 in hexanes ) 24.6 mg ( 17% ) of 1k and 67 mg ( 46% ) of 2k as colorless oils . spectroscopic data for 1k : h nmr ( 500 mhz , cdcl3 ) 7.58 ( m , 4 h ) , 7.44 ( m , 4 h ) , 7.33 ( m , 1 h ) , 5.83 ( m , 2 h ) , 4.79 ( t , j = 6.0 hz , 1 h ) , 4.06 ( m , 1 h ) , 2.45 ( m , 2 h ) , 0.12 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.2 , 141.0 , 140.1 , 128.7 ( 2 c ) , 128.2 , 127.1 , 127.08 ( 2 c ) , 127.05 ( 2 c ) , 126.97 ( 2 c ) , 120.0 , 72.1 , 70.0 , 30.2 , 2.9 ; ir ( film ) 3028 , 2955 , 1248 , 1070 , 841 cm ; hrms ( ei ) m / z 308.1584 [ ( m ) , calcd for c20h24osi , 308.1596 ] . spectroscopic data for 2k : h nmr ( 500 mhz , cdcl3 ) 7.59 ( m , 4 h ) , 7.44 ( m , 4 h ) , 7.43 ( m , 1 h ) , 5.85 ( m , 2 h ) , 4.47 ( dd , j = 3.5 , 10.0 hz , 1 h ) , 4.22 ( s , 1 h ) , 2.332.20 ( m , 2 h ) , 0.12 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.1 , 141.1 , 139.9 , 128.7 ( 2 c ) , 128.1 , 127.1 ( 3 c ) , 126.9 ( 2 c ) , 126.1 ( 2 c ) , 121.1 , 75.2 , 71.7 , 34.1 , 4.0 ; ir ( film ) 3030 , 2957 , 1246 , 1070 , 841 cm ; hrms ( ei ) m / z 308.1594 [ ( m ) , calcd for c20h24osi , 308.1596 ] . applying general procedure g to syn - s3-l ( 102 mg , 0.328 mmol , 1 equiv ) and second - generation grubbs catalyst ( 11.2 mg , 0.013 mmol , 0.04 equiv ) in benzene ( 4.1 ml ) for 1 h at 80 c afforded after column chromatography ( 40% ch2cl2 in hexanes ) 83.4 mg ( 90% ) of 1l as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.81 ( m , 4 h ) , 7.54 ( dd , j = 1.8 , 8.4 hz , 1 h ) , 7.45 ( m , 2 h ) , 5.87 ( m , 1 h ) , 5.81 ( m , 1 h ) , 4.91 ( t , j = 6.0 hz , 1 h ) , 4.04 ( quintet , j = 3.0 hz , 1 h ) , 2.52 ( m , 2 h ) , 0.12 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 139.5 , 133.2 , 132.8 , 128.2 , 128.0 , 127.9 , 127.6 , 125.9 , 125.7 , 125.2 , 125.1 , 120.0 , 72.5 , 69.9 , 30.2 , 3.0 ; ir ( film ) 3028 , 2955 , 2897 , 1248 , 841 cm ; hrms ( ei ) m / z 282.1436 [ ( m ) , calcd for c18h22osi , 282.1440 ] . applying general procedure g to anti - s3-l ( 110 mg , 0.3543 mmol , 1 equiv ) and second - generation grubbs catalyst ( 12 mg , 0.014 mmol , 0.04 equiv ) in benzene ( 4.4 ml ) for 1 h at 80 c afforded after column chromatography ( 10% ch2cl2 in hexanes ) 93 mg ( 93% ) of 2l as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.81 ( m , 4 h ) , 7.48 ( dd , j = 1.5 , 8.5 hz , 1 h ) , 7.44 ( m , 2 h ) , 5.84 ( m , 2 h ) , 4.56 ( dd , j = 3.5 , 9.5 hz , 1 h ) , 4.23 ( m , 1 h ) , 2.342.22 ( m , 2 h ) , 0.12 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.4 , 133.3 , 132.8 , 128.1 , 127.9 , 127.8 , 127.6 , 125.8 , 125.5 , 124.3 , 124.1 , 121.1 , 75.5 , 71.7 , 34.0 , 4.0 ; ir ( film ) 3028 , 2957 , 2772 , 1246 , 1072 , 841 cm ; hrms ( ei ) m / z 267.1217 [ ( m ch3 ) , calcd for c17h19osi , 267.1205 ] . applying general procedure f to syn / anti - s3-m ( 13:1 ratio , 99 mg , 0.307 mmol , 1 equiv ) and second - generation grubbs catalyst ( 10 mg , 0.0123 mmol , 0.04 equiv ) in ch2cl2 ( 3.5 ml ) at room temperature afforded after column chromatography ( 10% and 30% ch2cl2 in hexanes ) 80.4 mg ( 89% ) of 1 m and 2.5 mg ( 3% ) of 2 m as colorless oils . spectroscopic data for 1 m : h nmr ( 500 mhz , cdcl3 ) 7.58 ( m , 2 h ) , 7.397.32 ( m , 7 h ) , 7.26 ( m , 1 h ) , 5.845.77 ( m , 2 h ) , 4.65 ( dd , j = 5.0 , 6.5 hz , 1 h ) , 4.29 ( m , 1 h ) , 2.422.31 ( m , 2 h ) 0.43 ( s , 3 h ) , 0.40 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.0 , 136.9 , 134.1 , 129.3 , 128.2 , 127.8 , 127.2 , 126.6 , 120.5 , 72.3 , 69.8 , 30.2 , 4.4 , 4.6 ; ir ( film ) 3071 , 2960 , 1113 , 724 cm ; hrms ( ei ) m / z 294.1436 [ ( m ) , calcd for c19h22osi , 294.1440 ] . applying general procedure f to anti - s3-m ( 119.8 mg , 0.3714 mmol , 1 equiv ) and second - generation grubbs catalyst ( 12.6 mg , 0.0148 mmol , 0.04 equiv ) in ch2cl2 ( 4 ml ) at room temperature afforded after column chromatography ( 20% ch2cl2 in hexanes ) 99 mg ( 91% ) of 2 m as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.68 ( m , 2 h ) , 7.437.38 ( m , 7 h ) , 7.31 ( t , j = 7 hz , 1 h ) , 5.84 ( m , 2 h ) , 4.48 ( m , 2 h ) , 2.332.19 ( m , 2 h ) , 0.47 ( s , 3 h ) , 0.45 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.9 , 136.7 , 134.2 , 129.2 , 128.1 , 127.8 , 127.7 , 127.0 , 125.6 , 121.5 , 75.5 , 71.3 , 34.0 , 5.1 , 5.9 ; ir ( film ) 3071 , 2959 , 1428 , 1115 , 724 cm ; hrms ( ei ) m / z 294.1440 [ ( m ) , calcd for c19h22osi , 294.1440 ] . applying general procedure g to syn / anti - s3-n ( 1:1.7 ratio , 312 mg , 0.811 mmol , 1 equiv ) and second - generation grubbs catalyst ( 27.5 mg , 0.032 mmol , 0.04 equiv ) in benzene ( 11.6 ml ) at room temperature afforded after column chromatography ( 20% and 30% ch2cl2 in hexanes ) 75 mg ( 26% ) of 1n and 101 mg ( 35% ) of 2n as colorless oils . spectroscopic data for 1n : h nmr ( 500 mhz , cdcl3 ) 7.63 ( m , 2 h ) , 7.59 ( m , 2 h ) , 7.497.23 ( m , 11 h ) , 5.875.80 ( m 2 h ) , 4.63 ( m , 2 h ) , 2.432.32 ( m , 2 h ) , 0.66 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.7 , 135.3 , 135.13 ( 2 c ) , 135.08 ( 2 c ) , 134.97 , 129.5 , 129.4 , 128.1 ( 2 c ) , 127.9 ( 2 c ) , 127.8 ( 2 c ) , 127.7 , 127.2 , 126.7 ( 2 c ) , 121.1 , 72.1 , 68.5 , 29.7 , 5.5 ; ir ( film ) 3071 , 2975 , 1111 , 724 cm ; hrms ( ei ) m / z 356.1579 [ ( m ) , calcd for c24h24osi , 356.1596 ] . spectroscopic data for 2n : h nmr ( 500 mhz , cdcl3 ) 7.65 ( m , 4 h ) , 7.417.30 ( m , 10 h ) , 7.24 ( m , 1 h ) , 5.80 ( m , 2 h ) , 4.76 ( m , 1 h ) , 4.49 ( dd , j = 3.0 , 10.0 hz , 1 h ) , 2.26 ( m , 1 h ) , 2.19 ( m , 1 h ) , 0.64 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.8 , 135.22 ( 2 c ) , 135.16 , 135.1 ( 2 c ) , 134.8 , 129.5 , 129.4 , 128.1 ( 2 c ) , 127.8 ( 2 c ) , 127.7 ( 2 c ) , 127.6 , 126.9 , 125.6 ( 2 c ) , 122.1 , 75.8 , 70.9 , 33.9 , 6.4 ; ir ( film ) 3069 , 2963 , 1427 , 788 , 696 cm ; hrms ( ei ) m / z 356.1587 [ ( m ) , calcd for c24h24osi , 356.1596 ] . applying general procedure f to syn - s3-o ( 73.5 mg , 0.243 mmol , 1 equiv ) and second - generation grubbs catalyst ( 8.3 mg , 0.01 mmol , 0.04 equiv ) in ch2cl2 ( 3 ml ) at room temperature afforded after column chromatography ( 25% ch2cl2 in hexanes ) 55.9 mg ( 84% ) of 1o as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.37 ( d , j = 7.2 hz , 2 h ) , 7.32 ( t , j = 7.2 hz , 2 h ) , 7.24 ( m , 1 h ) , 5.76 ( m , 2 h ) , 4.74 ( t , j = 5.4 hz , 1 h ) , 4.14 ( m , 1 h ) , 2.44 ( m , 1 h ) , 2.39 ( m , 1 h ) , 0.97 ( t , j = 7.8 hz , 9 h ) , 0.64 ( q , j = 7.8 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.0 , 128.9 , 128.1 ( 2 c ) , 127.1 , 126.6 ( 2 c ) , 119.4 , 72.3 , 67.7 , 30.0 , 7.5 , 2.6 ; ir ( film ) 3030 , 2955 , 1454 , 1072 cm ; hrms ( ei ) m / z 274.1737 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure g to anti - s3-o ( 102 mg , 0.337 mmol , 1 equiv ) and second - generation grubbs catalyst ( 11.4 mg , 0.013 mmol , 0.04 equiv ) in benzene ( 4.8 ml ) at 80 c for 1 h afforded after column chromatography ( 10% ch2cl2 in hexanes ) 88 mg ( 95% ) of 2o as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.33 ( m , 4 h ) , 7.24 ( m , 1 h ) , 5.84 ( m , 1 h ) , 5.76 ( m , 1 h ) , 4.39 ( dd , j = 3.5 , 9.5 hz , 1 h ) , 4.33 ( m , 1 h ) , 2.282.16 ( m , 2 h ) , 1.01 ( t , j = 8.0 hz , 9 h ) , 0.68 ( q , j = 8.0 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 144.2 , 128.7 , 128.1 ( 2 c ) , 126.9 , 125.6 ( 2 c ) , 120.7 , 75.7 , 70.2 , 34.1 , 7.5 , 1.9 ; ir ( film ) 3028 , 2953 , 1454 , 1072 cm ; hrms ( ei ) m / z 274.1746 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure f to syn - s3-p ( 174 mg , 0.4936 mmol , 1 equiv ) and second - generation grubbs catalyst ( 17 mg , 0.0197 mmol , 0.04 equiv ) in ch2cl2 ( 5 ml ) at room temperature for 3 h afforded after column chromatography ( 40% ch2cl2 in hexanes ) 137.4 mg ( 86% ) of 1p as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.57 ( m , 2 h ) , 7.397.34 ( m , 3 h ) , 7.24 ( t , j = 8.4 hz , 1 h ) , 6.89 ( m , 2 h ) , 6.80 ( m , 1 h ) , 5.835.77 ( m , 2 h ) , 4.62 ( t , j = 5.4 hz , 1 h ) , 4.30 ( m , 1 h ) , 3.79 ( s , 3 h ) , 2.402.31 ( m , 2 h ) , 0.42 ( s , 3 h ) , 0.39 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 159.5 , 143.7 , 136.9 , 134.1 ( 2 c ) , 129.3 , 129.1 , 127.8 ( 2 c ) , 127.7 , 120.5 , 118.9 , 112.7 , 112.2 , 72.2 , 69.9 , 55.1 , 30.2 , 4.4 , 4.6 ; ir ( film ) 3071 , 2954 , 1492 , 1244 , 814 cm ; hrms ( ei ) m / z 324.1536 [ ( m ) , calcd for c20h24o2si , 324.1546 ] . applying general procedure g to anti - s3-p ( 95 mg , 0.269 mmol , 1 equiv ) and second - generation grubbs catalyst ( 9.2 mg , 0.011 mmol , 0.04 equiv ) in benzene ( 3.8 ml ) at 80 c for 1 h afforded after column chromatography ( 30% ch2cl2 in hexanes ) 75.4 mg ( 87% ) of 2p as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.63 ( m , 2 h ) , 7.37 ( m , 3 h ) , 7.26 ( t , j = 7.8 hz , 1 h ) , 6.96 ( m , 1 h ) , 6.94 ( dd , j = 0.6 , 7.2 hz , 1 h ) , 6.81 ( ddd , j = 1.2 , 3.0 , 8.4 hz , 1 h ) , 5.79 ( m , 2 h ) , 4.42 ( m , 2 h ) , 3.82 ( s , 3 h ) , 2.25 ( m , 1 h ) , 2.16 ( m , 1 h ) , 0.41 ( s , 3 h ) , 0.39 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 159.5 , 145.6 , 136.7 , 134.2 ( 2 c ) , 129.2 , 129.1 , 127.7 , 127.6 ( 2 c ) , 121.5 , 118.0 , 112.4 , 111.2 , 75.3 , 71.3 , 55.1 , 33.9 , 5.2 , 5.9 ; ir ( film ) 3070 , 2959 , 1494 , 1249 , 815 cm ; hrms ( ei ) m / z 324.1546 [ ( m ) , calcd for c20h24o2si , 324.1546 ] . applying general procedure g to syn - s3-q ( 128 mg , 0.359 mmol , 1 equiv ) and second - generation grubbs catalyst ( 12.2 mg , 0.014 mmol , 0.04 equiv ) in benzene ( 5.1 ml ) at 80 c for 1 h afforded after column chromatography ( 25% ch2cl2 in hexanes ) 109 mg ( 92% ) of 1q as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.54 ( m , 2 h ) , 7.397.33 ( m , 3 h ) , 7.27 ( m , 2 h ) , 7.21 ( m , 2 h ) , 5.805.75 ( m , 2 h ) , 4.58 ( t , j = 5.4 hz , 1 h ) , 4.22 ( m , 1 h ) , 2.352.27 ( m , 2 h ) , 0.39 ( s , 3 h ) , 0.37 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 140.5 , 136.7 , 134.1 ( 2 c ) , 132.9 , 129.3 , 128.3 ( 2 c ) , 128.0 ( 2 c ) , 127.9 , 127.8 ( 2 c ) , 120.3 , 71.5 , 69.6 , 30.0 , 4.4 , 4.7 ; ir ( film ) 3068 , 2957 , 1490 , 1249 , 809 cm ; hrms ( ei ) m / z 328.1035 [ ( m ) , calcd for c19h21osicl , 328.1050 ] . applying general procedure g to syn - s3-r ( 94.6 mg , 0.281 mmol , 1 equiv ) and second - generation grubbs catalyst ( 9.5 mg , 0.012 mmol , 0.04 equiv ) in benzene ( 4 ml ) at 80 c for 1 h afforded after column chromatography ( 25% ch2cl2 in hexanes ) 71.9 mg ( 83% ) of 1r as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.29 ( m , 4 h ) , 5.75 ( m , 2 h ) , 4.71 ( t , j = 5.4 hz , 1 h ) , 4.09 ( m , 1 h ) , 2.44 ( m , 1 h ) , 2.32 ( m , 1 h ) , 0.96 ( t , j = 7.8 hz , 9 h ) , 0.62 ( q , j = 7.8 hz , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 140.5 , 132.8 , 129.0 , 128.3 ( 2 c ) , 128.0 ( 2 c ) , 119.1 , 71.6 , 67.4 , 29.8 , 7.4 , 2.5 ; ir ( film ) 3030 , 2953 , 2878 , 1493 , 1014 , 819 , 715 cm ; hrms ( ei ) m / z 308.1349 [ ( m ) , calcd for c17h25osicl , 308.1363 ] . applying general procedure g to syn - s3-s ( 99.5 mg , 0.282 mmol , 1 equiv ) and second - generation grubbs catalyst ( 9.6 mg , 0.0113 mmol , 0.04 equiv ) in benzene ( 4 ml ) at 80 c for 1 h afforded after column chromatography ( 25% ch2cl2 in hexanes ) 85.7 mg ( 94% ) of 1s as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.85 ( m , 4 h ) , 7.57 ( dd , j = 1.5 , 8.5 hz , 1 h ) , 7.49 ( m , 2 h ) , 5.85 ( m , 2 h ) , 4.96 ( t , j = 5.0 hz , 1 h ) , 4.20 ( m , 1 h ) , 2.632.52 ( m , 2 h ) , 1.03 ( t , j = 8.0 hz , 9 h ) , 0.70 ( dq , j = 1.5 , 8.0 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.4 , 133.2 , 132.7 , 129.0 , 128.1 , 127.8 , 127.6 , 125.8 , 125.6 , 125.3 , 125.1 , 119.4 , 72.4 , 67.6 , 29.9 , 7.5 , 2.6 ; ir ( film ) 3055 , 2953 , 2874 , 1458 , 1018 , 817 , 719 cm ; hrms ( ei ) m / z 324.1902 [ ( m ) , calcd for c21h28osi , 324.1909 ] . applying general procedure g to anti - s3-s ( 102.2 mg , 0.29 mmol , 1 equiv ) and second - generation grubbs catalyst ( 9.8 mg , 0.0116 mmol , 0.04 equiv ) in benzene ( 4.1 ml ) at 80 c for 1 h afforded after column chromatography ( 10% ch2cl2 in hexanes ) 74.8 mg ( 79% ) of 2s as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.81 ( m , 4 h ) , 7.49 ( dd , j = 1.0 , 8.0 hz , 1 h ) , 7.44 ( m , 2 h ) , 5.88 ( m , 1 h ) , 5.81 ( m , 1 h ) , 4.55 ( dd , j = 4.5 , 9.5 hz , 1 h ) , 4.39 ( m , 1 h ) , 2.31 ( m , 2 h ) , 1.04 ( t , j = 8.0 hz , 9 h ) , 0.70 ( dq , j = 1.5 , 8.0 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.6 , 133.3 , 132.7 , 128.7 , 128.0 , 127.8 , 127.6 , 125.8 , 125.5 , 124.3 , 124.1 , 120.7 , 75.9 , 70.3 , 34.0 , 7.5 , 1.9 ; ir ( film ) 3028 , 2951 , 2874 , 1458 , 1072 , 815 , 715 cm ; hrms ( ei ) m / z 324.1894 [ ( m ) , calcd for c21h28osi , 324.1909 ] . applying general procedure g to syn - s3-t ( 36 mg , 0.119 mmol , 1 equiv ) and second - generation grubbs catalyst ( 4 mg , 0.0048 mmol , 0.04 equiv ) in benzene ( 2.4 ml ) at 80 c for 1 h afforded after column chromatography ( 25% ch2cl2 in hexanes ) 27.9 mg ( 85% ) of 1 t as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.41 ( m , 1 h ) , 7.17 ( m , 3 h ) , 5.86 ( dq , j = 3.5 , 10.0 hz , 1 h ) , 5.81 ( m , 1 h ) , 4.97 ( t , j = 5.5 hz , 1 h ) , 3.90 ( quintet , j = 3.0 hz , 1 h ) , 2.722.60 ( m , 2 h ) , 2.422.31 ( m , 2 h ) , 1.61 ( m , 2 h ) , 0.98 ( t , j = 7.0 hz , 3 h ) , 0.07 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.6 , 139.3 , 129.2 , 128.1 , 127.2 , 126.9 , 125.6 , 120.5 , 69.4 , 69.1 , 34.6 , 30.7 , 24.9 , 14.3 , 3.1 ; ir ( film ) 3028 , 2959 , 2872 , 1248 . 