These results demonstrate that five-year-olds exhibit “strategic prosociality,” behaving differentially generous as a function of the amount of information available to the recipient about their actions. These findings suggest that long before they develop a rich understanding of the social significance of reputation or are conscious of complex strategic reasoning, children behave more generously when the details of their prosocial actions are available to others.Adults frequently employ reputation-enhancing strategies when engaging in prosocial acts, behaving more generously when their actions are likely to be witnessed by others and even more so when the extent of their generosity is made public. This study examined the developmental origins of sensitivity to cues associated with reputationally motivated prosociality by presenting five-year-olds with the option to provide one or four stickers to a familiar peer recipient at no cost to themselves. We systematically manipulated the recipient’s knowledge of the actor’s choices in two different ways: (1) occluding the recipient’s view of both the actor and the allocation options and (2) presenting allocations in opaque containers whose contents were visible only to the actor. Children were consistently generous  Human adults are unique in that they perform what appears to be an inordinate amount of generous behavior Why do children show prosocial behavior from such an early age? To date, prosocial behavior in children has primarily been explained in terms of intrinsic motivations such as empathy, other-regarding preferences, or a desire for fair outcomes e.g., . Under tTo answer this question, it may be helpful to look at the factors associated with self-presentational motivations and prosocial behavior in adults in an effort to track the developmental trajectory of these tendencies. Recent research suggests that, at least for adults, prosocial actions stem in part from an implicit evolutionarily selfish motivation–to promote one’s reputation Labroides dimidiatus) Cebus apella) audience cues– appear to be extremely important in determining the extent of people’s prosocial behavior across a number of situations, although it is not clear that people are consciously considering their reputations in cases in which they are affected by these cues.Although the presence of an audience clearly affects people’s decisions about when to act prosocially, it is not clear that adults realize the extent to which audiences influence their behavior. Indeed, research suggests that people’s prosocial tendencies are impacted by audience cues that are even incidentally presented. Merely exposing people to eyespots or other subtle audience cues can increase prosociality in adults transparency cues– signals that indicate that people know which kind of action has occurred. As with audience cues, there is much work suggesting that adults are more generous when the specifics of their generosity are made available to others ungenerous ways In addition to being sensitive to whether an audience is present, adults’ generosity also seems to be affected by a second set of cues: those related to the amount of information other parties have regarding the presence, absence, or degree of a prosocial action Although there is reason to suspect that audience cues are conceptually distinct from transparency cues, both types of cues are often inextricably linked in real world situations. Indeed, the presence of transparency cues in non-experimental situations is often contingent on the fact that there is an audience likely to become aware of one’s actions. However, it should be noted that the two sets of cues are in no way mutually exclusive. As noted above, people are sensitive to audience cues (eyespots) in the absence of any true agents gathering information Given that adults clearly modify their behavior in response to both audience and transparency cues, is it possible that young children do the same? Existing developmental literature suggests that children’s behavior more generally is influenced by the presence or opinions of others  and the transparency of the allocations to the recipient . We chose to test five-year-olds because previous research has shown that children at this age can successfully represent the goals and beliefs of others  and recipients . Actors were 32  five year-old children from preschools in the New England area; each actor was paired with another child , who was designated the recipient. In total, 16 actors participated in the visible condition  and 16 actors participated in the occluded condition . During the course of the experiment, the actor made all decisions regarding allocations, while the recipient merely watched and collected whatever resources the actor gave to her. Each child participated in only one session and the children never switched roles (i.e. the recipient never became the actor); both children were made aware of the fact that they would never switch roles, thus eliminating possible confounds associated with concerns about direct reciprocity within the experiment. Interaction between the participants was limited as they were instructed not to talk to one another. All children were paired with individuals from the same classroom and, therefore, were familiar with one another prior to testing.We divided our participants into two separate roles: We constructed a testing apparatus that allowed the actor to choose between two allocation distributions; each distribution included an allocation for the actor and an allocation for the recipient see . In desiyou get if you pull the left side?”). Overall, children seemed to find the apparatus quite intuitive and learned how to operate it almost immediately upon interacting with it. Testing commenced once both participants demonstrated an understanding of the outcomes associated with both sides of the apparatus; the training usually lasted about five minutes.Both individuals were trained on the mechanics of the apparatus using differently colored “bouncy balls” and their understanding of the apparatus was confirmed through probe questions  as allocations, and were told that each sticker was worth a point that they could cash in at the end of the test for a final grand prize. The grand prize for all participants was a large sticker of their choice; this served to eliminate possible social repercussions associated with one child getting an obviously superior prize than the other once they returned to class.Each test session consisted of 16 trials. Two experimenters ran each session. The first experimenter presented the allocation options to the actor while the second experimenter sat next to the recipient and ensured that she paid attention throughout the session. During each trial, the first experimenter presented each container individually by showing the actor the contents and then allowing her to track the placement of the container in one of the four wells on top of the apparatus. The experimenter presented opaque containers in such a way that the actor, but not the recipient, could see the contents. Once the actor was aware of the container’s contents, the experimenter closed the container and placed it in the appropriate location. The order in which the containers were presented was randomized. Once all the containers were in place, the actor had a chance to pull one of the two bars in order to deliver either the left or right distribution. As soon as the actor committed to a distribution and pulled a bar, the experimenter removed the remaining two containers to eliminate additional pulling. Each test session consisted of eight trials using the opaque containers and eight trials using the transparent containers; blocks of opaque/transparent containers were broken up into alternating groups of four trials. Subjects were assigned to one of six different counterbalanced distribution orders.not acting prosocially To ensure that the actor’s choices were motivated by the recipient’s allocation and not their own, the actor’s allocation was constant within trials; actors always received the same number of stickers regardless of which distribution they chose. Research shows that young children often tend to act in ways that maximize their own self-interest when presented with sharing tasks in which they stand to gain from While the allocation options for the actor were kept constant within trials, the allocation options for the recipient were varied. Each trial consisted of one of four different conditions, with each kind of trial presented four times during the test session (twice in opaque containers and twice in transparent containers). For simplicity, distributions will be denoted as a pair, with the actor’s allocation denoted first and the recipient’s allocation second. For example, if the actor chose a distribution in which she got one sticker and the recipient got four stickers, this will be represented as .Precautions were taken to eliminate social cueing from the recipient, assure that the actor’s choices could not be seen in the relevant conditions, and minimize the possibility for conflict or punishment once the children returned to the classroom. First, participants were told the experiment was a “quiet game” and asked not to communicate with one another during the duration of the test session; this eliminated any verbal and non-verbal cuing that took place and freed the actor from having to answer questions about her allocation choices. Second, both children were asked to place their allocations into separate, opaque cloth bags after each trial to minimize distractions and also to reduce the ability of the recipient to directly compare her own allocation to the one received by the actor. At the end of the test session, children were taken to opposite ends of the testing room with different experimenters to count their stickers. All participants were told they had “just enough sticker points to get a big sticker,” which removed any evidence of inequity that may have occurred during the experiment. Taken together, these precautions reduced extraneous social factors that may have caused the actor to behave in a more prosocial manner for reasons other than those for which we were testing.visible condition, in which the actor and recipient were able to maintain visual contact throughout the test session. The other half of actors were tested in an occluded condition, in which a large, opaque piece of foam-board was placed into the apparatus on the end closest to the recipient, eliminating the recipient’s visual access to the actor and the allocation options for the duration of the test session.Finally, to examine the influence of audience cues on allocation decisions, we varied the visibility of the recipient between subjects. Half of our actors were tested in a Each test session consisted of eight critical trials and eight non-critical trials that were presented in a counterbalanced order; as a result, there is no reason to believe that participation in non-critical trials influenced children’s behavior in critical trials. Critical trials were the focus of data analysis, as they were the only trials in which the actor had a choice between a more and less generous allocation for the recipient. Non-critical trials were included in test sessions to ensure that the recipient received a mix of one and four sticker allocations, and therefore, was less able to deduce what the actor’s options had been on any given trial.High Value Choice trials and Low Value Choice trials. In High Value Choice trials, the actor received a high-value allocation (four stickers) for herself and chose between an allocation of four stickers  or one sticker  for the recipient; the  distribution was the prosocial (more generous) choice. The actor received two High Value Choice trials in opaque containers and two high value choice trials in transparent containers over the course of the test session. In Low Value Choice trials, the actor received a low-value allocation (1 sticker) for herself and chose between an allocation of four stickers  or one sticker  for the recipient; the  distribution was the prosocial (more generous) choice. The actor received two Low Value Choice trials in opaque containers and two low value choice trials in transparent containers over the course of the test session.Critical trials were comprised of two types of choices: All Equal trials and Unequal Identical trials. In All Equal trials, both the actor and recipient received the same allocation and that allocation was equal; the actor had the choice between distributions of either  vs.  or  vs. . The actor received one  trial and one  trial in opaque containers and one  trial and one  trial in transparent containers over the course of the test session. In Unequal Identical trials, the actor received either one sticker or four stickers and the recipient received the oppositely valued allocation. Specifically, the actor always received one sticker while the recipient always received four stickers,  vs. , or vice versa  vs. . The actor received one  trial and one  trial in opaque containers and one  trial and one  trial in transparent containers over the course of the test session.Non-critical trials were also comprised of two different types of choices: ps>.10. Within each test session, the actor had eight critical trials: four were high-value choices  and four were low-value choices . We were interested in how often the actor chose the prosocial option (four stickers) over the antisocial option (one sticker).Preliminary tests showed no significant effects for or interactions with gender so the authors collapsed across these groups for the remainder of the analyses; all M = 48%) than when the recipient was occluded from view (M = 22%), F = 15.32, p<.001, suggesting that the lack of a visible audience led to more ungenerous behavior in this task. In addition, we observed a main effect of opacity; actors were significantly more ungenerous when allocations were presented in opaque containers (M = 22%), than when they were presented in transparent containers (M = 48%), F = 18.77, p<.001, suggesting that the amount of information available to the recipient regarding the allocation options also played a role in children’s allocation decisions. Lastly, the main effect of the actor’s own allocation value shows no overall significant difference in prosocial behavior regardless of whether the actor received four stickers (M = 38%) or one sticker (M = 33%), F = 0.72, p = .402.A mixed-model repeated-measures ANOVA was conducted with visibility  as a between-subjects factor and actor’s personal allocation  and container opacity  as within-subjects factors . We founF = 3.18, p = .085. Paired t-tests indicate that children in visible condition were more generous when the containers were transparent (M = 67%) than when the containers were opaque (M = 30%), t = 3.77, p = .002. Similarly, children in the occluded condition were more generous when the containers were transparent (M = 30%) than when they were opaque (M = 14%), t = 2.18, p = .046.Furthermore, the interaction between actor-recipient visibility and container opacity was nearing significance, M = 67%), t = 2.20, p = .044. In contrast, actors selected ungenerous options more often than expected by chance (50%) when the recipient was visible and allocations were in opaque containers (M = 30%), t = 3.11, p = .007, when the recipient was occluded from view but allocations were in transparent containers (M = 30%), t = 3.11, p = .007, and when the recipient was occluded from view and allocations were opaque containers (M = 14%), t = 7.90, p<.001.We next examined whether allocations in each of these four primary conditions differed from chance using one-sample t-tests. Actors distributed allocations more generously than would be expected by chance (50%) on trials in which the recipient was visible and the allocations were presented in clear containers  to the recipient when they received four stickers themselves (M = 72%), t(15) = 2.41, p = .029, but not when they received only one sticker (M = 63%), t(15) = 1.29, p = .216. In contrast, when the recipient was visible but allocations were presented in opaque containers  actors were significantly more likely to give only one sticker (the ungenerous option); this was true regardless of whether the actor received four stickers (M = 31%), t(15) = 3.00, p = .009, or one sticker (M = 28%), t(15) = 2.41, p = .029, themselves.No interactions involving the actor’s personal allocation (1 vs. 4 stickers) approached significance, all M = 25%), t(15) = 2.74, p = .015, but not when they received four stickers, (M = 34%), t(15) = 1.78, p = .096, themselves. Additionally, when visibility between the actor and the recipient was occluded and allocations were presented in opaque containers, rates of giving four stickers were the lowest (and therefore rates of giving one sticker were the highest) and therefore most different from chance; this was true when actors received four stickers (M = 13%), t(15) = 6.71, p<.001, or one sticker (M = 16%), t(15) = 4.57, p<.001, themselves. Finally, the fact that children behaved in patterns that differed significantly from chance for all but one of these conditions suggests that confusion about the experimental paradigm and the apparatus can be ruled out as possible explanations for our pattern of results.When visibility between the actor and the recipient was occluded and, thus, the recipient could not see either the actor or the allocation distributions, overall rates of generosity were very low. When allocations were presented in transparent containers, actors were significantly more likely than chance to distribute one sticker to the recipient when they received one sticker  the tokens cannot be kept and are used to get a pre-determined prize ungenerous behavior.The potential for children to have construed our task in a competitive framework highlights another potential framing of our results. We initially interpreted our findings as showing evidence that children exhibit more generosity when transparency and audience cues are present. However, our results are also consistent with the possibility that transparency and audience cues work the opposite way– rather than increasing children’s generosity, such cues might instead inhibit children’s Under either framing of the results, the upshot is the same: children modify their behavior in response to the presence of audience and transparency cues. When audience and transparency cues were present, children were substantially more willing to give resources to another recipient. Competitive motives may have reduced children’s overall rates of generosity in our study, but children restricted these competitive impulses in cases where the recipient could see them and when the recipient would know if she was given less. We further found that both the presence of an audience and transparency cues independently influenced children’s behavior. Based only on the present results, it is difficult to know whether children acted more generously in the presence of audience and transparency cues, or whether they inhibited their ungenerous tendencies in the presence of such cues.why children exhibit more generous behavior in the presence of audience and transparency cues. One possibility is that children behave more generously because seeing the recipient allows them to have more empathy for that individual Our results also leave open the question of Overall, our results illustrate that children’s prosocial decisions are sensitive to both audience and transparency cues years before children seem to have an explicit understanding of the concept of reputation (for a review see