839 cm ; hrms ( ei ) m / z 274.1755 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure g to anti - s3-t ( 57 mg , 0.188 mmol , 1 equiv ) and second - generation grubbs catalyst ( 6.4 mg , 0.0075 mmol , 0.04 equiv ) in benzene ( 3.8 ml ) at 80 c for 1 h afforded after column chromatography ( 12% ch2cl2 in hexanes ) 38.6 mg ( 75% ) of 2 t as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.43 ( dd , j = 2.0 , 7.0 hz , 1 h ) , 7.20 ( m , 2 h ) , 7.15 ( m , 1 h ) , 5.83 ( m , 2 h ) , 4.58 ( dd , j = 3.0 , 10.0 hz , 1 h ) , 4.18 ( m , 1 h ) , 2.62 ( m , 2 h ) , 2.25 ( m , 1 h ) , 2.17 ( m , 1 h ) , 1.63 ( m , 2 h ) , 0.99 ( t , j = 7.0 hz , 3 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.4 , 139.3 , 128.9 , 128.0 , 126.9 , 126.1 , 126.0 , 121.6 , 72.7 , 71.9 , 34.5 , 33.4 , 24.6 , 14.3 , 3.9 ; ir ( film ) 3028 , 2959 , 2872 , 1248 , 841 cm ; hrms ( ei ) m / z 274.1740 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure f to syn / anti - s3-u ( 1:1 ratio , 309 mg , 1.126 mmol , 1 equiv ) and second - generation grubbs catalyst ( 38 mg , 0.045 mmol , 0.04 equiv ) in ch2cl2 ( 13 ml ) at room temperature for 3 h afforded after column chromatography ( 10% and 25% ch2cl2 in hexanes ) 159 mg ( 57% ) of 1u and 100 mg of 2u ( 36% ) as colorless oils . spectroscopic data for 1u : h nmr ( 500 mhz , cdcl3 ) 7.367.30 ( m , 4 h ) , 7.24 ( tt , j = 1.5 , 6.5 hz , 1 h ) , 5.47 ( m , 1 h ) , 4.54 ( dd , j = 5.0 , 7.5 hz , 1 h ) , 4.01 ( d , j = 1.0 hz , 1 h ) , 2.332.29 ( m , 2 , h ) , 1.68 ( m , 3 h ) , 0.18 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.8 , 135.4 , 128.2 ( 2 c ) , 127.1 , 126.0 ( 2 c ) , 115.8 , 75.0 , 72.9 , 32.1 , 20.4 , 1.4 ; ir ( film ) 3029 , 2957 , 1250 , 839 cm ; hrms ( ei ) m / z 246.1440 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 2u : h nmr ( 500 mhz , cdcl3 ) 7.32 ( m , 4 h ) , 7.22 ( m , 1 h ) , 5.51 ( m , 1 h ) , 4.29 ( dd , j = 3.0 , 8.5 hz , 1 h ) , 4.05 ( m , 1 h ) , 2.20 ( m , 1 h ) , 2.10 ( m , 1 h ) , 1.66 ( m , 3 h ) , 0.13 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 144.0 , 135.3 , 128.1 ( 2 c ) , 126.8 , 125.6 ( 2 c ) , 117.0 , 75.0 , 74.4 , 34.2 , 20.0 , 2.6 ; ir ( film ) 3030 , 2957 , 1248 , 839 cm ; hrms ( ei ) m / z 246.1432 [ ( m ) , calcd for c15h22osi , 246.1440 ] . applying general procedure f to syn - s3-v ( 94.6 mg , 0.311 mmol , 1 equiv ) and second - generation grubbs catalyst ( 10.6 mg , 0.0124 mmol , 0.04 equiv ) in ch2cl2 ( 3.5 ml ) at room temperature for 3 h afforded after column chromatography ( 60% ch2cl2 in hexanes ) 61.5 mg ( 72% ) of 1v as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.28 ( m , 2 h ) , 6.87 ( m , 2 h ) , 5.47 ( m , 1 h ) , 4.49 ( dd , j = 4.5 , 9.0 hz , 1 h ) , 3.99 ( m , 1 h ) , 3.78 ( s , 3 h ) , 2.372.24 ( m , 2 h ) , 1.64 ( m , 3 h ) , 0.15 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 158.7 , 135.3 , 134.9 , 127.3 ( 2 c ) , 115.9 , 113.6 ( 2 c ) , 74.9 , 72.5 , 55.2 , 32.0 , 20.4 , 1.4 ; ir ( film ) 2959 , 1248 , 838 cm ; hrms ( ei ) m / z 276.1549 [ ( m ) , calcd for c16h24o2si , 276.1546 ] . applying general procedure f to anti - s3-v ( 116.9 mg , 0.3839 mmol , 1 equiv ) and second - generation grubbs catalyst ( 13 mg , 0.0154 mmol , 0.04 equiv ) in ch2cl2 ( 4 ml ) at room temperature for 3 h afforded after column chromatography ( 35% ch2cl2 in hexanes ) 91 mg ( 86% ) of 2v as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.25 ( m , 2 h ) , 6.85 ( m , 2 h ) , 5.49 ( m , 1 h ) , 4.23 ( dd , j = 3.0 , 10.8 hz , 1 h ) , 4.04 ( m , 1 h ) , 3.78 ( s , 3 h ) , 2.16 ( m , 1 h ) , 2.07 ( m , 1 h ) , 1.65 ( d , j = 1.2 hz , 3 h ) , 0.11 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 158.6 , 136.3 , 135.3 , 126.8 ( 2 c ) , 117.1 , 113.5 ( 2 c ) , 74.6 , 74.4 , 55.2 , 34.2 , 20.0 , 2.6 ; ir ( film ) 2964 , 1248 , 839 cm ; hrms ( ei ) m / z 276.1551 [ ( m ) , calcd for c16h24o2si , 276.1546 ] . applying general procedure g to syn / anti - s3-w ( 1.5:1 ratio , 159 mg , 0.551 mmol , 1 equiv ) and second - generation grubbs catalyst ( 17.6 mg , 0.021 mmol , 0.04 equiv ) in benzene ( 7 ml ) at 80 c for 1 h afforded after column chromatography ( 15% and 35% ch2cl2 in hexanes ) 86 mg ( 60% ) of 1w and 55 mg ( 38% ) of 2w as colorless oils . spectroscopic data for 1w : h nmr ( 600 mhz , cdcl3 ) 7.26 ( d , j = 8.4 hz , 2 h ) , 7.14 ( d , j = 7.8 hz , 2 h ) , 5.49 ( m , 1 h ) , 4.52 ( dd , j = 4.2 , 9.0 hz , 1 h ) , 4.02 m , 1 h ) , 2.34 ( s , 3 h ) , 2.31 ( m , 2 h ) , 1.65 ( m , 3 h ) , 0.16 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 139.8 , 136.7 , 135.3 , 128.9 ( 2 c ) , 125.9 ( 2 c ) , 115.9 , 74.9 , 72.8 , 32.2 , 21.1 , 20.4 , 1.4 ; ir ( film ) 3026 , 2959 , 1250 , 839 cm ; hrms ( ei ) m / z 260.1583 [ ( m ) , calcd for c16h24osi , 260.1596 ] . spectroscopic data for 2w : h nmr ( 600 mhz , cdcl3 ) 7.25 ( d , j = 7.8 hz , 2 h ) , 7.14 ( d , j = 7.8 hz , 2 h ) , 5.52 ( m , 1 h ) , 4,28 ( dd , j = 3.0 , 4.2 hz , 1 h ) , 4.07 ( m , 1 h ) , 2.34 ( s , 3 h ) , 2.21 ( m , 1 h ) , 2.12 ( m , 1 h ) , 1.68 ( m , 3 h ) , 0.15 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 141.1 , 136.4 , 135.3 , 128.7 ( 2 c ) , 125.6 ( 2 c ) , 117.1 , 74.8 , 74.3 , 34.2 , 21.1 , 20.0 , 2.6 ; ir ( film ) 3028 , 2963 , 1248 , 843 cm ; hrms ( ei ) m / z 260.1590 [ ( m ) , calcd for c16h24osi , 260.1596 ] . applying general procedure f to syn - s3-x ( 201 mg , 0.738 mmol , 1 equiv ) and second - generation grubbs catalyst ( 25 mg , 0.03 mmol , 0.04 equiv ) in ch2cl2 ( 8 ml ) at room temperature for 12 h afforded after column chromatography ( 4% etoac in hexanes ) 125 mg ( 62% ) of 1x as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.377.31 ( m , 4 h ) , 7.25 ( tt , j = 1.5 , 7 hz , 1 h ) , 5.83 ( m , 1 h ) , 4.92 ( s , 1 h ) , 4.82 ( s , 1 h ) , 4.65 ( q , j = 2.0 hz , 1 h ) , 4.56 ( dd , j = 6.0 , 8.5 hz , 1 h ) , 2.42 ( m , 2 h ) , 1.89 ( t , j = 0.5 , hz , 3 h ) , 0.10 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.8 , 141.5 , 140.5 , 128.3 ( 2 c ) , 127.3 , 125.8 ( 2 c ) , 117.8 , 112.3 , 72.8 , 71.8 , 32.7 , 21.4 , 0.8 ; ir ( neat ) 3089 , 3031 , 2955 , 2895 , 1450 , 1248 , 1028 , 839 cm ; hrms ( ei ) m / z 272.1590 [ ( m ) , calcd for c17h24osi , 272.1596 ] . applying general procedure f to anti - s3-x ( 291.7 mg , 1.07 mmol , 1 equiv ) and second - generation grubbs catalyst ( 74.4 mg added in two portions , 0.0154 mmol , 0.07 equiv ) in ch2cl2 ( 11 ml ) at room temperature for 27 h afforded after column chromatography ( 4% etoac in hexanes ) 141.8 mg ( 49% ) of 2x as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.34 ( m , 4 h ) , 7.24 ( tt , j = 1.5 , 7.0 hz , 1 h ) , 5.88 ( dt , j = 1.5 , 7.5 hz , 1 h ) , 4.85 ( s , 2 h ) , 4.63 ( t , j = 2,0 hz , 1 h ) , 4.28 ( dd , j = 3.0 , 10.0 hz , 1 h ) , 2.32 ( ddt , j = 3.0 , 7.0 , 17.0 hz , 1 h ) , 2.19 ( dddd , j = 2.5 , 4.0 , 12.5 , 16.5 hz , 1 h ) , 1.90 ( s , 3 h ) , 0.09 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 143.7 , 142.7 , 140.7 , 128.1 ( 2 c ) , 127.0 , 125.7 ( 2 c ) , 118.5 , 111.5 , 74.3 , 71.5 , 34.7 , 21.7 , 2.3 ; ir ( neat ) 3088 , 3030 , 2953 , 2895 , 1452 , 1246 , 1028 , 841 cm ; hrms ( ei ) m / z 272.1586 [ ( m ) , calcd for c17h24osi , 272.1596 ] . applying general procedure g to syn - s3-y ( 102 mg , 0.3716 mmol , 1 equiv ) and second - generation grubbs catalyst ( 12.6 mg , 0.015 mmol , 0.04 equiv ) in benzene ( 10 ml ) at 80 c for 1.5 h afforded after column chromatography ( 30% ch2cl2 in hexanes ) 58 mg ( 63% ) of 1y as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.377.31 ( m , 4 h ) , 7.26 ( tt , j = 2.0 , 7.5 hz , 1 h ) , 5.47 ( m , 1 h ) , 4.73 ( t , j = 5.5 hz , 1 h ) , 3.95 ( m , 1 h ) , 2.29 ( m , 2 h ) , 1.78 ( m , 3 h ) , 0.08 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 142.2 , 128.2 , 127.5 , 127.2 , 126.6 , 121.2 , 72.5 , 69.5 , 34.8 , 23.4 , 2.9 ; ir ( film ) 3030 , 2959 , 1248 , 1099 , 841 cm ; hrms ( ei ) m / z 246.1433 [ ( m ) , calcd for c15h22osi , 246.1440 ] . applying general procedure g to anti - s3-y ( 104.2 mg , 0.3796 mmol , 1 equiv ) and second - generation grubbs catalyst ( 13 mg , 0.0152 mmol , 0.04 equiv ) in benzene ( 5.4 ml ) at 80 c for 2 h afforded after column chromatography ( 10% ch2cl2 in hexanes ) 62 mg ( 67% ) of 2y as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.34 ( t , j = 7.5 hz , 2 h ) , 7.31 ( t , j = 7.5 hz , 2 h ) , 7.23 ( tt , j = 1.5 , 7.0 hz , 1 h ) , 5.47 ( d , j = 1.0 hz , 1 h ) , 4.38 ( dd , j = 3.5 , 10.0 hz , 1 h ) , 4.08 ( m , 1 h ) , 2.152.02 ( m , 2 h ) , 1.73 ( s , 3 h ) , 0.06 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 144.1 , 128.8 , 128.1 , 126.9 , 125.6 , 121 , 75.7 , 71.2 , 38.9 , 23.2 , 3.97 ; ir ( film ) 3035 , 2958 , 1248 , 1099 , 840 cm ; hrms ( ei ) m / z 246.1430 [ ( m ) , calcd for c15h22osi , 246.1440 ] . applying general procedure g to syn - s3-z ( 148.7 mg , 0.515 mmol , 1 equiv ) and second - generation grubbs catalyst ( 17.5 mg , 0.0206 mmol , 0.04 equiv ) in benzene ( 10.5 ml ) at 80 c for 1.5 h afforded after column chromatography ( 30% ch2cl2 in hexanes ) 110 mg ( 82% ) of 1z as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.23 ( d , j = 7.5 hz , 2 h ) , 7.13 ( d , j = 8.0 hz , 2 h ) , 5.44 ( m , 1 h ) , 4.69 ( t , j = 5.5 hz , 1 h ) , 3.92 ( quintet , j = 2.5 hz , 1 h ) , 2.33 ( s , 3 h ) , 2.27 ( m , 2 h ) , 1.77 ( m , 3 h ) , 0.06 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.1 , 136.8 , 128.9 ( 2 c ) , 127.6 , 126.6 ( 2 c ) , 121.2 , 72.4 , 69.3 , 34.7 , 23.5 , 21.1 , 3.0 ; ir ( film ) 3025 , 2959 , 2855 , 1248 , 841 cm ; hrms ( ei ) m / z 260.1583 [ ( m ) , calcd for c16h24osi , 260.1596 ] . applying general procedure g to anti - s3-z ( 122 mg , 0.4229 mmol , 1 equiv ) and second - generation grubbs catalyst ( 14.4 mg , 0.0169 mmol , 0.04 equiv ) in benzene ( 8.5 ml ) at 80 c for 1.5 h afforded after column chromatography ( 10% ch2cl2 in hexanes ) 66 mg ( 60% ) of 2z as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.24 ( d , j = 7.8 hz , 2 h ) , 7.13 ( d , j = 7.8 hz , 2 h ) , 5.46 ( d , j = 1.8 , hz , 1 h ) , 4.34 ( dd , j = 3.0 , 10.2 hz , 1 h ) , 4.07 ( m , 1 h ) , 2.33 ( s , 3 h ) , 2.11 ( m , 1 h ) , 2.02 ( dt , j = 3.0 , 16.8 hz , 1 h ) , 1.73 ( m , 3 h ) , 0.06 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 141.1 , 136.5 , 128.80 , 128.78 ( 2 c ) , 125.6 ( 2 c ) , 121.0 , 75.6 , 71.2 , 38.9 , 23.2 , 21.1 , 3.9 ; ir ( film ) 3019 , 2959 , 2766 , 1246 , 1101 , 841 cm ; hrms ( ei ) m / z 260.1602 [ ( m ) , calcd for c16h24osi , 260.1596 ] . applying general procedure f to syn / anti - s3-aa ( 2:1 ratio , 95 mg , 0.357 mmol , 1 equiv ) and second - generation grubbs catalyst ( 12 mg , 0.0143 mmol , 0.04 equiv ) in ch2cl2 ( 4 ml ) at room temperature for 3 h afforded after column chromatography ( 10% and 20% ch2cl2 in hexanes ) 53 mg ( 62% ) of 1aa and 27 mg ( 31% ) of 2aa as colorless oils . spectroscopic data for 1aa : h nmr ( 600 mhz , cdcl3 ) 7.23 ( dd , j = 1.8 , 5.4 hz , 1 h ) , 6.96 ( m , 1 h ) , 6.95 ( dd , j = 3.6 , 5.4 hz , 1 h ) , 5.77 ( m , 2 h ) , 5.05 ( t , j = 4.8 hz , 1 h ) , 4.00 ( m , 1 h ) , 2.58 ( m , 1 h ) , 2.42 ( m , 1 h ) , 0.08 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 145.5 , 128.3 , 126.3 , 124.7 , 124.5 , 119.3 , 68.5 , 68.1 , 30.2 , 3.3 ; ir ( neat ) 3031 , 2957 , 1248 , 1051 , 841 cm ; hrms ( ei ) m / z 238.0840 [ ( m ) , calcd for c12h18osis , 238.0848 ] . spectroscopic data for 2aa : h nmr ( 600 mhz , cdcl3 ) 7.21 ( dd , j = 1.8 , 5.4 hz , 1 h ) , 6.95 ( dd , j = 3.6 , 4.8 hz , 1 h ) , 6.92 ( m , 1 h ) , 5.815.74 ( m , 2 h ) , 4.64 ( t , j = 6.0 hz , 1 h ) , 4.18 ( m , 1 h ) , 2.33 ( m , 2 h ) , 0.07 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 147.6 , 128.1 , 126.2 , 124.1 , 122.4 , 120.4 , 71.96 , 71.94 , 33.9 , 4.1 ; ir ( neat ) 3030 , 2957 , 1248 , 1070 , 843 cm ; hrms ( ei ) m / z 238.0824 [ ( m ) , calcd for c12h18ossi , 238.0848 ] . applying general procedure f to syn - s3-bb ( 45.5 mg , 0.1817 mmol , 1 equiv ) and second - generation grubbs catalyst ( 6.1 mg , 0.007 mmol , 0.04 equiv ) in ch2cl2 ( 2 ml ) at room temperature for 3 h afforded after column chromatography ( 30% ch2cl2 in hexanes ) 33 mg ( 82% ) of 1bb as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.36 ( m , 1 h ) , 6.31 ( dd , j = 2.0 , 3.0 hz , 1 h ) , 6.26 ( m , 1 h ) , 5.76 ( m , 2 h ) , 4.86 ( t , j = 5.0 hz , 1 h ) , 3.86 ( m , 1 h ) , 2.492.37 ( m , 2 h ) , 0.07 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 154.7 , 141.9 , 128.1 , 119.2 , 109.9 , 107.1 , 68.1 , 66.4 , 27.7 , 3.4 ; ir ( neat ) 3030 , 2955 , 1248 , 1057 , 1012 , 841 cm ; hrms ( ei ) m / z 222.1081 [ ( m ) , calcd for c12h18o2si , 222.1076 ] . applying general procedure g to anti - s3-bb ( 108 mg , 0.431 mmol , 1 equiv ) and second - generation grubbs catalyst ( 14.6 mg , 0.017 mmol , 0.04 equiv ) in benzene ( 6 ml ) at 80 c for 1 h afforded after column chromatography ( 20% ch2cl2 in hexanes ) 83 mg ( 87% ) of 2bb as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.36 ( m , 1 h ) , 6.32 ( dd , j = 2.0 , 3.0 hz , 1 h ) , 6.23 ( m , 1 h ) , 5.77 ( m , 2 h ) , 4.42 ( dd , j = 3.0 , 5.5 hz , 1 h ) , 4.14 ( m , 1 h ) , 2.43 ( m , 1 h ) , 2.21 ( m , 1 h ) , 0.04 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 156.0 , 141.7 , 128.1 , 120.6 , 110.0 , 105.8 , 71.7 , 69.6 , 29.8 , 4.1 ; ir ( film ) 3030 , 2959 , 2776 , 1248 , 843 cm ; hrms ( ei ) m / z 222.1080 [ ( m ) , calcd for c12h18o2si , 222.1076 ] . applying general procedure h to 2a ( 67.2 mg , 0.289 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in cyclohexane , 0.65 ml , 3 equiv ) at 78 c for 3 h afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 40.4 mg ( 60% ) of 3a ( dr > 20:1 ) and 19.2 mg ( 29% ) of 4a ( dr > 20:1 ) as colorless oils . spectroscopic data for 3a : h nmr ( 300 mhz , cdcl3 ) 7.067.27 ( m , 5 h ) , 2.68 ( dd , j = 6.3 , 16.8 hz , 2 h ) , 1.65 ( dt , j = 5.1 , 9.3 hz , 1 h ) , 1.31 ( m , 1 h ) , 1 ( m , 1 h ) , 0.76 ( m , 1 h ) , 0.2 ( s , 9 h ) ; c nmr ( 62.8 mhz , cdcl3 ) 247.2 , 142.8 , 129.1 , 128.2 , 125.9 , 125.5 , 53.1 , 22.7 , 16.7 , 15.7 , 3.1 ; ir ( film ) 3028 , 2959 , 1711 , 1643 , 1604 , 1496 , 1250 , 846 cm ; hrms ( esi ) m / z 233.1362 [ ( m + h ) , calcd for c14h21osi , 233.1362 ] . spectroscopic data for 4a : h nmr ( 300 mhz , cdcl3 ) 7.197.31 ( m , 3 h ) , 7.38 ( m , 2 h ) , 5.98 ( dddd , j = 1.8 , 2.7 , 4.8 , 5.7 hz , 1 h ) , 5.76 ( dddd , j = 1.5 , 2.1 , 3.6 , 5.7 hz , 1 h ) , 3.43 ( dd , j = 8.4 , 10.5 hz , 1 h ) , 2.572.79 ( m , 2 h ) , 1.48 ( s , 1 h ) , 0.31 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.4 , 137.3 , 130.6 , 128.5 , 128.17 , 126.7 , 84.4 , 60 , 35.4 , 3.5 ; ir ( neat ) 3441 , 3059 , 2955 , 2928 , 2856 , 1496 , 1452 , 1246 cm ; hrms ( es ) m / z 215.1244 [ ( m oh ) , calcd for c14h19si , 215.1256 ] . applying general procedure h to 2b ( 76 mg , 0.2896 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in cyclohexane , 0.62 ml , 3 equiv ) at 78 c for 3 h afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 35.5 mg ( 47% ) of 3b ( dr = 18:1 ) as a colorless oil and 29.6 mg ( 39% ) of 4b ( dr > 20:1 ) as a white solid . spectroscopic data for 3b : h nmr ( 600 mhz , cdcl3 ) 6.92 ( ddd , j = 2.4 , 7.2 , 8.4 hz , 1 h ) , 6.66 ( m , 2 h ) , 6.62 ( d , j = 8.4 hz , 1 h ) , 2.84 ( dd , a of abx system , j = 6.0 , 16.8 hz , 1 h ) , 2.53 ( dd , b of abx system , j = 7.2 , 16.8 hz , 1 h ) , 1.91 ( dt , j = 4.8 , 8.4 hz , 1 h ) , 1.30 ( m , 1 h ) , 0.91 ( dt , j = 5.4 , 8.4 hz , 1 h ) , 0.72 ( dt , j = 5.4 , 9.0 hz , 1 h ) , 0.19 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 247.6 , 158.0 , 130.9 , 126.4 , 125.2 , 120.5 , 110.1 , 55.4 , 53.3 , 16.7 , 15.3 , 14.7 , 3.2 ; ir ( film ) 2955 , 1645 , 1248 , 1047 , 843 cm ; hrms ( esi ) 263.1464 [ ( m + h ) , calcd for c15h23o2si , 263.1467 ] . spectroscopic and melting point data for 4b : mp 4647 c ; h nmr ( 600 mhz , cdcl3 ) 7.37 ( dd , j = 1.8 , 7.8 hz , 1 h ) , 7.23 ( m , 1 h ) , 6.97 ( dt , j = 0.6 , 7.8 hz , 1 h ) , 6.88 ( dd , j = 1.2 , 8.4 hz , 1 h ) , 5.90 ( m , 1 h ) , 5.74 ( m , 1 h ) , 3.87 ( s , 1 h , ome ) , 3.87 ( m , 1 h , overlapped with ome ) , 3.84 ( s , 1 h ) , 2.83 ( m , a of abx system , 1 h ) , 2.54 ( dddd , b of abx system , j = 1.2 , 3.0 , 8.4 , 15.6 hz , 1 h ) , 0.31 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 158.0 , 136.9 , 129.2 , 128.7 , 127.9 , 127.7 , 121.1 , 110.6 , 84.0 , 55.6 , 52.7 , 35.3 , 3.9 ; hrms ( esi ) 245.1357 [ ( m oh ) , calcd for c15h21osi , 245.1362 ] . applying general procedure h to 1c ( 70.4 mg , 0.268 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.2 ml , 1.2 equiv ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 22.9 mg ( 33% ) of 3c ( dr = 17:1 ) and 31.1 mg ( 44% ) of 4c ( dr > 20:1 ) as colorless oils . spectroscopic data for 3c : h nmr ( 500 mhz , cdcl3 ) 7.14 ( t , j = 8.0 hz , 1 h ) , 6.66 ( m , 2 h ) , 6.60 ( m , 1 h ) , 2.74 ( dd , a of abx system , j = 6.5 , 17.0 hz , 1 h ) , 2.57 ( dd , b of abx system , j = 7.0 , 17.0 hz , 1 h ) , 1.62 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 1.31 ( m , 1 h ) , 0.97 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.74 ( dt , j = 5.5 , 9.0 hz , 1 h ) , 0.19 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.1 , 159.7 , 144.6 , 129.2 , 118.4 , 111.7 , 110.9 , 55.1 , 53.1 , 22.8 , 16.8 , 15.8 , 3.2 ; ir ( film ) 2957 , 1645 , 1250 , 1157 , 1047 , 844 cm ; hrms ( esi ) 263.1467 [ ( m + h ) , calcd for c15h23o2si , 263.1467 ] . spectroscopic data for 4c : h nmr ( 500 mhz , cdcl3 ) 7.20 ( t , j = 7.5 hz , 1 h ) , 6.97 ( m , 2 h ) , 6.77 ( m , 1 h ) , 5.96 ( m , 1 h ) , 5.76 ( m , 1 h ) , 3.41 ( dd , j = 8.0 10.0 hz , 1 h ) , 2.70 ( m , 1 h ) , 2.62 ( m , 1 h ) , 1.24 ( s , 1 h ) , 0.3 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 159.5 , 142.1 , 137.2 , 130.5 , 129.1 , 120.9 , 114.4 , 112.0 , 84.3 , 60.0 , 55.2 , 35.4 , 3.4 ; ir ( film ) 3452 , 3055 , 2952 , 1246 , 839 cm ; hrms ( esi ) 245.1360 [ ( m oh ) , calcd for c15h21osi , 245.1362 ] . applying general procedure h to 1d ( 82 mg , 0.312 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.23 ml , 1.2 equiv ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and etoac in hexanes ) 53.2 mg ( 65% ) of 3d ( dr = 15:1 ) as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.01 ( d , j = 8.5 hz , 2 h ) , 6.77 ( d , j = 8.5 hz , 2 h ) , 2.70 ( dd , a of abx system , j = 6.5 , 17.0 hz , 1 h ) , 2.60 ( dd , b of abx system , j = 7.0 , 17.0 hz , 1 h ) , 1.59 ( dt , j = 4.5 , 9.0 hz , 1 h ) , 1.22 ( m , 1 h ) , 0.89 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.68 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.18 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.7 , 157.7 , 134.8 , 127.1 ( 2 c ) , 113.8 ( 2 c ) , 55.3 , 53.2 , 22.0 , 16.1 , 15.0 , 3.1 ; ir ( film ) 2958 , 1644 , 843 cm ; hrms ( esi ) m / z 263.1466 [ ( m + h ) , calcd for c15h23o2si , 263.1467 ] . applying general procedure h to 1e ( 109 mg , 0.442 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.33 ml , 1.2 equiv ) in thf ( 5.5 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 86.6 mg ( 80% ) of 3e ( dr > 20:1 ) and 16.3 mg ( 15% ) of 4e ( dr > 20:1 ) as colorless oils . spectroscopic data for 3e : h nmr ( 600 mhz , cdcl3 ) 7.137.04 ( m , 4 h ) , 2.84 ( dd , a of abx system , j = 6.0 , 16.8 hz , 1 h ) , 2.63 ( dd , b of abx system , j = 7.2 , 16.8 hz , 1 h ) , 2.37 ( s , 3 h ) , 1.64 ( dt , j = 5.4 , 10.2 hz , 1 h ) , 1.34 ( m , 1 h ) , 0.92 ( dt , j = 5.4 , 9.0 hz , 1 h ) , 0.74 ( dt , j = 4.8 , 8.4 hz , 1 h ) , 0.22 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 247.3 , 140.2 , 137.5 , 129.5 , 125.8 , 125.74 , 125.73 , 53.2 , 20.9 , 19.7 , 14.5 , 13.6 , 3.2 ; ir ( film ) 3065 , 2958 , 1643 , 1249 , 847 cm ; hrms ( ei ) m / z 246.1432 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 4e : h nmr ( 500 mhz , cdcl3 ) 7.35 ( m , 1 h ) , 7.167.09 ( m , 3 h ) , 6.00 ( ddd , j = 2.0 , 2.5 , 5.5 hz , 1 h ) , 5.72 ( ddd , j = 1.5 , 2.5 , 6.0 hz , 1 h ) , 3.76 ( t , j = 9.0 hz , 1 h ) , 2.77 ( ddt , a of abx system , j = 2.5 , 9.5 , 16.5 hz , 1 h ) , 2.71 ( dddd , b of abx system , j = 1.5 , 2.5 , 8.5 , 16.5 hz , 1 h ) , 2.49 ( s , 3 h ) , 1.40 ( s , 1 h ) , 0.28 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.5 , 138.2 , 136.5 , 131.3 , 130.7 , 127.2 , 126.4 , 125.4 , 86.2 , 55.0 , 38.1 , 21.0 , 3.4 ; ir ( film ) 3440 , 3060 , 2958 , 1247 , 839 cm ; hrms ( esi ) 229.1411 [ ( m oh ) , calcd for c15h21si , 229.1413 ] . applying general procedure h to 1f ( 91.4 mg , 0.371 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.28 ml , 1.2 equiv ) in thf ( 4.6 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 53.5 mg ( 59% ) of 3f ( dr > 20:1 ) and 27.6 mg ( 30% ) of 4f ( dr > 20:1 ) as colorless oils . spectroscopic data for 3f : h nmr ( 600 mhz , cdcl3 ) 7.12 ( t , j = 7.2 hz , 1 h ) , 6.94 ( d , j = 7.2 hz , 1 h ) , 6.88 ( s , 1 h ) , 6.86 ( d , j = 7.8 hz , 1 h ) , 2.75 ( dd , a of abx system , j = 6.0 , 16.8 hz , 1 h ) , 2.58 ( dd , b of abx system , j = 7.2 , 16.8 hz , 1 h ) , 2.30 ( s , 3 h ) , 1.61 ( dt , j = 4.8 , 9.0 hz , 1 h ) , 1.30 ( m , 1 h ) , 0.97 ( dt , j = 5.4 , 8.4 hz , 1 h ) , 0.73 ( dt , j = 5.4 , 8.4 hz , 1 h ) , 0.19 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 247.2 , 142.7 , 137.8 , 128.1 , 126.6 , 126.3 , 122.9 , 53.1 , 22.6 , 21.4 , 16.6 , 15.7 , 3.2 ; ir ( film ) 3066 , 2958 , 1643 , 1249 , 844 cm ; hrms ( ei ) m / z 246.1434 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 4f : h nmr ( 600 mhz , cdcl3 ) 7.12 ( m , 1 h ) , 7.17 ( m , 2 h ) , 7.04 ( m , 1 h ) , 5.98 ( ddd , j = 1.8 , 2.4 , 5.4 hz , 1 h ) , 5.75 ( ddd , j = 1.8 , 2.4 , 6.0 hz , 1 h ) , 3.40 ( ddt , j = 7.8 , 10.2 hz , 1 h ) , 2.72 ( ddt , a of abx system , j = 2.4 , 10.8 , 16.2 hz , 1 h ) , 2.62 ( dddd , j = 1.8 , 3.0 , 8.4 , 16.2 hz , 1 h ) , 2.33 ( s , 3 h ) , 1.48 ( s , 1 h ) , 0.30 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 140.3 , 137.6 , 137.2 , 130.6 , 129.4 , 128.0 , 127.4 , 125.3 , 84.4 , 59.9 , 35.4 , 21.4 , 3.5 ; ir ( film ) 3440 , 2957 , 1490 , 1247 , 838 cm ; hrms ( esi ) 229.1401 [ ( m oh ) , calcd for c15h21si , 229.1413 ] . applying general procedure h to 1 g ( 70.6 mg , 0.2865 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.21 ml , 1.2 equiv ) in thf ( 3.6 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 60.3 mg ( 86% ) of 3 g spectroscopic data for 3 g : mixture of tautomers ( keto / enol = 1:0.06 ) ; h nmr ( 500 mhz , cdcl3 ) 7.04 ( d , j = 7.5 hz , 2.12 h ) , 6.97 ( d , j = 8.0 hz , 2.12 h ) , 4.52 ( d , j = 7.0 hz , 0.06 h ) , 4.40 ( s , 0.06 h ) , 2.74 ( dd , a of abx system , j = 6.0 , 16.0 hz , 1 h ) , 2.58 ( dd , b of abx system , j = 7.0 , 17.0 hz , 1 h ) , 1.84 ( m , 0.06 h ) , 1.68 ( m , 0.06 h ) , 1.61 ( dt , j = 5.0 , 9.0 hz , 1 h ) , 1.27 ( m , 1 h ) , 1.19 ( m , 0.06 h ) , 0.99 ( m , 0.06 h ) , 0.93 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.72 ( dt , j = 5.5 , 8.5 hz , 1 h ) , 0.19 ( s , 9 h ) , 0.12 ( s , 0.55 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.3 , 139.7 , 135.0 , 128.9 ( 2c ) , 125.9 ( 2c ) , 53.2 , 22.4 , 20.9 , 16.5 , 15.4 , 3.2 ; ir ( film ) 3060 , 2958 , 1643 , 1248 , 844 cm ; hrms ( ei ) m / z 246.1442 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 4 g : h nmr ( 500 mhz , cdcl3 ) 7.26 ( d , j = 8.0 hz , 2 h ) , 7.09 ( d , j = 7.5 hz , 2 h ) , 5.97 ( m , 1 h ) , 5.75 ( ddd , j = 1.5 , 2.0 , 6.0 hz , 1 h ) , 3.39 ( dd , j = 8.0 , 10.5 hz , 1 h ) , 2.70 ( m , 1 h ) , 2.59 ( m , 1 h ) , 2.32 ( s , 3 h ) , 0.30 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 137.2 , 136.2 , 130.6 , 128.8 ( 2 c ) , 128.3 ( 2 c ) , 84.4 , 59.7 , 35.5 , 21.0 , 3.4 ; ir ( film ) 3443 , 2957 , 1491 , 1247 , 839 cm ; hrms ( esi ) 229.1407 [ ( m oh ) , calcd for c15h21si , 229.1413 ] . applying general procedure h to 1h ( 60.3 mg , 0.24 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.18 ml , 1.2 equiv ) in thf ( 3 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% etoac in hexanes ) 39.5 mg ( 66% ) of 3h ( dr > 20:1 ) and 6.7 mg ( 11% ) of 4h ( dr > 20:1 ) as colorless oils . spectroscopic data for 3h : h nmr ( 500 mhz , cdcl3 ) 7.03 ( m , 2 h ) , 6.90 ( m , 2 h ) , 2.67 ( m , 2 h ) , 1.60 ( dt , j = 4.5 , 9.0 hz , 1 h ) , 1.23 ( m , 1 h ) , 0.90 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.72 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.18 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.1 , 161.1 ( d , j = 243.4 hz ) , 138.3 ( d , j = 3.2 hz ) , 127.5 ( d , j = 7.7 hz , 2 c ) , 114.9 ( d , j = 21.3 hz , 2 c ) , 53.0 , 22.1 , 16.3 , 15.2 , 3.2 ; ir ( film ) 3071 , 2959 , 1645 , 1512 , 1250 , 844 cm ; hrms ( ei ) m / z 250.1181 [ ( m ) , calcd for c14h19osif , 250.1189 ] . spectroscopic data for 4h : h nmr ( 500 mhz , cdcl3 ) 7.35 ( m , 2 h ) , 6.98 ( m , 2 h ) , 5.97 ( ddd , j = 2.0 , 3.0 , 5.5 hz , 1 h ) , 5.75 ( ddd , j = 1.5 , 2.5 , 6.0 hz , 1 h ) , 3.40 ( t , j = 8.5 hz , 1 h ) , 2.702.59 ( m , 2 h ) , 0.30 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 161.9 ( d , j = 245.7 hz ) , 137.4 , 136.2 , 130.5 , 129.8 ( d , j = 7.8 hz , 2 c ) , 114.8 ( d , j = 20.8 hz , 2 c ) , 84.2 , 59.2 , 35.6 , 3.4 ; ir ( film ) 3431 , 3059 , 2922 , 1510 , 839 cm ; hrms ( ei ) m / z 232.1085 [ ( m h2o ) , calcd for c14h17sif , 232.1084 ] . applying general procedure h to 1i ( 93 mg , 0.349 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.24 ml , 1.1 equiv ) in thf ( 4.4 ml ) at 78 c for 5 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 26 mg ( 28% ) of 3i ( dr = 15:1 ) and 60.4 mg ( 65% ) of 4i ( dr > 20:1 ) as colorless oils . spectroscopic data for 3i : h nmr ( 500 mhz , cdcl3 ) 7.18 ( d , j = 8.5 hz , 2 h ) , 6.99 ( d , j = 9.0 hz , 2 h ) , 2.66 ( d , j = 7.0 hz , 2 h ) , 1.59 ( dt , j = 5.0 , 9.0 hz , 1 h ) , 1.25 ( m , 1 h ) , 0.92 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.74 ( dt , j = 5.5 , 8.5 hz , 1 h ) , 0.18 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.0 , 141.4 , 131.1 , 128.3 ( 2 c ) , 127.4 ( 2 c ) , 53.0 , 22.2 , 16.7 , 15.5 , 3.2 ; ir ( film ) 3072 , 2960 , 1645 , 1496 , 1250 , 846 cm ; hrms ( ei ) m / z 266.0891 [ ( m ) , calcd for c14h19siocl , 266.0894 ] . spectroscopic data for 4i : h nmr ( 500 mhz , cdcl3 ) 7.31 ( m , 2 h ) , 7.24 ( m , 2 h ) , 5.96 ( ddd , j = 2.0 , 2.5 , 5.5 hz , 1 h ) , 5.74 ( ddd , j = 1.0 , 2.0 , 5.5 hz , 1 h ) , 3.38 ( dd , j = 8.0 , 10.0 hz , 1 h ) , 2.66 ( ddt , a of abx system , j = 2.0 , 10.0 , 16.0 hz , 1 h ) , 2.60 ( dddd , b of abx system , j = 1.5 , 3.0 , 8.0 , 16.0 hz , 1 h ) , 1.50 ( s , 1 h ) , 0.30 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 139.0 , 137.4 , 132.4 , 130.4 , 129.8 ( 2 c ) , 128.2 ( 2 c ) , 84.2 , 59.3 , 35.3 , 3.4 ; ir ( film ) 3443 , 3054 , 2957 , 1491 , 1247 , 838 cm ; hrms ( esi ) m / z 249.0867 [ ( m oh ) , calcd for c14h18sicl , 249.0866 ] . applying general procedure h to 1j ( 42 mg , 0.1378 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.13 ml , 1.5 equiv ) in thf 1.5 ml ) at 78 c for 30 min afforded after workup and column chromatography ( 10% and 15% etoac in hexanes ) 38 mg ( 90% ) of 4j ( dr > 20:1 ) as a colorless oil : h nmr ( 600 mhz , cdcl3 ) 7.53 ( m , 4 h ) , 5.99 ( ddd , j = 1.8 , 3.0 , 6.0 hz , 1 h ) , 5.77 ( ddd , j = 1.2 , 2.4 , 6.0 hz , 1 h ) , 3.48 ( dd , j = 7.8 , 9.6 hz , 1 h ) , 2.73 ( ddt , a of abx system , j = 2.4 , 10.8 , 16.2 hz , 1 h ) , 2.65 ( dddd , b of abx system , j = 1.2 , 3.0 , 7.8 , 15.6 hz , 1 h ) , 1.53 ( s , 1 h ) , 0.31 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 144.7 , 137.4 , 130.3 , 129.0 ( q , j = 32.3 hz ) , 128.8 ( 2 c ) , 124.9 ( q , j = 3.8 hz , 2 c ) , 124.3 ( q , j = 272.6 hz ) , 84.2 , 59.7 , 35.2 , 3.5 ; ir ( neat ) 3441 , 1327 , 1248 , 1165 , 1126 , 1070 , 843 cm ; hrms ( esi ) m / z 283.1138 [ ( m oh ) , calcd for c15h18sif3 , 283.1130 ] . applying general procedure h to 2k ( 66.8 mg , 0.2165 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in cyclohexane , 0.46 ml , 3 equiv ) in thf ( 2.4 ml ) at 78 c for 3 h afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 3.5 mg ( 5% ) of 3k ( dr = 7:1 ) as colorless oil and 50.4 mg ( 75% ) of 4k ( dr > 20:1 ) as a white solid . spectroscopic data for 3k : h nmr ( 500 mhz , cdcl3 ) 7.54 ( m , 2 h ) , 7.46 ( m , 2 h ) , 7.40 ( m , 2 h ) , 7.30 ( m , 1 h ) , 7.13 ( m , 2 h ) , 2.76 ( dd , a of abx system , j = 6.0 , 16.5 hz , 1 h ) , 2.62 ( dd , b of abx system , j = 7.0 , 17.0 hz , 1 h ) , 1.68 ( dt , j = 5.0 , 9.0 hz , 1 h ) , 1.35 ( m , 1 h ) , 1.01 ( dt , j = 5.5 , 8.5 hz , 1 h ) , 0.78 ( dt , j = 5.5 , 8.5 hz , 1 h ) , 0.20 ( s , 9 h ) . spectroscopic and melting point data for 4k : mp 7576 c ; h nmr ( 500 mhz , cdcl3 ) 7.62 ( m , 2 h ) , 7.56 ( m , 2 h ) , 7.48 ( m , 2 h ) , 7.43 ( m , 2 h ) , 7.33 ( m , 1 h ) , 6.01 ( ddd , j = 1.5 , 2.5 , 5.5 hz , 1 h ) , 5.80 ( ddd , j = 1.5 , 2.5 , 6.0 hz , 1 h ) , 3.49 ( dd , j = 8.0 , 10.0 hz , 1 h ) , 2.78 ( ddt , a of abx system , j = 2.0 , 10.0 , 16.0 hz , 1 h ) , 2.68 ( dddd , b of abx system , j = 1.0 , 3.0 , 8.0 , 16.0 hz , 1 h ) , 1.56 ( s , 1 h ) , 0.25 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.9 , 139.6 , 137.3 , 130.5 , 128.9 ( 2 c ) , 128.7 ( 2 c ) , 127.1 , 126.9 ( 2 c ) , 126.7 ( 2 c ) , 84.5 , 59.7 , 35.4 , 3.4 ( one aromatic carbon could not be located ) ; ir ( film ) 3442 , 3060 , 2955 , 1246 , 839 cm ; hrms ( ei ) m / z 290.1481 [ ( m h2o ) , calcd for c20h22si , 290.1491 ] . applying general procedure h to 1l ( 83.4 mg , 0.295 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.22 ml , 1.2 equiv ) in thf ( 3.7 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 2.6 mg ( 3% ) of 3l ( dr > 20:1 ) as a colorless oil and 79.9 mg ( 96% ) of 4l ( dr > 20:1 ) as a white solid . spectroscopic data for 3l : h nmr ( 600 mhz , cdcl3 ) 7.73 ( m , 3 h ) , 7.51 ( s , 1 h ) , 7.41 ( t , j = 7.8 hz , 1 h ) , 7.36 ( t , j = 7.8 hz , 1 h ) , 7.20 ( d , j = 8.4 hz , 1 h ) , 2.77 ( dd , a of abx system , j = 6.0 , 17.0 hz , 1 h ) , 2.67 ( dd , b of abx system , j = 6.6 , 16.8 hz , 1 h ) , 1.80 ( m , 1 h ) , 1.42 ( m , 1 h ) , 1.09 ( dt , j = 4.8 , 8.4 hz , 1 h ) , 0.82 ( m , 1 h ) ; c nmr ( 151 mhz , cdcl3 ) 247.2 , 140.3 , 133.5 , 131.9 , 127.9 , 127.6 , 127.3 , 125.9 , 125.0 , 124.9 , 124.0 , 53.2 , 23.0 , 16.8 , 15.7 , 3.1 , 3.5 ; ir ( film ) 3053 , 2957 , 1645 , 1250 , 844 cm ; hrms ( ei ) m / z 282.1429 [ ( m ) , calcd for c18h22osi , 282.1440 ] . spectroscopic and melting point data for 4l : mp 5860 c ; h nmr ( 600 mhz , cdcl3 ) 7.81 ( m , 3 h ) , 7.77 ( d , j = 9.0 hz , 1 h ) , 7.60 ( dd , j = 1.8 , 8.4 hz , 1 h ) , 7.44 ( m , 2 h ) , 6.04 ( ddd , j = 2.4 , 3.0 , 6.0 hz , 1 h ) , 5.82 ( dq , j = 1.2 , 6.0 hz , 1 h ) , 3.61 ( dd , j = 8.4 , 10.8 hz , 1 h ) , 2.89 ( ddt , a of abx system , j = 1.8 , 10.8 , 16.2 hz , 1 h ) , 2.73 ( dddd , b of abx system , j = 1.2 , 2.4 , 7.8 , 15.6 hz , 1 h ) , 1.60 ( s , 1 h ) , 0.31 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 138.2 , 137.5 , 133.3 , 132.5 , 130.5 , 128.0 , 127.7 , 127.6 , 127.5 , 126.1 , 125.9 , 125.4 , 84.4 , 60.1 , 35.5 , 3.3 ; ir ( film ) 3437 , 3055 , 2957 , 2855 , 1246 , 839 cm ; hrms ( ei ) m / z 264.1326 [ ( m h2o ) , calcd for c18h20si , 264.1334 ] . applying general procedure h to 1 m ( 80.4 mg , 0.273 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.26 ml , 1.5 equiv ) in thf ( 2.9 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 55 mg ( 69% ) of 3 m ( dr > 20:1 ) as a colorless oil and 6 mg ( 6% ) of 4 m ( dr > 20:1 ) contaminated with desilylated 3 m as a colorless oil . spectroscopic data for 3 m : h nmr ( 500 mhz , cdcl3 ) 7.53 ( m , 2 h ) , 7.427.36 ( m , 3 h ) , 7.21 ( m , 2 h ) , 7.11 ( m , 1 h ) , 7.00 ( m , 2 h ) , 2.70 ( dd , a of abx system , j = 6.5 , 17.0 hz , 1 h ) , 2.57 ( dd , b of abx system , j = 7.0 , 16.5 hz , 1 h ) , 1.54 ( dt , j = 4.5 , 9.0 hz , 1 h ) , 1.23 ( m , 1 h ) , 0.87 ( dt , j = 5.5 , 9.0 hz , 1 h ) , 0.64 ( dt , j = 5.5 , 9.0 hz , 1 h ) , 0.49 ( s , 3 h ) , 0.48 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 245.2 , 142.7 , 134.3 , 134.0 ( 2 c ) , 129.9 , 128.17 ( 2 c ) , 128.16 ( 2 c ) , 125.8 ( 2 c ) , 125.4 , 53.3 , 22.6 , 16.7 , 15.6 , 4.79 , 4.82 ; ir ( film ) 3089 , 2987 , 1642 , 1423 , 1113 , 698 cm ; hrms ( ei ) m / z 294.1440 [ ( m ) , calcd for c19h22osi , 294.1440 ] . spectroscopic data for a mixture of 4 m and desilylated 3 m ( 1:0.33 ) : h nmr ( 500 mhz , cdcl3 ) 9.82 ( m , 0.33 h ) , 7.30 ( m , 2.66 h ) , 7.267.20 ( m , 8 h ) , 7.14 ( m , 0.33 h ) , 7.07 ( m , 0.66 h ) , 5.98 ( m , 1 h ) , 5.70 ( m , 1 h ) , 3.44 ( t , j = 9.0 hz , 1 h ) , 2.592.48 ( m , 2.33 h ) , 2.43 ( m , 0.33 h ) , 1.75 ( m , 0.33 h ) , 1.55 ( s , 1 h ) , 1.32 ( m , 1 h ) , 1.06 ( m , 0.33 h ) , 0.87 ( m , 0.33 h ) , 0.00 ( s , 3 h ) , 0.11 ( s , 3 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.0 , 137.3 , 134.5 ( 3 c ) , 131.0 , 128.9 , 128.7 ( 2 c ) , 128.0 ( 2 c ) , 127.3 ( 2 c ) , 126.8 , 125.9 , 84.2 , 59.8 , 35.4 , 4.9 , 5.4 ; hrms ( esi ) m / z 277.1402 [ ( m oh ) , calcd for c19h21si , 277.1413 ] . applying general procedure h to 1n ( 70 mg , 0.196 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.15 ml , 1.2 equiv ) in thf ( 2.5 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% etoac in hexanes ) 48.6 mg ( 70% ) of 3n ( dr = 20:1 ) as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.57 ( m , 4 h ) , 7.43 ( m , 2 h ) , 7.37 ( m , 4 h ) , 7.20 ( t , j = 7.5 hz , 2 h ) , 7.11 ( tt , j = 1.5 , 7.5 hz , 1 h ) , 6.98 ( m , 2 h ) , 2.77 ( dd , a of abx system , j = 6.0 , 17.5 hz , 1 h ) , 2.66 ( dd , b of abx system , j = 7.0 , 17.0 hz , 1 h ) , 1.52 ( m , 1 h ) , 1.26 ( m , 1 h ) , 0.88 ( m , 1 h ) , 0.74 ( s , 3 h ) , 0.63 ( dt , j = 5.5 , 9.0 hz , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 243.6 , 142.8 , 135.0 ( 4 c ) , 132.61 , 132.59 , 130.1 ( 2 c ) , 128.22 ( 4 c ) , 128.18 ( 2 c ) , 125.9 ( 2 c ) , 125.4 , 54.1 , 22.6 , 16.7 , 15.6 , 5.3 ; ir ( film ) 3089 , 2990 , 1643 , 1429 , 1113 , 698 cm ; hrms ( ei ) m / z 356.1605 [ ( m ) , calcd for c24h24osi , 356.1596 ] . applying general procedure h to 1o ( 91.4 mg , 0.333 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.25 ml , 1.5 equiv ) in thf ( 4.2 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 85.2 mg ( 93% ) of 3o ( dr > 20:1 ) and 4 mg ( 5% ) of 4o ( dr > 20:1 ) as colorless oils . spectroscopic data for 3o : h nmr ( 600 mhz , cdcl3 ) 7.22 ( m , 2 h ) , 7.12 ( m , 1 h ) , 7.06 ( m , 2 h ) , 2.73 ( dd , a of abx system , j = 6.6 , 17.4 hz , 1 h ) , 2.56 ( dd , b of abx system , j = 7.2 , 16.8 hz , 1 h ) , 1.62 ( dt , j = 4.8 , 9.0 hz , 1 h ) , 1.33 ( m , 1 h ) , 0.96 ( m , heavily overlapped , 1 h ) , 0.96 ( t , j = 7.8 hz , 9 h ) , 0.73 ( m , heavily overlapped , 1 h ) , 0.73 ( q , j = 7.8 hz , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 247.0 , 142.9 , 128.2 ( 2 c ) , 125.9 ( 2 c ) , 125.4 , 54.8 , 22.6 , 16.5 , 15.7 , 7.2 , 2.1 ; ir ( film ) 3033 , 2952 , 1644 , 1011 , 730 cm ; hrms ( ei ) m / z 274.1753 [ ( m ) , calcd for c17h26osi , 274.1753 ] . spectroscopic data for 4o : h nmr ( 600 mhz , cdcl3 ) 7.40 ( d , j = 7.8 hz , 2 h ) , 7.28 ( t , j = 7.8 hz , 2 h ) , 7.23 ( m , 1 h ) , 5.95 ( m , 1 h ) , 5.83 ( m , 1 h ) , 3.40 ( t , j = 8.4 hz , 1 h ) , 2.74 ( m , 1 h ) , 2.64 ( m , 1 h ) , 1.41 ( s , 1 h ) , 0.81 ( t , j = 7.8 hz , 9 h ) , 0.27 ( dq , j = 4.8 , 7.8 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 140.1 , 138.1 , 130.5 , 128.5 ( 2 c ) , 128.1 ( 2 c ) , 126.8 , 85.9 , 