Defective Ribosomal Products (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from  To defend the host from an infection, the immune system continuously scans cell surfaces for foreign objects. Specifically, a virus inside a cell exploits the host to make copies of its proteins; viral proteins are broken up into peptide fragments; and the fragments are displayed on the infected cell's surface, thereby allowing detection and cell-killing. How these peptide fragments for cell-surface presentation are generated remains unknown. An understanding of this step will lead to rational design of vaccines and insights into tumor immunosurveillance and autoimmunity. One possible mechanism is that the peptide fragments come from defective proteins missing either the beginning or end regions, which may result in a bias. Here, we analyzed locations of a large set of known viral epitopes, peptide fragments recognized by the immune system, within their proteins. We find that all regions of proteins are represented well by the immune system. However, there is a statistically significant bias in the central regions of proteins, which correlate with a pattern of conservation spanning the length of viral proteins. Our results suggest a combined effect of conservation and enhancement of immune responses through repeated exposures in shaping the distribution of known viral epitopes. For example, a dominance of downstream initiation would result in more epitopes detected from the C-terminal ends; conversely, a dominance of premature termination would result in more epitopes detected from the N-terminal regions.Although the DRiPs hypothesis is well into its teens, surprisingly little is known about the nature of the DRiPs To overcome the limitation of the current experimental approaches, we have investigated if a data-driven approach can provide insights into the nature of DRiPs. Namely, the availability of a large repository of immune epitopes stored at the Immune Epitope Database (IEDB) vaccinia virus contributed the greatest number of tested peptides, followed by hepatitis C Virus, lymphocytic choriomeningitis virus, human herpesvirus 5/4 and influenza A virus.Because viruses exploit the translational machinery of the host to synthesize their proteins and are thus relevant in the context of the DRiPs hypothesis, we retrieved all MHC-I restricted T-cell epitopes of viruses for which reference proteomes are available. Top 20 viruses based on number of tested peptides are shown in After determining positions of viral peptides in their reference antigens and calculating normalized positions, we constructed distributions of normalized positions for positives (i.e. epitopes) and negatives as shown in To show positional bias of epitopes, a probability ratio plot (i.e. p(x|positive)/p(x|negative), see Table S1). Using a binomial test for each bin, it was determined whether counts of positives significantly deviate from the expected fraction of positives / = 2394/12974). Out of 5 bins, 4 deviated from the expected .The positional bias of epitopes observed is supported by results of statistical analyses on the corresponding contingency table . After grouping predictions based on supertypes, for each supertype, we generated distributions of normalized positions for predicted ‘binders’ (i.e. those peptides with predicted binding affinities <500 nM) and ‘non-binders’ (i.e. those with affinities >500 nM).Our prediction strategy uses recently developed peptide:MHC-I binding algorithms, which have achieved high accuracies in benchmarks Probability ratio plots derived from the distributions for 12 HLA supertypes are shown in residue positions are used instead of normalized positions (data not shown).The presented results largely reflect results reported in A DRiP-independent factor that could explain the positional bias of viral epitopes is positional bias of protein conservation. Specifically, if ends of proteins are less conserved than the middle region and epitopes tend to be more conserved than non-epitopes, positional bias of epitopes may result. To test this possibility, we calculated conservation scores at the residue-level for proteins of the viruses (See In −8). In To determine if sequence conservation alone can explain positional bias of epitopes, we first had to determine the relationship between conservation and immune recognition. In Next, we combined our estimates of conservation bias over the length of a protein with our estimate of correlation between conservation and immune recognition. Using conservation as a function of normalized position in There are two possible explanations for the observed correlation between immune recognition and conservation of peptides. One explanation is that the immune recognition machinery has evolved to preferably recognize epitopes that are conserved, as evidenced by an overlap of MHC binding motifs on conserved sequence regions found in peptide immunization, rather than with viral infection. To determine whether there is an intrinsic enhanced immune recognition for peptides that are conserved in viral species, we retrieved viral epitopes identified in the context of By leveraging a large set of experimentally determined epitopes from a wide range of viruses stored in the IEDB, we determined positional bias of epitopes in source antigens. The shape of positional bias curve Fig. 1 sThe connection between positional epitope biases with protein conservation is reasonable in the context of boosting effects associated with repeated vaccine administrations. The principle idea behind boosting is that those epitopes already exposed to the immune system tend to dominate in the following exposure In addition to explanations discussed above, there are a number of recent ones that may be relevant. First, Calis et al. have reported correlations between G+C content and potential MHC-I binders Other investigators have also reported epitope conservation for HIV Regarding the MHC-motif specific biases and conservation, it has been reported that predicted binding affinities of HLA molecules positively correlate with conserved regions of a wide range of viruses In conclusion, to better understand mechanistic details of antigen processing steps involving DRiPs, positional bias of MHC-I restricted viral T-cell epitopes was measured. Our findings indicate that there is indeed such bias in antigens, where epitopes at N- and C-termini are under-represented. Although mechanisms associated with translational errors such as downstream initiation and premature termination may contribute to observed positional bias, our data indicate that differential conservation spanning protein length is an alternative explanation.http://www.iedb.org), which is the largest publicly available database of epitopes for infectious agents For each virus listed in First, the query should retrieve epitopes derived from proteins newly expressed in a host cell, rather than epitopes recognized after peptide or protein immunization. Only for newly synthesized epitopes can defective ribosomal products skew the positional distribution of epitopes in antigens. To meet this requirement, we query the IEDB for epitopes identified using assays in which the ‘Immunogen Type’ is a whole ‘Organism’ . Second, we further limit the query to epitopes restricted by MHC class I molecules. Third, we limit the query to epitopes with ‘virus’ as the source organism.We then grouped the epitopes retrieved with the query by viral species. As described below, we want to map all epitopes from one species to a single reference proteome. Therefore, we excluded all viruses for which we do not have reference proteomes available, resulting in a total of 93 different viruses.To ensure consistent calculation of positional bias, we mapped all epitopes onto antigens from a single complete reference genome for each species based on sequence similarity rather than using the source antigens listed in the IEDB, which are those specified by the author mapping the epitope and are derived from different strains and are of divergent quality. For example, an author may have used truncated versions of an antigen, or epitopes may come from a polyprotein of Dengue virus, which later gets cleaved into individual products. Consequently, epitope positions can be made relative to the polyprotein or to final cleavage products.To carry out the mapping, we used NCBI's BLAST with a default setting to search for presence of epitopes in antigens and to retrieve only those hits with exact matches in the reference genome. In addition, we required homology between the originally curated source antigen of the epitope and the antigen in the reference genome using BLAST searches with an E-value cutoff of 0.001, thereby ensuring meaningful mapping. Lastly, we required that there is only a single best match of the epitope in the reference genome to ensure that the position of the epitope in the antigen can be uniquely determined. We did not consider ties because of associated uncertainty in mapping.x, is defined as follows:peptide_start’ indicates the position of the first residue from the peptide mapped onto the protein sequence; ‘protein_length’ and ‘peptide_length’ are lengths of protein and peptide, respectively. The first residue of a protein has a position of 1, rather than 0. Our measure of normalized position, x, has the property that if a peptide contains the first residue of the protein , then its value is zero; if the peptide contains the last residue, then the value is 1.0. Consequently, if all regions of a protein are equally likely to contain epitopes, then one would obtain a uniform distribution of normalized positions in the range . This property has been verified using randomly sampled peptide positions and uniformly sampled peptides from a set of proteins, followed by mapping the peptides onto the proteins with BLAST.To derive a measure of epitope position that is independent of protein length, a normalized position, x, given that only positives were considered.After mapping peptides with positive (i.e. epitopes) and negative T-cell assay outcomes onto their corresponding antigens, we calculated their normalized positions, x, as described in To indicate positional bias, we calculated ratios of probability masses for positive and negative PMFs: p(x|positive)/p(x|negative). Absence of positional bias corresponds to a probability ratio of 1.0 for all bins. A probability ratio less than 1 indicates under-representation of epitopes while greater than 1 indicates over-representation.To determine whether differences in conservation over the normalized position in a protein contribute to positional bias of epitopes, we estimated conservations at the residue level for proteins from the viruses using Rate4site algorithm Figure S1Positional bias curves of epitopes after removing varying amounts of data. For reference, the positional bias curve using all data is shown . The curve labeled ‘v’ refers to exclusion of data from Vaccinia virus. The curve labeled ‘v+h’ refers to exclusion of data from vaccinia virus and Hepatitis C virus. Vaccinia virus had the largest amount of immune epitope data, followed by Hepatitis C virus.(TIFF)Click here for additional data file.Figure S2Weighted combination of supertype-specific positional bias curves of predicted binders based on the frequencies of MHC restrictions observed for the known immune epitope data.(TIFF)Click here for additional data file.Figure S3Scatter plots of predicted binding affinities and their normalized positions for the known immune epitope data. Top panels: normalized positions vs. predicted binding affinities. Bottom panels: bar plots where for each bar, a median of predicted binding affinities is shown. Red line indicates 500.0 nM cutoff. Orange line indicates a median of predicted binding affinities.(TIFF)Click here for additional data file.Table S1Contingency table of number of peptides with positive and negative T-cell assay outcomes after binning based on their normalized positions. The bins cover the range [0..1] and each bin has a fixed width of 0.2.(XLS)Click here for additional data file.Table S2HLA supertypes and their member alleles used for peptide binding predictions. Only those HLA alleles for which IEDB's SMMPMBEC has 9-mer predictors available are shown.(DOCX)Click here for additional data file.


The past year has been an interesting one for those interested in reproducible research. There have been great examples of replicability ,2 in resWhen we launched the call for papers for Open Research Computation in late 2010 we saw a clear need for higher standards. Computational research should stand out as an exemplar of just how reproducible research can be, yet it falls short more often than not. With modern computational tools it is entirely possible to provide packages which allow direct replication of results. It is possible to provide data and code in the form of a functional virtual machine image along with automated tests to ensure everything is working as expected. But alongside this we can support the reader’s ability to modify and re-purpose tools, to run them against new data, indeed to support efforts to deliberately break the system to identify its limitations. In short, to do what we are supposed to do as scientists – replicate, reproduce, and test the limits of our models and understanding.We deliberately set the bar high, because we felt it should be high, and because we felt that current standards were, in general, not high enough. Over the past year commentaries -6 have sAs we looked at papers for ORC we set higher standards. We demanded that testing of the code be described. We required that we be able to fork the relevant code before formal acceptance of the paper. We looked hard at the documentation. Are the papers and the code being published today perfect? No. Are they an improvement over the average? Absolutely. Indeed a big part of that improvement is in ensuring that those imperfections can be identified, and worked on for the future.But arguably we may have set the bar too high. Creating code and documentation to the level that we wanted to see is hard work. And the bottom line is that relatively few groups or projects are willing to put in that work, particularly for publication in an, as yet unproven, journal. So we struggled to get submissions at the level we wanted. And here we ran into the second problem.In approaching our goals in the form of a journal we made an implicit compromise. We wanted to do something radical, but we did it in a form that was familiar and conventional. This was a deliberate tactical move. But problems arise from the straightjacket that a conventional journal form creates. The first is the issue of indexing. To get indexed requires a journal to publish in each month of a calendar year. If you’re trying something new this isn’t so straightforward.that means in a web based world) is a definite no-no.In the end we have decided to fold ORC into another BMC journal, Source Code in Biology and Medicine, as a thematic series. This has a lot of advantages. It means there is less pressure to immediately get the submission numbers up, making it possible to take a longer term approach, and adapt over time as interest, demand and standards change. We can pull papers in from across the BMC portfolio and offer the “badge” of ORC certification as an extra bonus. In this way we get most of the advantages of a journal but avoid many of the pitfalls. But part of our strategy was the thinking that the journal could have a high Journal Impact Factor (JIF) if a reasonable proportion of papers, being highly used tools, got significant numbers of citations. But you only get JIF’s for individual journals – not for collections of papers. And re-publishing articles that have “already been published” (whatever Many of us have felt for a long time that the construct of the journal places artificial constrictions on what we can do in research communication. The restrictions placed on what counts as a journal, what is allowed in a journal, by indexing services are a real drag on innovation. This raises the question of what is required to place innovation at the heart of the system we use to communicate research. How can we build the systems and infrastructure that we use in a way that actively encourages innovation?In part, the answer lies with the papers that we are publishing today, and that we will continue to review for acceptance into this thematic series. Show our working, use open tools, enable others to replicate and to fork our work and our tools and our systems. Embed within those systems the measures of reputation and use and re-use that support the most successful open source projects and arguably also the research projects that are most successful at using the web as a resource.Raising the standards of computational research is an important task, and one that we will continue to pursue by identifying and celebrating papers describing code that reaches those high standards that we have set. But equally we hope to keep learning from the process about what we can apply both to research more generally beyond pure computation and to the process of communicating that research.We need more than just reproducible computational research, we desperately need a step change in our expectations and in the incentives for communicating research in a reproducible form more generally. We need educators and the materials to support them in raising awareness and experience. And we need the development of policy and standards that help us move towards a world where reproducibility and replicability are minimum standards not aspirations. ORC will not be doing this as a separate journal, but through the thematic series we will continue to promote the principles and the lessons we can learn. The problems are real and we need to tackle them.