60.6 , 35.6 , 7.8 , 2.2 ; ir ( film ) 3465 , 2951 , 2878 , 1012 , 729 cm ; hrms ( ei ) m / z 256.1641 [ ( m h2o ) , calcd for c17h24si , 256.1647 ] . applying general procedure h to 1p ( 75.4 mg , 0.232 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.17 ml , 1.2 equiv ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 41.3 mg ( 44% ) of 3p ( dr = 17:1 ) and 14.3 mg ( 20% ) of 4p ( dr > 20:1 ) . spectroscopic data for 3p : h nmr ( 600 mhz , cdcl3 ) 7.51 ( m , 2 h ) , 7.427.34 ( m , 3 h ) , 7.12 ( t , j = 7.8 hz , 1 h ) , 6.66 ( dd , j = 3.0 , 7.8 hz , 1 h ) , 6.58 ( d , j = 7.2 hz , 1 h ) , 6.52 ( t , j = 2.4 hz , 1 h ) , 3.75 ( s , 3 h ) , 2.69 ( dd , a of abx system , j = 6.0 , 16.8 hz , 1 h ) , 2.53 ( dd , b of abx system , j = 7.2 , 17.4 hz , 1 h ) , 1.51 ( dt , j = 4.8 , 9.0 hz , 1 h ) , 1.22 ( m , 1 h ) , 0.86 ( dt , j = 5.4 , 8.4 hz , 1 h ) , 0.62 ( dt , j = 5.4 , 8.4 hz , 1 h ) , 0.48 ( s , 3 h ) , 0.47 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 245.2 , 159.6 , 144.5 , 134.3 , 134.0 ( 2 c ) , 129.9 , 129.2 , 128.2 ( 2 c ) , 118.3 , 111.5 , 110.9 , 55.1 , 53.2 , 22.7 , 16.8 , 15.8 , 4.80 , 4.83 ; ir ( film ) 2957 , 1645 , 844 cm ; hrms ( ei ) m / z 324.1546 [ ( m ) , calcd for c20h24o2si , 324.1546 ] . spectroscopic data for 4p : h nmr ( 600 mhz , cdcl3 ) 7.30 ( m , 3 h ) , 7.23 ( m , 2 h ) , 7.14 ( t , j = 7.8 hz , 1 h ) , 6.87 ( m , 1 h ) , 6.79 ( s , 1 h ) , 6.75 ( dd , j = 2.4 , 8.4 hz , 1 h ) , 5.97 ( m , 1 h ) , 5.71 ( m , 1 h ) , 3.70 ( s , 3 h ) , 3.42 ( dd , j = 8.4 , 10.2 hz , 1 h ) , 2.582.48 ( m , 2 h ) , 1.59 ( s , 1 h ) , 0.05 ( s , 3 h ) , 0.05 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 159.3 , 141.6 , 137.3 , 137.0 , 134.5 ( 2 c ) , 131.0 , 128.92 , 128.89 , 127.3 ( 2 c ) , 121.0 , 114.5 , 112.3 , 84.1 , 59.7 , 55.1 , 35.4 , 4.9 , 5.2 ; ir ( film ) 3440 , 3030 , 2957 , 1250 , 819 cm ; hrms ( ei ) m / z 306.1436 [ ( m h2o ) , calcd for c20h22osi , 306.1440 ] . applying general procedure h to 1q ( 83 mg , 0.2524 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.17 ml , 1.1 equiv ) at 78 c for 5 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 38.4 mg ( 47% ) of 3q ( dr > 20:1 ) and 36.1 mg ( 44% ) of 4q ( dr > 20:1 ) . spectroscopic data for 3q : h nmr ( 600 mhz , cdcl3 ) 7.51 ( m , 2 h ) , 7.41 ( m , 1 h ) , 7.36 ( m , 2 h ) , 7.15 ( m , 2 h ) , 6.90 ( m , 2 h ) , 2.61 ( m , 2 h ) , 1.48 ( dt , j = 4.8 , 9.0 hz , 1 h ) , 1.16 ( m , 1 h ) , 0.82 ( dt , j = 5.4 , 9.0 hz , 1 h ) , 0.63 ( dt , j = 5.4 , 9.0 hz , 1 h ) , 0.48 ( s , 3 h ) , 0.47 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 245.1 , 141.3 , 134.3 , 134.0 ( 2c ) , 131.0 , 129.9 , 128.23 ( 2c ) , 128.20 ( 2 c ) , 127.3 ( 2 c ) , 53.1 , 22.1 , 16.8 , 15.5 , 4.8 , 4.9 ; ir ( film ) 3030 , 2958 , 1641 , 1490 , 1012 , 823 , 734 cm ; hrms ( ei ) m / z 328.1049 [ ( m ) , calcd for c19h21osicl , 328.1050 ] . spectroscopic data for 4q : h nmr ( 600 mhz , cdcl3 ) 7.31 ( m , 1 h ) , 7.27 ( m , 2 h ) , 7.23 ( m , 2 h ) , 7.15 ( m , 4 h ) , 5.98 ( ddd , j = 1.8 , 3.0 , 6.0 hz , 1 h ) , 5.73 ( ddd , j = 1.2 , 2.4 , 6.0 hz , 1 h ) , 3.39 ( dd , j = 7.2 , 10.2 hz , 1 h ) , 2.55 ( dddd , a of abx system , j = 1.2 , 3.0 , 7.8 , 15.6 hz , 1 h ) , 2.45 ( ddt , b of abx system , j = 2.4 , 10.2 , 18.0 hz , 1 h ) , 1.56 ( s , 1 h ) , 0.04 ( s , 3 h ) , 0.03 ( s , 3 h ) ; c nmr ( 151 mhz , cdcl3 ) 138.5 , 137.4 , 136.5 , 134.3 ( 2 c ) , 132.4 , 131.0 , 129.9 ( 2 c ) , 129.0 , 128.0 ( 2 c ) , 127.4 ( 2 c ) , 84.2 , 59.0 , 35.3 , 4.8 , 5.2 ; ir ( film ) 3440 , 2950 , 1246 , 820 cm ; hrms ( ei ) m / z 310.0946 [ ( m h2o ) , calcd for c19h19sicl , 310.0945 ] . applying general procedure h to 1r ( 69.5 mg , 0.225 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.15 ml , 1.1 equiv ) in thf ( 2.8 ml ) at 78 c for 5 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 43.7 mg ( 63% ) of 3r ( dr > 20:1 ) and 15.7 mg ( 23% ) of 4r ( dr > 20:1 ) . spectroscopic data for 3r : h nmr ( 600 mhz , cdcl3 ) 7.18 ( m , 2 h ) , 7.00 ( m , 2 h ) , 2.682.60 ( m , 2 h ) , 1.57(dt , j = 5.6 , 9.0 hz , 1 h ) , 1.27 ( m , 1 h ) , 0.95 ( t , j = 7.8 hz , 9 h ) , 0.92 ( m , 1 h , overlapped ) , 0.73 ( m , 1 h , heavily overlapped ) , 0.72 ( q , j = 7.8 hz , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 246.9 , 141.4 , 131.0 , 128.3 ( 2 c ) , 127.4 ( 2 c ) , 54.7 , 22.2 , 16.5 , 15.5 , 7.2 , 2.1 ; ir ( film ) 2955 , 2878 , 1641 , 1495 , 1012 , 823 , 734 cm ; hrms ( ei ) m / z 308.1357 [ ( m ) , calcd for c17h25osicl , 308.1363 ] . spectroscopic data for 4r : h nmr ( 600 mhz , cdcl3 ) 7.34 ( m , 2 h ) , 7.24 ( m , 2 h ) , 5.94 ( m , 1 h ) , 5.82 ( ddd , j = 1.2 , 2.4 , 6.0 hz , 1 h ) , 3.35 ( t , j = 8.4 hz , 1 h ) , 2.692.60 ( m , 2 h ) , 0.82 ( t , j = 7.8 hz , 9 h ) , 0.28 ( m , 6 h ) ; c nmr ( 151 mhz , cdcl3 ) 139.3 , 138.2 , 132.3 , 130.4 , 129.8 ( 2 c ) , 128.1 ( 2 c ) , 85.7 , 59.9 , 35.6 , 7.8 , 2.3 ; ir ( film ) 3439 , 3053 , 2953 , 2878 , 1493 , 1093 , 1014 , 825 , 717 cm ; hrms ( ei ) m / z 290.1249 [ ( m h2o ) , calcd for c17h23sicl , 290.1258 ] . applying general procedure h to 2s ( 133.5 mg , 0.411 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in cyclohexane , 1.5 ml , 5 equiv ) at 78 c , and then at 0 c for 6 h , afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 58.8 mg ( 45% ) of 3s ( dr = 3.2:1 ) and 11.8 mg ( 9% ) of 4s ( dr > 20:1 ) as colorless oils . spectroscopic data for 3s ( major diastereomer ( trans ) ) : h nmr ( 500 mhz , cdcl3 ) 7.767.70 ( m , 3 h ) , 7.51 ( s , 1 h ) , 7.437.34 ( m , 2 h ) , 7.20 ( dd , j = 2.0 , 8.5 hz , 1 h ) , 2.77 ( dd , a of abx system , j = 6.0 , 17.0 hz , 1 h ) , 2.62 ( dd , b of abx system , j = 7.0 , 17.4 hz , 1 h ) , 1.79 ( quintet , j = 4.5 hz , 1 h ) , 1.44 ( m , 1 h ) , 1.10 ( m , 1 h , 0.96 ( t , j = 7.5 hz , 9 h ) , 0.81 ( m , 1 h ) , 0.74 ( q , j = 7.5 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.1 , 140.4 , 135.5 , 131.9 , 127.8 , 127.5 , 127.3 , 125.9 , 125.0 , 124.9 , 124.0 , 54.8 , 22.9 , 16.5 , 15.7 , 7.3 , 2.1 ; ir ( film ) 3055 , 2955 , 2876 , 1639 , 1018 , 910 , 740 cm ; hrms ( ei ) m / z 324.1901 [ ( m ) , calcd for c21h28osi , 324.1909 ] . spectroscopic data for 4s : h nmr ( 500 mhz , cdcl3 ) 7.80 ( m , 3 h ) , 7.76 ( d , j = 8.5 hz , 1 h ) , 7.61 ( dd , j = 1.0 , 8.5 hz , 1 h ) , 7.44 ( m , 2 h ) , 6.00 ( m , 1 h ) , 5.88 ( m , 1 h ) , 3.57 ( m , 1 h ) , 2.87 ( m , 1 h ) , 2.73 ( m , 1 h ) , 0.77 ( t , j = 8.0 hz , 9 h ) , 0.26 ( dq , j = 1.5 , 8.0 hz , 6 h ) ; c nmr ( 126 mhz , cdcl3 ) 138.5 , 138.3 , 133.3 , 132.5 , 130.5 , 127.9 , 127.7 , 127.6 , 127.4 , 126.2 , 125.8 , 125.3 , 85.9 , 60.7 , 35.8 , 7.8 , 2.3 ; ir ( film ) 3053 , 2951 , 2876 , 1458 , 1012 , 819 , 727 cm ; hrms ( esi ) m / z 307.1869 [ ( m oh ) , calcd for c21h27si , 307.1882 ] . applying general procedure h to 1 t ( 26.6 mg , 0.097 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 73 l , 1.2 equiv ) in thf ( 1.2 ml ) at 78 c for 10 min afforded after workup and column chromatography ( 5% etoac in hexanes ) 15.4 mg ( 64% ) of 3 t spectroscopic data for 3 t : h nmr ( 500 mhz , cdcl3 ) 7.09 ( m , 3 h ) , 7.01 ( m , 1 h ) , 2.86 ( dd , a of abx system , j = 5.5 , 16.5 hz , 1 h ) , 2.69 ( m , 2 h ) , 2.57 ( dd , b of abx system , j = 7.5 , 17.0 hz , 1 h ) , 1.68 ( dt , j = 5.0 , 9.0 hz , 1 h ) , 1.62 ( m , 2 h ) , 1.34 ( m , 1 h ) , 0.98 ( t , j = 7.0 hz , 3 h ) , 0.92 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.74 ( dt , j = 5.5 , 8.5 hz , 1 h ) , 0.20 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.3 , 141.7 , 139.8 , 128.7 , 125.9 , 125.7 , 125.6 , 53.3 , 35.2 , 23.8 , 20.3 , 14.9 , 14.3 , 14.2 , 3.2 ; ir ( film ) 3064 , 2959 , 2872 , 1645 , 1250 , 844 cm ; hrms ( ei ) m / z 274.1739 [ ( m ) , calcd for c17h26osi , 274.1753 ] . spectroscopic data for 4 t : h nmr ( 500 mhz , cdcl3 ) 7.34 ( m , 1 h ) , 7.167.10 ( m , 3 h ) , 6.02 ( dt , j = 2.5 , 5.5 hz , 1 h ) , 5.74 ( dt , j = 2.0 , 6.0 hz , 1 h ) , 3.81 ( t , j = 8.5 hz , 1 h ) , 3.11 ( ddd , j = 6.5 , 8.5 , 14.0 hz , 1 h ) , 2.802.69 ( m , 2 h ) , 2.54 ( ddd , j = 7.0 , 9.5 , 14.0 hz , 1 h ) , 1.58 ( m , 2 h ) , 0.96 ( t , j = 7.0 hz , 3 h ) , 0.28 ( s , 9 h ) ; ir ( film ) 3444 , 3058 , 2957 , 1490 , 1246 , 838 cm ; hrms ( ei ) m / z 274.1739 [ ( m ) , calcd for c17h26osi , 274.1753 ] . applying general procedure h to 2u ( 200 mg , 0.81 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in cyclohexane , 1.8 ml , 3 equiv ) at 78 c for 7 h afforded after workup and column chromatography ( 10% etoac in hexanes ) 157.7 mg ( 79% ) of 4u ( dr = 20:1 ) as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.38 ( m , 2 h ) , 7.27 ( m , 2 h ) , 7.22 ( m , 1 h ) , 5.64 ( m , 1 h ) , 3.41 ( m , 1 h ) , 2.63 ( m , 1 h ) , 2.43 ( m , 1 h ) , 1.76 ( m , 3 h ) , 1.40 ( s , 1 h ) , 0.30 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 145.7 , 140.9 , 128.7 ( 2 c ) , 128.1 ( 2 c ) , 126.8 , 124.8 , 84.8 , 61.8 , 32.9 , 14.6 , 2.2 ; ir ( film ) 3440 , 2957 , 1243 , 838 cm ; hrms ( esi ) m / z 229.1401 [ ( m oh ) , calcd for c15h21si , 229.1413 ] . applying general procedure h to 1v ( 61.5 mg , 0.2225 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.17 ml , 1.2 equiv ) at 78 c for 35 min afforded after workup and column chromatography ( 10% etoac in hexanes ) 44 mg ( 72% ) of 4v ( dr = 20:1 ) as a white solid : mp 9697 c ; h nmr ( 500 mhz , cdcl3 ) 7.29 ( m , 2 h ) , 6.82 ( m , 2 h ) , 5.62 ( m , 1 h ) , 3.78 ( s , 3 h ) , 3.34 ( dd , j = 7.5 , 10.5 hz , 1 h ) , 2.58 ( m , 1 h ) , 2.41 ( m , 1 h ) , 1.75 ( dt , j = 1.5 , 3.0 hz , 3 h ) , 1.37 ( s , 1 h ) , 0.29 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 158.5 , 145.6 , 132.9 , 129.5 ( 2 c ) , 124.8 , 113.5 ( 2 c ) , 84.8 , 61.0 , 55.2 , 33.1 , 14.6 , 2.1 ; ir ( film ) 3435 , 2952 , 1240 , 838 cm ; hrms ( esi ) m / z 259.1507 [ ( m oh ) , calcd for c16h23osi , 259.1518 ] . applying general procedure h to 1w ( 66.6 mg , 0.2557 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.19 ml , 1.2 equiv ) at 78 c for 15 min afforded after workup and column chromatography ( 10% etoac in hexanes ) 60.2 mg ( 91% ) of 4w ( dr = 20:1 ) as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.26 ( d , j = 8.0 hz , 2 h ) , 7.08 ( d , j = 8.0 hz , 2 h ) , 5.63 ( quintet , j = 1.5 hz , 1 h ) , 3.36 ( dd , j = 8.0 , 11.0 hz , 1 h ) , 2.61 ( m , 1 h ) , 2.41 ( m , 1 h ) , 2.32 ( s , 3 h ) , 1.76 ( m , 3 h ) , 1.37 ( s , 1 h ) , 0.30 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 145.6 , 137.7 , 136.2 , 128.8 ( 2 c ) , 128.5 ( 2 c ) , 124.8 , 84.8 , 61.4 , 33.0 , 21.0 , 14.6 , 2.2 ; ir ( film ) 3434 , 2953 , 1246 , 830 cm ; hrms ( esi ) m / z 243.1568 [ ( m oh ) , calcd for c16h23si , 243.1569 ] . applying general procedure h to 1x ( 120 mg , 0.445 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.56 ml , 2 equiv ) at 78 c for 1.5 h afforded after workup and column chromatography ( 4% etoac in hexanes ) 16.3 mg ( 13% ) of unreacted 1x and 90 mg ( 75% ) of 4x ( dr = 20:1 ) as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 7.45 ( m , 2 h ) , 7.30 ( m , 2 h ) , 7.24 ( tt , j = 1.5 , 7.0 hz , 1 h ) , 5.96 ( dd , j = 2.5 , 3.5 hz , 1 h ) , 5.63 ( d , j = 1.0 hz , 1 h ) , 4.98 ( s , 1 h ) , 3.53 ( dd , j = 8.0 , 12.0 hz , 1 h ) , 2.71 ( dd , j = 11.5 , 16.5 hz , 1 h ) , 2.49 ( ddd , j = 3.5 , 7.5 , 16.5 hz , 1 h ) , 1.94 ( s , 3 h ) , 1.67 ( s , 1 h ) , 0.32 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 148.5 , 140.1 , 139.0 , 128.7 ( 2 c ) , 128.1 ( 2 c ) , 126.9 , 115.1 , 85.2 , 62.2 , 31.7 , 23.1 , 1.7 ; ir ( film ) 3443 , 3029 , 2956 , 1242 , 833 cm ; hrms ( esi ) m / z 255.1562 [ ( m oh ) , calcd for c17h23si , 255.1569 ] . applying general procedure h to 1y ( 51.3 mg , 0.208 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.16 ml , 1.2 equiv ) at 78 c for 30 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 22.5 mg ( 44% ) of 3y ( dr = 7:1 ) and 19.6 mg ( 38% ) of 4y ( dr = 20:1 ) as colorless oils . spectroscopic data for 3y : h nmr ( 500 mhz , cdcl3 ) 7.27 ( m , 4 h ) , 7.16 ( m , 1 h ) , 2.91 ( d , j = 17.5 hz , 1 h ) , 2.52 ( d , j = 17.5 hz , 1 h ) , 1.89 ( dd , j = 6.5 , 8.5 hz , 1 h ) , 0.81 ( m , 2 h ) , 0.73 ( s , 3 h ) , 0.20 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 247.9 , 139.5 , 129.4 , 128.9 , 127.9 , 125.8 , 58.6 , 28.2 , 18.8 , 18.5 , 17.1 , 3.3 ; ir ( film ) 3061 , 2959 , 1647 , 1250 , 844 cm ; hrms ( ei ) m / z 246.1436 [ ( m ) , calcd for c15h22osi , 246.1440 ] . spectroscopic data for 4y : h nmr ( 500 mhz , cdcl3 ) 7.37 ( m , 2 h ) , 7.28 ( m , 2 h ) , 7.21 ( m , 1 h ) , 5.38 ( m , 1 h ) , 3.47 ( dd , j = 8.0 , 10.0 hz , 1 h ) , 2.68 ( m , 1 h ) , 2.51 ( dd , j = 8.0 , 15.5 hz , 1 h ) , 1.84 ( m , 3 h ) , 1.42 ( s , 1 h ) , 0.33 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 141.2 , 140.8 , 130.7 , 128.4 ( 2c ) , 128.1 ( 2c ) , 126.7 , 84.4 , 60.4 , 39.6 , 17.2 , 3.3 ; ir ( film ) 3437 , 3034 , 2912 , 1244 , 837 , 758 cm ; hrms ( esi ) m / z 229.1413 [ ( m oh ) , calcd for c15h21si , 229.1412 ] . applying general procedure h to 1z ( 50.3 mg , 0.193 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.145 ml , 1.2 equiv ) at 78 c for 15 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 21.8 mg ( 44% ) of 3z ( dr = 9:1 ) and 21.5 mg ( 43% ) of 4z ( dr = 20:1 ) as colorless oils . spectroscopic data for 3z : h nmr ( 600 mhz , cdcl3 ) 7.16 ( d , j = 7.8 hz , 2 h ) , 7.07 ( d , j = 7.8 hz , 2 h ) , 2.88 ( d , j = 17.4 hz , 1 h ) , 2.52 ( d , j = 16.8 hz , 1 h ) , 2.30 ( s , 3 h ) , 1.85 ( t , j = 7.2 hz , 1 h ) , 0.76 ( m , 2 h ) , 0.72 ( s , 3 h ) , 0.20 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 248.0 , 136.3 , 135.2 , 129.3 ( 2c ) , 128.6 ( 2c ) , 58.7 , 27.8 , 21.0 , 18.8 , 18.3 , 17.0 , 3.2 ; ir ( film ) 3051 , 2957 , 2868 , 1647 , 1250 , 844 cm ; hrms ( ei ) m / z 260.1599 [ ( m ) , calcd for c16h24osi , 260.1596 ] . spectroscopic data for 4z : h nmr ( 600 mhz , cdcl3 ) 7.25 ( d , j = 7.8 hz , 2 h ) , 7.08 ( d , j = 7.8 hz , 2 h ) , 5.37 ( s , 1 h ) , 3.42 ( t , j = 8.4 hz , 1 h ) , 2.65 ( dd , a of abx system , j = 9.8 , 15.0 hz , 1 h ) , 2.49 ( dd , b of abx system , j = 7.2 , 15.6 hz , 1 h ) , 2.31 ( s , 3 h ) , 1.83 ( s , 3 h ) , 1.40 ( s , 1 h ) , 0.32 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 141.2 , 137.7 , 136.1 , 130.6 , 128.8 ( 2c ) , 128.3 ( 2c ) , 84.5 , 60.0 , 39.7 , 21.0 , 17.2 , 3.3 ; ir ( film ) 3434 , 3020 , 2961 , 2853 , 1244 , 837 cm ; hrms ( ei ) m / z 242.1502 [ ( m h2o ) , calcd for c16h22si , 242.1491 ] . applying general procedure h to 1aa ( 108 mg , 0.453 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.34 ml , 1.2 equiv ) at 78 c for 10 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 78.8 mg ( 73% ) of 3aa ( dr = 2.1:1 ) and 25.9 mg ( 24% ) of 4aa ( dr = 3.2:1 ) as colorless oils . spectroscopic data for 3aa : mixture of diastereomers ( 1.0:0.6 ) ; h nmr ( 500 mhz , cdcl3 ) 7.05 ( dd , j = 1.0 , 5.0 hz , 0.6 h ) , 7.01 ( dd , j = 1.0 , 5.5 hz , 1 h ) , 6.87 ( dd , j = 3.0 , 5.0 hz , 0.6 h ) , 6.85 ( dd , j = 3.5 , 5.0 hz , 1 h ) , 6.75 ( m , 1 h ) , 6.67 ( dt , j = 1.0 , 3.5 hz , 0.6 h ) , 2.71 ( dd , a of abx system , j = 6.0 , 16.5 hz , 1 h ) , 2.60 ( dd , b of abx system , j = 7.0 , 17.5 hz , 1 h ) , 2.45 ( dd , c of cdx system , j = 8.0 , 18.5 hz , 0.6 h ) , 2.39 ( dd , d of cdx system , j = 6.0 , 18.5 hz , 0.6 h ) , 2.23 ( m , 0.6 h ) , 1.81 ( m , 1 h ) , 1.48 ( m , 0.6 h ) , 1.33 ( m , 1 h ) , 1.17 ( dt , j = 5.5 , 8.5 hz , 0.6 h ) , 0.99 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.77 ( dt , j = 5.0 , 8.5 hz , 1 h ) , 0.63 ( q , j = 5.5 hz , 0.6 h ) , 0.19 ( s , 9 h ) , 0.04 ( s , 5.4 h ) ; c nmr ( 126 mhz , cdcl3 ) ( major diastereomer ) 246.9 , 147.2 , 126.7 , 122.8 , 122.1 , 52.7 , 17.9 , 17.5 , 16.4 , 3.2 ; ( minor diastereomer ) 247.5 , 143.5 , 126.6 , 125.5 , 123.4 , 47.6 , 14.9 , 13.2 , 11.9 , 3.4 ; ir ( film ) 3071 , 2959 , 1645 , 1250 , 846 cm ; hrms ( esi ) m / z 239.0922 [ ( m + h ) , calcd for c12h19osis , 239.0926 ] . spectroscopic data for 4aa : h nmr ( 500 mhz , cdcl3 ) 6.35 ( dd , j = 2.0 , 6.5 hz , 1 h ) , 6.07 ( m , 1 h ) , 5.83 ( ddd , j = 3.5 , 8.5 , 12.0 hz , 1 h ) , 5.66 ( ddd , j = 1.0 , 3.0 , 6.5 hz , 1 h ) , 5.42 ( dd , j = 3.0 , 7.0 hz , 1 h ) , 4.40 ( m , 1 h ) , 2.81 ( m , 1 h ) , 2.46 ( quintet , j = 3.5 hz , 1 h ) , 1.59 ( s , 1 h ) , 0.15 ( s , 9 h ) ; c nmr ( 600 mhz , cdcl3 ) 140.8 , 131.8 , 129.1 , 126.3 , 123.1 , 122.7 , 67.8 , 57.2 , 26.4 , 2.3 ; ir ( film ) 3418 , 3017 , 2955 , 1246 , 839 cm ; hrms ( esi ) m / z 221.0821 [ ( m oh ) , calcd for c12h17sis , 221.0820 ] . applying general procedure h to 1bb ( 93.8 mg , 0.422 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.29 ml , 1.2 equiv ) at 78 c for 10 min afforded after workup and column chromatography ( 10% etoac in hexanes ) 76 mg ( 81% ) of 4bb ( dr = 20:1 ) as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 6.50 ( dd , j = 2.0 , 3.0 hz , 1 h ) , 5.84 ( dddd , j = 0.5 , 3.5 , 7.5 , 12.0 hz , 1 h ) , 5.63 ( ddd , j = 0.5 , 3.0 , 12.0 hz , 1 h ) , 5.46 ( m , 1 h ) , 5.15 ( m , 1 h ) , 4.11 ( m , 1 h ) , 2.82 ( d of quintets , a of abx system , j = 3.5 , 20.0 hz , 1 h ) , 2.50 ( dt , b of abx system , j = 7.5 , 20.0 hz , 1 h ) , 0.13 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 157.4 , 145.3 , 132.1 , 131.1 , 103.8 . 100.4 , 64.9 , 50.3 , 24.5 , 2.6 ; ir ( film ) 3499 , 3013 , 2955 , 1246 , 1151 , 839 cm ; hrms ( ei ) m / z 222.1068 [ ( m ) , calcd for c12h18o2si , 222.1076 ] . applying general procedure h to 2bb ( 56.6 mg , 0.255 mmol , 1 equiv ) and sec - butyllithium ( 1.6 m in cyclohexane , 0.55 ml , 3 equiv ) at 78 c for 3.5 h afforded after workup and column chromatography ( 5% etoac in hexanes ) 21.2 mg ( 37% ) of 3bb ( dr = 1:0.7 ) as a colorless oil and 12.9 ( 23% ) of unreacted 2bb ( single diastereomer ) : mixture of diastereomers ( 1:0.7 ) ; h nmr ( 600 mhz , cdcl3 ) 7.23 ( m , 1 h ) , 7.20 ( m , 0.7 h ) , 6.24 ( m , 1 h ) , 6.22 ( m , 0.7 h ) , 5.94 ( m , 1 h ) , 5.92 ( m , 1 0.7 h ) , 2.70 ( dd , a of abx system , j = 6.6 , 17.4 hz , 0.7 h ) , 2.54 ( dd , b of abx system , j = 7.2 , 16.8 hz , 0.7 h ) , 2.50 ( dd , c of cdx system , j = 7.2 , 18.0 hz , 1 h ) , 2.39 ( dd , d of cdx system , j = 6.0 , 18.0 hz , 1 h ) , 2.05 ( dt , j = 6.0 , 9.0 hz , 1 h ) , 1.64 ( dt , j = 4.8 , 8.0 hz , 0.7 h ) , 1.43 ( m , 1 h ) , 1.38 ( m , 0.7 h ) , 1.10 ( dt , j = 4.8 , 8.4 hz , 1 h ) , 1.03 ( dt , j = 4.8 , 9.0 hz , 0.7 h ) , 0.66 ( m , 1.7 h ) , 0.18 ( s , 6.3 h ) , 0.08 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) ( major diastereomer ) 247.3 , 154.4 , 141.0 , 110.1 , 106.5 , 47.7 , 13.3 , 12.6 , 10.1 , 3.2 ; ( minor diastereomer ) 246.9 , 156.2 , 140.4 , 110.2 , 103.6 , 52.5 , 15.8 , 14.5 , 13.3 , 3.1 ; ir ( film ) 2961 , 1645 , 1250 , 844 cm ; hrms ( ei ) m / z 222.1086 [ ( m ) , calcd for c12h18o2si , 222.1076 ] . a high - pressure glass tube was charged with racemic -(trimethylsilyl)allyl alcohol s2-a ( 2.4 g , 18.43 mmol , 1 equiv ) , vinyl acetate ( 2.4 g , 27.65 mmol , 1.5 equiv ) , and pentane ( 10 ml ) . powdered 4 molecular sieves ( 1.4 g ) and lipase candida antarctica ( 15 mg / mmol of alcohol ) were added . the tube was sealed and heated in an oil bath at 40 c . after 34 h the reaction was cooled at room temperature , filtered through a plug of celite , and rinsed with pentane , and the filtrate was carefully concentrated . column chromatography ( 5% , 10% and 15% et2o in pentane ) afforded 1.06 g ( 34% ) of ( s)-1-(trimethylsilyl)allyl acetate ( ee not determined ) and 786 mg ( 31% ) of ( r)-s2-a ( [ ]d = 6.1 ( c = 1 , chcl3 , 76% ee ) ) . the enantiomeric excess ( ee ) was determined by hplc ( oj column , hexanes , 0.6 ml / min , 10 c ) . applying general procedure d to s2-a ( 76% ee , 180 mg , 1.382 mmol , 1 equiv ) , the trichloroacetimidate of 1-phenylbut-3-en-1-ol ( 606 mg , 2.07 mmol , 1.5 equiv ) , and ( tms)otf ( 25 l , 0.138 mmol , 0.1 equiv ) in hexane ( 8 ml ) afforded after column chromatography ( 7% ch2cl2 in hexanes ) a total of 222.5 ( 62% ) of ( + ) -syn - s3a/(+)-anti - s3-a distributed in three fractions : 79.2 mg of ( + ) -antis3-a ( [ ]d = + 112 ( c = 0.96 , chcl3 ) ) , 87.3 mg of ( + ) -syn - s3-a ( [ ]d = + 2 ( c = 0.92 , chcl3 ) ) , and 56 mg of a mixture of ( + ) -syn - s3a/(+)-anti - s3-a as a colorless oil . the enantiomeric excess of both diastereomers could not be determined by chiral hplc or gc . applying general procedure f to ( + ) -syn - s3a/(+)-anti - s3-a ( 1.7:1 ratio , 90 mg , 0.346 mmol , 1 equiv ) and second - generation grubbs catalyst ( 11.7 mg , 0.0138 mmol , 0.04 equiv ) in ch2cl2 ( 4 ml ) for 3 h afforded after column chromatography ( 10% and 30% ch2cl2 in hexanes ) 42.9 mg ( 53% ) of ( )-1a ( [ ]d = 93.3 ( c = 1.28 , chcl3 , ee not determined ) ) and 25 mg ( 31% ) of ( + ) -2a ( [ ]d = + 66.2 ( c = 0.72 , chcl3 , 76% ee ) ) as colorless oils . the enantiomeric excess of ( + ) -2a was determined by hplc ( oj column , hexanes , 0.4 ml / min , 10 c , tr(minor ) = 14.9 min , tr(major ) = 17.6 min ) . the enantiomeric excess of ( )-1a could not be obtained by hplc but was inferred to be 76% ee on the basis of that of ( + ) -2a . applying general procedure h to ( )-1a ( ca . 76% ee , 42.9 mg , 0.1846 mmol , 1 equiv ) and n - butyllithium ( 1.6 m in hexanes , 0.17 ml , 1.5 equiv ) in thf ( 2.3 ml ) at 78 c for 5 min afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 24.1 mg ( 56% ) of ( )-3a ( dr = 20:1 ) in 62% ee ( [ ]d = 25.6 ( c = 0.83 , chcl3 ) ) and 9.6 mg ( 22% ) of ( + ) -4a ( dr > 20:1 ) in 73% ee ( [ ]d = + 73.4 ( c = 0.96 , chcl3 ) ) . chiral hplc conditions for ( )-3a : oj column , hexanes , 0.7 ml / min , 20 c , tr(major ) = 12.7 min , tr(minor ) = 25.8 min . chiral hplc conditions for ( + ) -4a : oj column , 0.5% i - proh / hexanes , 0.4 ml / min , 20 c , tr(minor ) = 22.8 min , tr(major ) = 25.0 min . applying general procedure h to ( + ) -2a ( 76% ee , 25 mg , 0.1076 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in cyclohexane , 0.23 ml , 3 equiv ) in thf ( 1.4 ml ) at 78 c for 3 h afforded after workup and column chromatography ( 5% and 10% etoac in hexanes ) 15 mg ( 56% ) of ( + ) -3a ( dr = 20:1 ) in 56% ee ( [ ]d = + 27 ( c = 0.79 , chcl3 ) ) and 5.2 mg ( 21% ) of ( )-4a ( dr > 20:1 ) in 74% ee ( [ ]d = 52.7 ( c = 0.48 , chcl3 ) ) . the relative stereochemistries of cyclic ethers 1 ( trans ) and 2 ( cis ) were assigned by comparison with the h nmr spectra of compounds 1a and 2a , whose relative stereochemistries have been determined by noe studies . the relative stereochemistries of precursors s3 ( syn / anti ) were assigned on the basis of the assignment of the corresponding cyclic ethers 1 and 2 . the relative and absolute stereochemistries of -wittig product ( )-3a were assigned by derivatization to the known aldehyde ( )-5a . the relative and absolute stereochemistries of -wittig product ( + ) -4a were determined by x - ray crystallographic analysis of its 3,5-dinitrobenzoate derivative ( )-7 . the relative stereochemistries of all other - and -wittig products were assigned by comparison of their h nmr spectra with those of compounds 3a and 4a , respectively . further support for the relative stereochemistries of -wittig products was obtained by x - ray crystallographic analysis of compound 4s . compound ( )-3a was derivatized to the known aldehyde ( )-5 ( scheme 14 ) . enolate formation / benzoylation : to a solution of freshly distilled diisopropylamine ( 41 l , 0.29 mmol , 1.3 equiv ) in thf ( 1 ml ) at 78 c was added n - butyllithium ( 1.6 m in hexanes , 0.17 ml , 0.266 mmol , 1.2 equiv ) slowly . after 10 min the reaction was warmed to 0 c for 15 min and cooled to 78 c . compound ( )-3a ( 62% ee , 51.5 mg , 0.222 mmol , 1 equiv ) in thf ( 2 ml ) was added via syringe quickly to give a yellow solution . after 1 h benzoyl chloride ( 52 l , 0.444 mmol , 2 equiv ) was added , and the temperature kept at 78 c for 30 min and then raised to room temperature . the aqueous phase was extracted with diethyl ether ( 3 5 ml ) . combined organic extracts were washed with brine , dried over mgso4 , and concentrated . column chromatography ( 5% etoac in hexanes ) afforded 40 mg ( 54% ) of enol benzoate as an oil . ozonolysis : a solution of the benzoate ( 19 mg , 0.056 mmol , 1 equiv ) in ch2cl2 was cooled at 78 c . bubbling of ozone was applied for 15 min ( a slightly blue color persisted ) , and then dimethyl sulfide ( 0.1 ml , excess ) was added . after an additional 15 min , the cold bath was removed and the solvent was removed by rotary