In 156 years of existence, the prestigious school has given the country and the world some very valuable personalities, among whom we should mention Carol Davila, Nicolae Kretzulescu, Victor Babes, Thoma Ionescu, George Marinescu, Ion Cantacuzino, Francisc Rainer, George Emil Palade.“Carol Davila” University of Medicine and Pharmacy has gained a well deserved prestige in the elite of the Romanian medicine and pharmacy schools thanks to the existence of a high academic, didactic and scientific teaching staff, the excellent professional level of the generations of graduates, recognized both in Romania and abroad, the performances in Romania, the international performances and also the impressive editorial and publishing activity. From the 9400 students, over 1400 are foreigners and over 1300 are PhDs, benefiting from the guidance and experience of over 1600 professors. The General Mayor of Bucharest, Prof. Dr. Sorin Oprescu and the Ministry of National Education, Prof. Dr. Remus Pricopie have honored with their presence in the Congress. Very interesting subjects have been debated in the congress: “From the cells of the cardiovascular system to cardiovascular prevention”; “Urology and nephrology – a perfect relationship”; “Pediatric medicine: challenges of the year 2013”; “Arterial pulmonary hypertension and the systemic diseases: what is new?”;” New challenges in oncology”; “From the molecular investigations to the therapeutic challenges in current clinical practice”; “Cardio-neurology: what is new?”; “Infectious diseases with multidisciplinary implications”; “Dentistry and systemic diseases”, “Pharmacology in current clinical practice”, “Obesity – agony and ecstasy”; “Pregnancy and the systemic diseases”, papers which have generated an alive interest for the specialists. The congress has also included some satellite symposiums and an extremely useful session of posters presentation. Moreover, on the occasion of the congress, two Doctor Honoris Causa ceremonies have taken place and 6 Doctor Honoris Causa titles have been awarded. On the 30th of May, Prof. Antonio Federico, Prof. Martin Burian and Prof. Piero Portincasa have been awarded the honorable title Doctor Honoris Causa. Prof. ANTONIO FEDERICO, a valuable and prominent personality in the field of Neurology and Neurosciences, the author of over 500 scientific articles and of numerous treaties and chapters in important neurology treaties, published in some of the most important international medical publishing houses. His articles have 6000 citations in journals which are indexed in Thomson ISI, his Hirsch Index being 36. Prof. Antonio Federico is the Editor-in-chief of the prestigious publication “Neurological Sciences”. Prof. MARTIN BURIAN is the Chief of Otorhinolaryngology and Surgery of Head and Neck Department in Barmherzingen Schwestern Linz Hospital, Austria, has done 3400 surgeries/ year and the medical assistance of over 20000 patients/ year in ambulatory regimen, is a wonderful surgeon with an inexhaustible thirst for knowledge, but, he is also a great professor. The scientific, didactic and surgical preoccupations of Prof. Martin Burian are relevant in the field of head and neck oncological surgery, as well as reported to the endoscopic and Laser surgical approach of the nasal pathology, the functional and cosmetic surgery in the otorhinolaryngology field. The publishing and editorial activity bears the mark of the fundamental preoccupation for the oncological surgery of the head and neck, for the tumors localized in the ORL field, as well as for the complex chemotherapy and radiotherapy treatment of this pathology, including the publishing of some original personal studies regarding the innervation and the electromyography of the trapeze muscle, regarding the anatomy of the lachrymal canal, the electroneurophysiology of the face muscle, the physiopathology of the salivary glands, as well as the preoccupations regarding the gene expression in squamous-cellular carcinomas of the head and neck or some new chemotherapy protocols, diagnostic (photodynamic diagnosis) protocols or a treatment (electrophoresis) associated with the primary malign or recurrent tumors of the head and neck.Prof. PIERO PORTINCASA, professor of Internal Medicine in Bologna University, professor of Internal Medicine in the Medical School of Bari, President of the Internal Medicine Residency Program, Member of the Academy of Science in Apulia, responsible member of the ERASMUS Program for the University of Medicine in Bari, President of the European Society for Clinical Investigations, Co-editor of the European Journal for Clinical Investigations (Wiley-Blackwell), Member of Honor of the Romanian Society of Gastroenterology and Hepatology, Chief of the Diagnostic and Semiology Unit in “A Murri” Medical Clinic, University of Bari, vice-director of “A Murri” Medical Clinic, University of Bari. Professor Piero Portincasa is involved in the European mobility programs formation (Socrates-Erasmus Lifelong Learning) and is also the leader of a famous international research group in Bari, being involved in translational research, focusing on some aspects of the hepatobiliary diseases  and the physiopathology gastro-intestinal secretions, transport and motility. The ceremony of awarding the honorable title for Prof. Christopher Bull Granger, Prof. Mark Jean Cristophe Humbert and Prof. Frank M. Rümmele has taken place on the 31st of May 2013. Prof. CHRISTOPHER BULL GRANGER, from Duke University of Medicine, one of the most famous universities in the world, is currently director of Cardiac Care Unit in the cardiology division. Much of his activity has been dedicated to clinical research. The publishing activity of Prof. Granger is sensational: 562 articles published in international journals, most of them in New England Journal of Medicine, The Lancet, JAMA, Circulation and European Heart Journal. Among them, 426 are original articles, very important for the cardiologic research and 136 are reviews and editorials. Moreover, Prof. Granger has published 24 books and book chapters in important international treaties such as the ones edited by Rob Califf, Eric Topol, or Eugene Braunwald. Prof. Christopher Granger is a member of the editorial committees of 8 international cardiology journals. He is a fellow of the American College of Cardiology, American Heart Association and European Society of Cardiology. For his research and publishing activity he has received Top 10 prize twice, for research in USA, in 2007 and 2012. He had a decisive contribution in developing a research institute in Brazil, being a member of honor of the Brazilian Society of Internal Medicine. Prof. FRANK M. RÜMMELE, professor of pediatrics in Rene Descartes University of Medicine in Paris, responsible with the intestinal immunopathology program in the pediatrics department and is the Chief of the Pediatric Gastroenterology Clinic in Necker Enfants Malades Hospital. Prof. Frank Rümmele is currently the P-ECCO chief, GETAID president – the pediatric branch, vice-president of the Francophone Group of Gastroenterology, Hepatology and Nutrition (GFHGNP), vice-president of ESPGHAN working group on intestinal inflammatory diseases. He has gained lots of titles and accreditations, already having over 15 prizes and distinctions received for different scientific researchers presented and/ or published, the most recent distinction being received in 2011: Laureate of Sorbona University in Paris with the excellency prize for scientific research. He is the coordinator of numerous research grants and the author of over 100 original papers published in very prestigious journals and he is the author of many prestigious books in pediatrics gastroenterology . His preoccupations have been oriented towards the aspects concerning the anatomical and immunological modifications in the intestinal inflammatory affections which appear in pediatric pathology. Prof. MARC HUMBERT, is the president of South Paris Research Center for Care in Pulmonary Hypertension, Director of INSERM Unit 999 “Pulmonary hypertension: pathophysiological and innovative therapies”. His international consecration has brought him the title of President of the Social European Group for Respiration “Pulmonary circulation and vascular diseases”. His scientific