evaporation . column chromatography ( 15% etoac in hexanes ) afforded 4.2 mg ( 56% ) of the known compound ( )-(1r,2r)-5 as a colorless oil : h nmr ( 500 mhz , cdcl3 ) 9.30 ( d , j = 5.0 hz , 1 h ) , 7.28 ( m , 2 h ) , 7.21 ( m , 1 h ) , 7.08 ( m , 2 h ) , 2.61 ( m , 1 h ) , 2.16 ( m , 1 h ) , 1.17 ( dt , j = 5.5 , 10.0 hz , 1 h ) , 1.52 ( ddd , j = 5.0 , 7.0 , 8.5 hz , 1 h ) ; [ ]d = 183.4 ( c = 0.46 , chcl3 ) ( lit . [ ]d = 378 ( c = 0.374 , chcl3 ) ) . to a solution of ( + ) -4a ( 72% ee , 15 mg , 0.065 mmol ) in etoac ( 1 ml ) was added 10% pd / c ( 3 mg ) ( scheme 15 ) . the system was closed with a septum , evacuated , and filled with h2 . a h2 balloon was attached and the mixture vigorously stirred for 30 min . then the reaction mixture was filtered though a short plug of silica and concentrated . column chromatography ( hplc conditions : oj column , 0.5% i - proh / hexanes , 0.4 ml / min , 20 c ) afforded 15 mg ( 100% ) of ( )-6 as a colorless oil : [ ]d = 13.5 ( c = 1 , chcl3 , 72% ee ) ; h nmr ( 500 mhz , cdcl3 ) 7.24 ( m , 2 h ) , 7.17 ( m , 3 h ) , 3.20 ( dd , j = 5.0 , 8.0 hz , 1 h ) , 2.36 ( m , 1 h ) , 2.05 ( m , 2 h ) , 1.92 ( m , 2 h ) , 1.72 ( m , 1 h ) , 0.31 ( s , 9 h ) ; c nmr ( 151 mhz , cdcl3 ) 143.7 , 128.4 ( 2 c ) , 128.2 ( 2 c ) , 126.4 , 80.1 , 57.9 , 36.1 , 31.1 , 22.5 , 3.2 ; ir ( film ) 3430 , 2951 cm ; hrms ( esi ) m / z 217.1413 [ ( m oh ) , calcd for c14h21si , 217.1404 ] . to a solution of ( )-6 ( 15 mg , 0.064 mmol , 1 equiv ) in pyridine ( 2 ml ) was added 3,5-dinitrobenzoyl chloride ( 74.5 mg , 0.323 mmol , 5 equiv ) , and the mixture was vigorously stirred for 36 h. the reaction was quenched with water ( 2 ml ) and diluted with diethyl ether . the aqueous phase was extracted with diethyl ether ( 3 3 ml ) . combined organic extracts were washed with 0.1 m hcl ( 2 ml ) and brine , dried over mgso4 , and concentrated . column chromatography ( 4% etoac in hexanes ) followed by recrystallization from hexanes / ch2cl2 afforded 10.6 mg ( 39% ) of ( )-7 as white crystals suitable for x - ray analysis and 4.4 mg ( 29% ) of unreacted alcohol . characterization data for ( )-(1s,2s)-7 : [ ]d = 87 ( c = 1.06 , chcl3 ) ; mp 137 c dec ; h nmr ( 500 mhz , cdcl3 ) 9.21 ( t , j = 2.0 hz , 1 h ) , 9.10 ( d , j = 2.0 hz , 1 h ) , 7.327.22 ( m , 5 h ) , 4.05 ( dd , j = 5.5 , 7.0 hz , 1 h ) , 2.35 ( m , 1 h ) , 2.292.19 ( m , 2 h ) , 2.182.09 ( m , 2 h ) , 1.93 ( m , 1 h ) , 0.19 . c nmr ( 126 mhz , cdcl3 ) 162.2 , 148.7 , 142.2 , 135.2 , 129.2 , 129.1 , 128.3 , 127.1 , 122.1 , 96.2 , 53.4 , 35.5 , 32.1 , 23.7 , 0.8 ; ir ( film ) 3029 , 2955 , 1720 , 1625 , 910 cm ; hrms ( ei ) m / z 428.1404 [ ( m ) , calcd for c21h24n2o6si , 428.1404 ] . applying general procedure h to 2d ( 96 mg , 0.366 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in hexanes , 0.78 ml , 3 equiv ) in thf ( 4 ml ) at 78 c for 6 h afforded after quenching with d2o ( 1 ml ) , regular workup , and column chromatography ( 30% ch2cl2 in hexanes , then 5% and 10% etoac in hexanes ) 25.2 mg ( 26% ) of d-2d , 2.2 mg ( 2% ) of d2 - 1d , 28 mg ( 30% ) of d-3d , 6.9 mg ( 7% ) of d2 - 8 , and 13.2 mg ( 19% ) of d-9 ( dr > 20:1 ) as colorless oils ( scheme 16 ) . spectroscopic data for d2 - 8 : h nmr ( 500 mhz , cdcl3 ) 7.25 ( m , 2 h ) , 6.87 ( m , 1 h ) , 5.02 ( m , 1 h ) , 4.70 ( dd , j = 2.5 , 10.0 hz , 1 h ) , 3.79 ( s , 3 h ) , 2.18 ( m , 1 h ) , 2.00 ( dddd , j = 1.0 , 2.5 , 6.5 , 13.5 , hz , 1 h ) , 1.80 ( dt , j = 10.0 , 13.0 hz , 1 h ) , 0.10 ( s , 9 h ) ; c nmr ( 126 mhz , cdcl3 ) 160.4 , 135.5 , 126.9 ( 2 c ) , 126.8 ( 2 c ) , 113.6 , 109.7 , 75.8 , 55.3 , 30.2 , 20.9 ( t , j = 20.2 hz ) , 2.4 . spectroscopic data for d-9 : h nmr ( 500 mhz , cdcl3 ) 9.82 ( t , j = 2.0 hz , 1 h ) , 7.01 ( m , 2 h ) , 6.79 ( d , j = 9.0 hz , 1 h ) , 3.76 ( s , 3 h ) , 2.50 ( ddd , a of abx system , j = 2.0 , 7.0 , 17.5 hz , 1 h ) , 2.43 ( ddd , b of abx system , j = 2.0 , 7.5 , 17.5 hz , 1 h ) , 1.71 ( dt , j = 5.0 , 9.0 hz , 1 h ) , 1.23 ( m , 1 h ) , 0.98 ( dt , j = 5.5 , 8.5 hz , 1 h ) , 0.80 ( dt , j = 5.0 , 8.5 hz , 1 h ) ; c nmr ( 126 mhz , cdcl3 ) 201.7 , 157.8 , 127.1 , 127.0 , 113.9 , 55.3 , 48.2 , 21.9 , 15.5 , 14.6 . applying general procedure h to 2h ( 52.9 mg , 0.211 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in hexanes , 0.45 ml , 3 equiv ) in thf ( 2.6 ml ) at 78 c for 0.5 h afforded after quenching with d2o ( 1 ml ) , regular workup , and column chromatography ( 3% etoac in hexanes ) 22.7 mg ( 43% ) of d-2h , 10 mg ( 19% ) of d2 - 1h , and 7.9 mg ( 15% ) of d2 - 10 as colorless oils ( scheme 17 ) . spectroscopic data for d2 - 10 : h nmr ( 500 mhz , cdcl3 ) 7.31 ( m , 2 h ) , 7.03 ( m , 1 h ) , 5.06 ( m , 1 h ) , 4.75 ( dd , j = 2.5 , 10.0 hz , 1 h ) , 2.20 ( m , 1 h ) , 2.03 ( m , 1 h ) , 1.81 ( dt , j = 10.0 , 13.5 hz , 1 h ) , 0.13 ( s , 9 h ) . applying general procedure h to 2aa ( 16.8 mg , 0.07 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in hexanes , 0.15 ml , 3 equiv ) in thf ( 0.9 ml ) at 78 c for 3 h and quenching with d2o afforded following regular workup a mixture of d-2aa ( dr > 20:1 ) , d-3aa ( dr 1.7:1 ) , and enolic 3aa ( dr not determined ) in a 0.36:2.7:2.3 ratio , as determined from the crude mixture by h nmr ( scheme 18 ) . applying general procedure h to 2bb ( 9.8 mg , 0.044 mmol , 1 equiv ) and sec - butyllithium ( 1.4 m in hexanes , 94 l , 3 equiv ) in thf ( 0.6 ml ) at 78 c for 1 h and quenching with d2o afforded following regular workup mostly d-2bb ( dr > 20:1 ) and traces of d-3aa ( dr not determined ) , as determined from the crude mixture by h nmr ( scheme 19 ) . complete deuterium incorporation at the 5-position of the furyl group in d-2bb is observed . deuterium incorporation into d-3aa was not determined due to overlap with other signals . following general procedure h , to 1a ( 73 mg , 0.314 mmol , 1 equiv ) in thf ( 4 ml ) was added n - butyllithium ( 1.4 m in hexanes , 0.78 ml , 3 equiv ) quickly at 78 c , and immediately ( < 1 s ) d2o was added quickly via syringe . after regular workup the crude reaction was analyzed by h nmr . compounds d-1a , enolic 3aa , 4a , and 11 were present in a 1.0:0.33:0.17:0.11 ratio . in the early stage of this study involving the rearrangement of diastereomers 1a and 2a , we were unable to obtain reproducible isolated yields of both ( 3a ) and ( 4a ) products . the ratios of 3a and 4a in the crude h nmr varied in each experiment , and the determination of the diastereomeric ratio of 3a was complicated by the presence of additional signals in the cyclopropyl region ( 1.80.6 ppm ) . in addition , the integration of these signals correlated well with a doublet around 4.52 ppm ( j = 7.5 hz ) and a singlet around 4.35 ppm . we later found that subjecting the reaction mixture to column chromatography immediately after workup ( avoiding additional manipulation of the crude for h nmr analysis ) provided compounds 3a and 4a reproducibly ; however , we could not isolate the additional cyclopropyl compound . additional spectral data ( c nmr , dept , cosy , and ir ) from the crude reaction mixture strongly suggest that this species is actually the enolic form of 3a . following general procedure h , to 1a ( 65 mg , 0.28 mmol , 1 equiv ) in thf ( 3 ml ) at 78 c was added n - butyllithium ( 0.27 ml , 0.42 mmol , 1.5 equiv ) dropwise . after 20 min the reaction was quenched with nh4cl(satd ) and diluted with water and et2o . the solution was dried over magnesium sulfate , filtered , and quickly concentrated in a rotovap at room temperature . an aliquot of the crude reaction mixture was dissolved in cdcl3 and immediately analyzed by h nmr and ir . the ir spectrum showed a large broad band at 3435 cm ( oh from enolic 3a and 4a ) and a small band at 1635 cm ( c = o from keto 3a ) . the h nmr spectrum shows a mixture of keto 3a / enolic 3a / alcohol 4a present in a 0.29:1.0:0.64 ratio . addition of d2o led to disappearance of the singlet at 4.35 ppm , which has been assigned tentatively to the enol oh proton of enolic 3a . the c nmr spectrum of the crude reaction mixture ( different run , same conditions ) shows peaks at 158 and 104 ppm , attributable to the polarized enol c c double bond . this assignment is consistent with the results of a dept experiment and also with the cosy 2d spectrum showing coupling of the enolic c h bond with the cyclopropyl ch methine . interestingly , after column chromatography the ( cyclopropyl)acylsilanes were isolated as a single tautomer ( keto form ) . in the case of 3a , the tautomeric mixture could be regenerated by enolization of keto 3a with lda , followed by regular workup . notice that in the h nmr spectrum of the crude reaction mixture there is no evidence of cyclopentenol 4a , ruling out isomerization of the lithium enolate ( -product ) to the corresponding alkoxide ( -product ) . analogously , deprotonation of the -wittig products 4a and 4i with n - butyllithium ( 1.11.5 equiv ) at 78 c for 3 h followed by regular workup gave only 4a and 4i , without a trace of the corresponding cyclopropyl products .

the regiodivergent ring contraction of diastereomeric 2-silyl-5,6-dihydro-6-aryl-(2h)-pyrans via [ 1,2]- and [ 1,4]-wittig rearrangements to the corresponding -silylcyclopentenols or ( -cyclopropyl)acylsilanes favor the [ 1,4]-pathway by ortho and para directing groups in the aromatic appendage and/or by sterically demanding silyl groups . the [ 1,2]-pathway is dominant with meta directing or electron - poor aromatic moieties . exclusive [ 1,2]-wittig rearrangements are observed when olefin substituents proximal to the silyl are present . cis and trans diastereomers exhibit different reactivities , but converge to a single [ 1,2]- or [ 1,4]-wittig product with high diastereoselectivity and yield .

Introduction Results and Discussion Conclusions Experimental Section

although certain dihydrothiopyrans have been shown to isomerize to the corresponding cyclopropyl thioenolates under basic conditions at low temperatures , we are aware of only two -wittig rearrangements of cyclic allylic ethers . reported by rautenstrauch in 1972 , these examples involve the isomerization of dihydropyran and nerol oxide to the corresponding -cyclopropylacetaldehydes ( scheme 2 ) . thus , through electronic tuning at the aromatic appendage , judicious choice of the silyl group , or selecting certain olefin substitution patterns , allows one to maneuver these ring contractions toward -silylcyclopentenols ( via [ 1.2]-wittig ) or ( -cyclopropyl)acylsilanes ( via -wittig ) , with excellent diastereoselectivities and in a stereoconvergent fashion . values in parentheses refer to the yield of a mixture of diastereomers 1 and 2 prepared from the corresponding mixture of syn / anti precursors s3 . given the ability of the silyl group to ( 1 ) allow a selective deprotonation step and ( 2 ) suppress the competitive -pathway , we envisioned that diastereomeric cyclic ethers 1a/2a ( trans and cis , respectively ) would be suitable substrates for wittig rearrangements ( scheme 6 ) . indeed , under optimized conditions ( thf , 78 c ) , the trans diastereomer 1a underwent fast and selective allylic deprotonation by n - butyllithium and rearrangement within 5 min to give a mixture of the trans -wittig ( -cyclopropyl)acylsilanes 3a and cis -wittig -silylcyclopentenol 4a in good overall yield ( 82% ) , albeit with modest selectivity ( 2.4:1 ) in favor of the -product . the full stereoconvergence in the - and -wittig rearrangements of trans ( 1a ) and cis ( 2a ) diastereomers is of significant importance because , in particular for the -wittig pathway , it challenges the known stereochemical outcome of this manifold . our study continued with the evaluation of the electronic effects on the aromatic appendage ( table 1 ) . nonetheless , the same reactivity trend was observed for these compounds : trans diastereomers 1b l ( entries 111 ) underwent complete rearrangement within 10 min , whereas cis diastereomers 2b l ( entries 1222 ) required at least 3 h for complete conversion . a second trend was clearly observed in the trans diastereomers series : electron - donating groups located at the ortho and para positions increased the -selectivity ( entries 3 , 4 , and 6 ) , with the p - methoxy - substituted compound 1d giving exclusive -selectivity . o - methoxy substrate 1b ( entry 1 ) is an exception that might be attributed to coordination of oxygen lone pairs to the lithium cation during rearrangement , leading to a slight decrease in : -selectivity relative to that of the unsubstituted analogue 1a . the inductively electron - withdrawing methoxy group located at the meta position ( entry 2 ) led to an evident decrease in : -selectivity , providing the -wittig product in slight excess over the -product . the weakly electron - donating methyl group located at the meta position ( entry 5 ) led to a negligible effect in product distribution relative to that of the model substrate 1a . in all pertinent cases , the -wittig product was obtained as a single diastereomer , whereas the -product was formed with high diastereoselection ( > 15:1 ) . the large values ( 3.10 and 4.37 ) indicate a high sensitivity to the nature of the substituent x , and a balance between resonance and inductive effects seems to play an important role in determining the : -selectivity . we cautiously interpret these observations as supporting evidence for a stepwise mechanism for both - and -pathways , in which a relatively slow c o bond homolysis is followed by rapid intramolecular recombination of the diradical anion , leading to the observed products . in fact , -cyclopropyl ketones bearing anion - stabilizing groups attached to the ring are known to undergo ring expansion to their cyclopentenol isomers under basic conditions , whereas some cyclopropyl thioenolates isomerize to the corresponding cyclopentenyl thiolates . however , independent subjection of selected products to the reaction conditions a or b ( table 1 ) did not lead to any /-interconversion ( or vice versa ) ( scheme 7 ) . we next looked to tackle the modest : -selectivity obtained in most cases , as well as the adverse -selectivity in electron - deficient substrates . gratifyingly , our hypothesis was right , and a gradual increase in the steric demand of the silyl group consistently led to greater : -product ratios ( table 2 ) . in the trans series , changing a sime3 group to a sime2ph group increased the selectivity from 2.4:1 to 10:1 ( entries 1 and 2 ) , and the even larger simeph2 group facilitated exclusive -wittig rearrangement in excellent yield ( entry 3 ) . we believe the improved regioselectivity is primarily dominated by the sterics of the silyl group with little electronic contribution . indeed , increasing the bulkiness of the silyl group only with alkyl groups ( siet3 , entry 4 ) also led to excellent : -selectivity . however , the more sterically demanding silyl groups were deleterious for the reactivity of these isomers . given that the reactivity of trans isomers was minimally affected , the severe loss of reactivity in cis diastereomers suggests bulkier silyl groups shift the conformational equilibrium to the less reactive conformation . accordingly , in the case of trans diastereomer 1 , conformers i and ii are expected to exist in approximately a 1:2.5 ratio , with a small predominance of conformer ii on the basis of the conformational a values for trimethylsilyl and phenyl groups in cyclohexane ( 2.5 and 2.9 , respectively ) . although an increase in the steric demand of the silyl group is expected to shift the conformational equilibrium to the less reactive conformer i , the extent of this perturbation is expected to be modest given the larger si c bond length ( 1.89 vs 1.54 for c c bonds ) and the absence of severe steric interactions of the pseudoaxial silyl group in conformer ii . the required conformation for deprotonation ( conformation iii ) presents a severe steric clash between these bulky groups , which is expected to get worse as the silyl group becomes more sterically demanding . because the conformational equilibrium was also expected to be dependent on the steric demand of the aryl group , we reasoned that installing a large ortho substituent would lead to an increase in the population of conformers ii and iv ( for the trans-1 and cis-2 diastereomers , respectively ) , leading to a further increase of reactivity of the trans diastereomer ( 1 ) and a decrease in the reactivity of the cis diastereomer ( 2 ) . in the trans series the : ratio gradually increased from 2.4:1 for the unsubstituted substrate 1a to 5.3:1 for o - methyl analogue 1e and to 11:1 for o - propyl analogue 1s . for this purpose , we chose substrates that were electronically biased toward the -wittig rearrangement and studied the effect of silyl groups larger than sime3 , which were expected to shift the selectivity in favor of the -pathway ( table 3 ) . following this trend , in the trans - substituted ( p - chlorophenyl)dihydropyran series , the : -selectivity was gradually reversed from 1:2.3 with the sime3 analogue 1i ( entry 4 ) to 1:1 for the bulkier sime2ph group in 1q ( entry 5 ) and to 2.8:1 for the siet3 derivative 1r ( entry 6 ) . analysis of the crystal structure of -wittig product 4s ( figure 2 ) bearing the 2-naphthyl and siet3 groups in a cis relationship suggests a significant steric clash between these groups exists and thus the : -selectivity is higher for 1s vs 1l . one possible explanation is that under these reaction conditions the initially formed -wittig alkoxide isomerizes to the corresponding -enolate . alkyl or alkenyl substitution proximal to the silyl group veered the selectivity exclusively to the -wittig rearrangement . we attempted to shift the selectivity in favor of the -pathway by electronic modification of the aromatic ring ( introducing para electron - donating groups ) , but only the -products were obtained , and no cyclopropyl compounds were observed in these cases . in contrast to the previous examples , alkyl olefin substitution distal to the silyl group provided low : -product selectivity , but with good overall yields ( table 5 ) . the diastereoselectivity of the -shift remained high as in previous examples , but that of the -product , bearing an all - carbon quaternary center , was modest . conditions ( c ) ( 1y , 1z ) n - buli ( 1.2 equiv ) , 15 min ; ( d ) ( 2y , 2z ) : s - buli ( 3 equiv ) , 6 h. the exclusive -selectivity observed in substrates having olefin substitution proximal to the silyl group ( but not when distal ) can be attributed to an unfavorable steric interaction between the olefin substituent and the silyl group . diastereomeric 2-thiophene - yl ( 1aa/2aa ) and 2-furyl ( 1bb/2bb ) cyclic ethers were subjected to our standard reaction conditions for cis and trans diastereomers ( scheme 11 ) . on the contrary , the corresponding cis diasteromers 2aa ( 2-thiophene - yl ) and 2bb ( 2-furyl ) underwent exclusive -wittig rearrangements to give ( cyclopropyl)acylsilanes 3aa and 3bb ( > 20:1 selectivity ) in 84% and 48% yields ( based on recovered starting material ( brsm ) ) , respectively , both with low diastereoselectivity . additional control experiments discarded the potential isomerization of the -alkoxide to the -enolate within the reaction time ( 3 h ) at 78 c . although this product ratio is slightly higher than that observed when the reaction was stopped after 10 min , the lower yield of the reaction suggests some product decomposition took place during the extended time , thereby influencing the measured product distribution . although the -wittig shift is known to occur with high retention of stereochemistry at the migrating carbon , in both acyclic and cyclic ethers , the stereochemical course of the competing -wittig pathway has only been studied in one acyclic instance . it is important to note that the rearrangement of species b is in accord with the normal stereochemical course at the lithium - bearing carbon in the -wittig pathway ( assuming a localized character of ( -silylallyl)lithium b ) involving inversion of stereochemistry . in summary , we have developed a method to access ( -cyclopropyl)acylsilanes and -silylcyclopentenol structures with high diastereoselectivities and overall high efficiency via - and -wittig rearrangements of 2-silyl-6-aryldihydropyrans . the regioselective allylic deprotonations that trigger these rearrangements are made possible by the silyl appendage . on the other hand , electron - deficient migrating centers favor the -pathway , and olefin substitution at the olefin proximal to the silyl group leads to exclusive -migration . taken together , our results make it reasonable to conclude that the primary mechanism of the -wittig migration in these cyclic ethers involves a stepwise process analogous to the -pathway . to a solution of the corresponding homoallylic alcohol ( 110 mmol ) in diethyl ether ( 12 ml ) was added slowly sodium hydride ( 0.15 equiv , dispersion in mineral oil , 60% ( w / w ) ) . the filtrate was concentrated by rotary evaporation , and the crude product could be used without further purification in the next step . to a solution of the corresponding homoallylic alcohol ( 16 mmol ) in dichloromethane ( 80 ml ) was added dbu ( 0.18 equiv ) . a solution of the corresponding allylic alcohol in thf was cooled at 78 c , and n - butyllithium ( 1.6 m in hexanes ) was added dropwise over 5 min . to a solution of o - trimethylsilyl -(trimethylsilyl)allylic alcohol ( 10 mmol ) in dichloromethane ( 50 ml ) were added allyltrimethylsilane ( 1.1 equiv ) and the desired benzaldehyde derivative ( 1.1 equiv ) . filtration and concentration furnished the crude product s2-a ( 65% ) as a yellow thf solution ( 90.1% pure ) , which was used in the next step without further purification : h nmr ( 500 mhz , cdcl3 ) 6.73 ( ddd , j = 5.5 , 11.0 , 17.0 hz , 1 h ) , 5.77 ( dt , j = 2.0 , 17.0 hz , 1 h ) , 5.69 ( dt , j = 2.0 , 11.0 hz , 1 h ) , 4.72 ( m , 1 h ) 0.76 ( s , 9 h ) ; c nmr ( 62.8 mhz , cdcl3 ) 140.1 , 109.6 , 69.3 , 4.05 ; ir ( neat ) 3430 , 2959 , 1634 , 1250 cm . filtration and evaporation of solvent gave the crude product ( 80% ) , which was almost pure and was used in the next step without further purification . to a solution of syn / anti - s3-z ( 1:1 , 342 mg , 1.01 mmol , 1 equiv ) in thf ( 2.2 ml ) was added s - phos ( 4.2 mg , 0.02 mmol , 0.02 equiv ) , phenylboronic acid ( 184 mg , 1.512 mmol , 1.5 equiv ) , and k3po42h2o ( 500 mg , 2.016 mmol , 2 equiv ) . the relative stereochemistries of precursors s3 ( syn / anti ) were assigned on the basis of the assignment of the corresponding cyclic ethers 1 and 2 . compound ( )-3a was derivatized to the known aldehyde ( )-5 ( scheme 14 ) . enolate formation / benzoylation : to a solution of freshly distilled diisopropylamine ( 41 l , 0.29 mmol , 1.3 equiv ) in thf ( 1 ml ) at 78 c was added n - butyllithium ( 1.6 m in hexanes , 0.17 ml , 0.266 mmol , 1.2 equiv ) slowly . to a solution of ( + ) -4a ( 72% ee , 15 mg , 0.065 mmol ) in etoac ( 1 ml ) was added 10% pd / c ( 3 mg ) ( scheme 15 ) . to a solution of ( )-6 ( 15 mg , 0.064 mmol , 1 equiv ) in pyridine ( 2 ml ) was added 3,5-dinitrobenzoyl chloride ( 74.5 mg , 0.323 mmol , 5 equiv ) , and the mixture was vigorously stirred for 36 h. the reaction was quenched with water ( 2 ml ) and diluted with diethyl ether . in the early stage of this study involving the rearrangement of diastereomers 1a and 2a , we were unable to obtain reproducible isolated yields of both ( 3a ) and ( 4a ) products . the ratios of 3a and 4a in the crude h nmr varied in each experiment , and the determination of the diastereomeric ratio of 3a was complicated by the presence of additional signals in the cyclopropyl region ( 1.80.6 ppm ) . addition of d2o led to disappearance of the singlet at 4.35 ppm , which has been assigned tentatively to the enol oh proton of enolic 3a . the c nmr spectrum of the crude reaction mixture ( different run , same conditions ) shows peaks at 158 and 104 ppm , attributable to the polarized enol c c double bond . interestingly , after column chromatography the ( cyclopropyl)acylsilanes were isolated as a single tautomer ( keto form ) . notice that in the h nmr spectrum of the crude reaction mixture there is no evidence of cyclopentenol 4a , ruling out isomerization of the lithium enolate ( -product ) to the corresponding alkoxide ( -product ) .