papers and research have offered him the possibility to publish papers in fields such as asthma, pulmonary tension and pulmonary inflammation. These accomplishments have brought the author over 290 reviews of the articles in the most famous journals in the world , as well as in top journals in the field of problems with breathing and cardiovascular medicine . He has accumulated 13000 citations with 40 papers cited over 100 times, two of them being cited for more than 600 times. His Hirsch Index is 61. In 2006, he has been awarded the title European Respiratory Society Cournand Lecture (Course on the theme “The burden of pulmonary hypertension”), a special distinction for a young scientific personality. In 2009, the Royal Academy of Arts and Sciences in Netherlands has offered him the Descartes-Huygens Prize. In 2010 he was awarded the title of vice-president of Assistance Publique Hopitaux de Paris, responsible for the research department and, currently, he is the president of AP-HP Research Committee. The members of the Students Choir of “Carol Davila” University of Medicine and Pharmacy have brought their important contribution in intensifying these emotional ceremonies.The congress had an exceptional scientific value and the presence of over 1700 participants is an alive proof of the success of the event. The generous efforts of the University together with the rigorous and efficient effort of the organizers have given value and academic performance to the congress. Purcarea VL, MD, PhD, EngExecutive Editor


Consensus has been lacking as to how to reconstruct the posterolateral corner (PLC) of the knee in patients with posterolateral instability. We describe a new reconstructive technique for PLC based on Larson's method, which reflects the physiological load-sharing pattern of the lateral collateral ligament (LCL) and popliteofibular ligament (PFL).Semitendinosus graft is harvested, and one limb of the graft comprises PFL and the other comprises LCL. Femoral bone tunnels for the LCL and popliteus tendon are made at their anatomical insertions. Fibular bone tunnel is prepared from the anatomical insertion of the LCL to the proximal posteromedial portion of the fibular head, which corresponds to the insertion of the PFL. The graft end for popliteus tendon is delivered into the femoral bone tunnel and secured on the medial femoral condyle. The other end for LCL is passed through the fibular tunnel from posterior to anterior. While the knee is held in 90 of flexion, the graft is secured in the fibular tunnel using a 5 mm interference screw. Then, the LCL end is passed into the femoral bone tunnel and secured at the knee in extension.Differential tension patterns between LCL and PFL is critical when securing these graft limbs. Intrafibular fixation of the graft using a small interference screw allows us to secure these two graft limbs independently with intended tension at the intended flexion angle of the knee. Generally, posterolateral corner (PLC) reconstruction is performed to treat chronic posterolateral instability in patients with PLC injury. However, consensus has been lacking as to how to reconstruct the PLC. Abundant surgical procedures for PLC have been accumulated, and can be broadly divided into two types: anatomical and non-anatomical. Non-anatomical reconstructions include biceps tenodesis ,2, arcuaTM  is carried used at the both ends of the graft, and one end of the popliteus tendon is connected to an EndobuttonTM , then placed within antibiotic-soaked gauzes and set aside for later use.The patient is positioned supine on the operating table with an arthroscopic leg holder, after precise diagnosis of posterolateral instability and concomitant injuries. Arthroscopy is performed to identify lateral drive-through sign as well as concomitant disruption of the anterior cruciate ligament (ACL) or posterior cruciate ligament (PCL). If the decision has been made to reconstruct either of the cruciate ligaments, this should be performed first. After cruciate ligament reconstruction, PLC reconstruction is initiated. Semitendinosus (ST) tendon is normally harvested ipsilaterally using a smooth tendon stripper, but if ipsilateral ST tendon is planned for use as either PCL or ACL graft, the graft for PLC is harvested from the contralateral ST tendon. The appropriate length of ST graft for PLC is 16–19 cm, which typically reflects the distance between the femoral and fibular insertions of the PLC plus an additional 30 mm, as both ends of the graft require at least 15 mm each, corresponding to the insertion into bone tunnels. One limb of the graft comprises PFL and the other comprises LCL. A baseball glove suture using FiberWireA lateral incision is started just proximal to the lateral femoral epicondyle and followed distally to the midpoint between Gerdy’s tubercle and the fibular head Figure . FurtherThe posterolateral aspect of the fibular head is exposed, and attachments of the LCL and PFL are identified. The peroneal nerve should be identified, marked with tape, and retracted with the biceps muscle. To prepare the fibular tunnel, the starting point is set at the distal anterolateral portion of the fibular head, corresponding to the anatomical insertion of the LCL, and the guide pin should exit the proximal posteromedial portion of the fibular head, which corresponds to the insertion of the PFL. When the patient is of short stature and the fibular head is small, the location of the transfibular tunnel, particularly the LCL insertion site, should be changed from the anatomical site slightly anteriorly to avoid any risk of avulsing the fibular head with the reamer Figure A. At thiTM is delivered into the femoral bone tunnel and flipped on the cortex of the medial femoral condyle. The other end for LCL is then delivered under the iliotibial band and biceps femoris and is passed through the fibular tunnel from posterior to anterior  with 10 N force applied to the graft at the knee in extension and neutral rotation by use of a ligament tensioner (Smith & Nephew Endoscopy)  exercises are initiated using a continuous passive motion (CPM) device and are permitted from 0° to 90° of flexion during weeks 3 and 4. From week 4 on, >90 ° of flexion is permitted. The knee is maintained at 0° except during ROM exercises. A hinged functional brace is used for 3 months postoperatively. Partial weight-bearing with the brace locked in extension is initiated at 2 weeks postoperatively, with gradual progression to full weight-bearing by 4 weeks postoperatively.Written informed consent was obtained from the patient for publication of this report and any accompanying images.Injuries to the PLC of the knee can result in severe disability due to both instability and articular cartilage degeneration. These injuries do not commonly occur in isolation, but are usually found in the setting of other injuries, such as ACL or PCL ruptures. Most authorities recommend surgical reconstruction of the PLC in combination with ACL or PCL reconstruction -13, sincHistorically, numerous techniques for PLC reconstruction have been described, but which technique represents the best method for reconstructing physiologically functional PLC remains controversial. According to the distal insertion site of grafts for PLC, two surgical techniques are available: fibular-based techniques and combined tibial-fibular-based techniques. Larson’s procedure was one of the first fibular-based techniques, and reconstructs the LCL and PFL with distal insertion sites located at the fibula . Larson’Tibial-fibular-based techniques have gained increasing attention due to their nature of more anatomical reconstruction capable of reconstructing all three major PLC components at each precise insertion site, but certain investigations have reported that these techniques potentiate overconstraint of posterolateral instability ,15. We bAlthough favorable short-term results of tibial-fibular-based techniques have been reported ,8, furthThe authors declare that they have no competing interests.YN and HM have conceived and established the surgical techniques. YN and HE have performed the surgery. YN, HM, and TO have contributed to drafting of the manuscript. YS and YT have contributed to proof check of English. All authors have read and approved the final manuscript.


Mediterranean Journal of Hematology and Infectious Diseases (MJHID) is a new open access, peer-reviewed, online journal, which encompasses different aspects of clinical and translational research providing an insight into the relationship between acute and chronic infections and hematological diseases. MJHID will be a topical journal on subjects of current importance in clinical haematology and infectious diseases. Every issue should have, beside the editor in chief, a guest editor. Both editor in chief and guest editor provide to invite experts in the selected topic to performe a complete update of the arguments readily available for practising phisicians. The journal will have also a section devoted to original papers, case reports and letters to editor and Editorial comment mostly focusing on the arguments treated in the previous topical issues. Reciprocal interdependence between infectious and haematologic diseases  is well known.27Splenectomy frequently performed autoimmune diseases as well in lymphoproliferative diseases is a further cause of immunodeficiency.28helicobacter pylori and B lymphoproliferative diseases are well known65Nevertheless, the main cause of immunodeficiency at present is due to HIV infection in developed as well in developing countries.Urgent pragmatic and efficient measures are needed both at international and national levels. Most of the peoples affected by neglected infectious diseases live in Central Africa, South Asia This should be possible bridging the health system gap between the south and the north borders sides of Mediterranean Sea while improving scientific collaboration.Case report should have educational character. Clinical trials of hematologic and infectious diseases present of Mediterranean area will have their natural home in this Journal. Also case studies relative to important health care intervention in developing countries of south Mediterranean areas will be accepted. All that should contribute to disseminate updated informations among physicians of developing countries on the more frequent diseases of the Mediterranean area.The results of relevant studies in the field should be made available to scientists and clinicians, in particular those in developing countries where the fight against pathogen related cancers is of the utmost importance.Every accepted manuscript will be available for reading on its date of acceptance by any person who has access to the web in web-friendly XML. Considering that every issue will be devoted to a topic, when all articles of the same topic will be available they will be formatted and the typeset PDF version will be available. The first issue of the MJHID has been dedicated to “Update in Diagnosis and Treatment of Thalassemia and related Disorders” Guest Editor Prof. Maria Domenica Cappellini.


One of the great challenges for an HIV vaccine is to elicit broadly neutralizing antibodies specific for conserved epitopes from which the virus cannot easily escape. The CD4 binding site is one such epitope against which several antibodies  have been isolated. In macaques infected with SHIV, passive immunization with these CD4-directed neutralizing antibodies fails to control the virus, but prophylactic administration is highly protective. Similarly, patients who generate neutralizing antibodies over the course of an HIV infection derive no clinical benefit from them, but eliciting such antibodies prophylactically by vaccination may prevent the virus from establishing its lethal foothold.Yeast surface display is a powerful method for rapidly engineering complex glycoproteins. We have developed a stripped core gp120 that presents a functional CD4 binding site when displayed on yeast, and an accompanying suite of tools with which to map conformational epitopes of neutralizing antibodies, design novel immunogens, and monitor the specificity of serum following immunization.We map the epitopes of the anti-gp120 antibodies VRC01, b12, and b13, and uncover subtle energetic differences in their nearly-overlapping epitopes that are not obvious from existing crystal structures. With this information, we design novel gp120 immunogens that share the VRC01 epitope but whose other surface amino acids are highly diverse. When mice are immunized with these immunogens in a heterologous prime-boost format they elicit VRC01-competitive antibodies and not the competing immunodominant specificities seen with a single immunogen. The antisera are tested for their binding to a panel of gp120, and their neutralization potential is assayed.We have engineered a novel set of immunogens that elicit CD4 binding site-directed antibodies upon immunization. The yeast display tools may be used to design future immunogens.


Lung cancer cells express different chemokines and chemokine receptors that modulate leukocyte infiltration within tumor microenvironment. In this study we screened several mediators/growth factors on CXCL1 release in human carcinoma epithelial cells. Of the tested mediators, VEGF was found to have a robust increase in causing CXCL1 release. VEGF stimulated CXCL1 release and mRNA expression in a time- and concentration-dependent manner. The release was inhibited by the VEGF receptor antagonists and the JNK, PI-3K, tyrosine kinase, and transcription inhibitors. In parallel, VEGF induced JNK, PI3K and Akt activation. Strikingly, among these inhibitors only the JNK inhibitor could reduce VEGF-induced CXCL1 mRNA expression, suggesting that JNK participated in VEGF-induced CXCL1 synthesis, whereas PI-3K was responsible for cellular CXCL1 secretory process. In addition, the steroid dexamethasone and TGF-β suppressed CXCL1 release through a transcriptional regulation. We also showed that cells stimulated with VEGF significantly attracted monocyte migration, which could be abolished by CXCL1 B/N Ab, CXC receptor 2 antagonist, TGF-β, and dexamethasone. In summary, we provide here evidence showing JNK activation for VEGF-induced CXCL1 DNA transcription and PI-3K pathway for extracellular CXCL1 release in human carcinoma epithelial cells. The released CXCL1 was functionally linked to recruiting monocytes into lung cancer cell microenvironment. These chemokines are potent promoters of angiogenesis, as the recruited neutrophils are known to synthesize and store angiogenic molecules like vascular endothelial growth factors (VEGFs) [CXCL1, also known as growth-related oncogene protein-α (GRO-α) or melanoma growth stimulatory activity factor (MGSA), is a polypeptide which was initially isolated from Hs294 human melanoma cells. CXCL1 is one of the members of chemokines (chemotactic cytokines), which are small heparin-binding proteins that generally direct the movement of circulating leukocytes to sites of inflammation or injury [ (VEGFs) ,4.in vitro and in vivo in Lewis lung carcinomas (LLC) [VEGF represents a family of homodimeric glycoproteins which are critical for the embryonic development of the blood vascular system (vasculogenesis), lymphatic system (lymphangiogenesis) and in the formation of new blood vessels from pre-existing vessels (angiogenesis) in physiological and pathological conditions . VEGF bias (LLC) .In human airway epithelium and bronchoalveolar macrophages, monocyte chemoattractant protein-1 (MCP-1) and CXCL1 were constitutively expressed and upregulated by TNF-α but not by lipopolysaccharide (LPS) . In pathConsidering the significance of CXCL1 in human airway epithelium and in pathological processes such as chronic inflammation and lung cancer, in this study we screened several proinflammatory mediators and growth factors in inducing CXCL1 release in human A549 lung carcinoma epithelial cells. We found a marked enhancing effect by VEGF. Therefore, the effects on CXCL1 release in A549 cells by VEGF were further investigated. We showed that VEGF induced CXCL1 expression through a transcriptional regulation in A549 cells. The possible underlying mechanisms were determined, which showed that VEGF regulated CXCL1 production through JNK- and PI-3K-dependent pathways.To investigate which proinflammatory cytokines or growth factors affected CXCL1 release in A549 lung epithelial cells, an ELISA for measuring CXCL1 in A549 culture medium was performed. Next, we examined the concentration- and time-effect of VEGF on CXCL1 release in A549 lung epithelial cells. As shown in To further examine whether VEGF induced CXCL1 mRNA expression, A549 cells were treated with VEGF and CXCL1 and β-actin mRNA expression was evaluated by RT-PCR. As shown in To investigate the possible signaling pathways involved in the induction of CXCL1 by VEGF, signaling inhibitors targeting MAPKs, PI-3K, protein kinases, NF-κB signaling pathway, and DNA transcription were used. Among these inhibitors, it was found that the CXCL1 release by VEGF was significantly affected by the following inhibitors, including the VEGFR antagonists (Sunitinib and SU5416), JNK inhibitor , PI-3K inhibitor , and tyrosine kinase inhibitor  . MoreoveWe next examined whether SP and LY had a similar effect on VEGF-induced CXCL1 mRNA expression. Surprisingly, the real-time PCR analysis indicated that only SP reduced VEGF-induced CXCL1 mRNA expression, whereas LY had no such inhibitory effect . The RT-As JNK and PI-3K inhibitors reduced VEGF-induced CXCL1 release, we next examined whether VEGF could directly activate related signaling pathways in A549 cells. To evaluate the functional role of CXCL1 secretion by A549 cells in recruiting monocyte migration, a modified Boyden chamber transmigration system was used. The lower chamber was seeded with/without monolayered A549 cells, which assembled with the upper chamber added with U937 monocytes to form a coculture system. In the absence of A549 cells but presence of VEGF, there were no migrated monocytes, indicating that VEGF alone was not sufficient to cause monocyte migration. However, a slight increase of migrated monocytes was observed in the presence of seeded A549 cells in the lower chamber and a robust increase of monocytes in the presence of A549 cells stimulated with VEGF , suggestAlterations in TGF-β signaling are linked to a variety of human diseases, including cancer and inflammation. Disruption of TGF-β homeostasis occurs in several human cancers such as lung cancer –19. TGF-in vitro model  are also highly expressed in lung tumors in mice and humans [Some of the chemokines and cytokines have been found to be regulated in the d humans ,24,25. IIt has been shown that NF-κB mediates IL-1/TNF-α induction of CXCL1 in human fibroblasts  and protin vitro, including phospholipase Cγ (PLCγ), PI-3K, ERK1/2 and etc. However, VEGFR1 has been shown to regulate endothelial cells (ECs) via cross-talk with VEGFR-2. VEGFR-2 is the principal mediator of several physiological and pathological effects of VEGF-A on ECs. The intracellular signaling pathways mediating these effects downstream of VEGFR-2 activation include PLCγ, p38 MAPK, PI-3K, ERK1/2 and etc. [CXCL1 DNA transcription needs to be further investigated as Tanshinone IIA (an AP-1 inhibitor) did not significantly affect VEGF-induced CXCL1 release , wortmanin, and actinomycin D were purchased from Sigma Chemical Co. . SU3327 was from Tocris Bioscience . Human recombinant VEGF-A was purchased from Prospec Biotech . Human EGF, IGF, and bFGF were from Invitrogen Life Technologies . The antibody (Ab) raised against phospho-ERK1/2 was from Santa Cruz Biotechnology . The Abs raised against total p38 MAPK, phospho-p38 MAPK, and phospho-JNK were from Cell Signaling Technology, Inc. . Human IP-10, SDF-1, PDGF, TNF-α, and the Abs for total p38, ERK1/2, and CXCL1 blocking/neutralizing Ab (B/N Ab) were from R & D systems, Inc. . ATP and ADP were purchased from Affymetrix USB Products . U46619 was from Enzo Life Sciences, Inc. . Sunitinib malate was from Biovision  and SU5416 was from Cayman Chemical Co. . SIS3 and α-tubulin Ab were purchased from Calbiochem EMD Bioscience Inc. .l-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and 10% FBS.A549 cells, a human pulmonary epithelial carcinoma cell line with type II alveolar epithelial cell differentiation, were from Food Industry Research and Development Institute . The cells were cultured in DMEM/Ham’s F-12 nutrient mixture containing 10% FBS , penicillin (100 units/mL), streptomycin (100 μg/mL) and fungizone (250 ng/mL) . U937 monocytes were also from Food Industry Research and Development Institute and cultured in RPMI 1640 medium with 2 mM Secreted CXCL1 in culture medium was determined by human CXCL1 ELISA Development kit  according to the manufacturer’s protocol. Briefly, A549 cells were treated with vehicle or stimulators. The culture media were collected and centrifuged and CXCL1 release in culture medium was measured. The product of this enzymatic reaction was yellowish color and absorbs strongly at 412 nm. The intensity of this color is proportional to the amount of CXCL1 present in the well after the incubation. The CXCL1 concentrations in A549 cell culture medium were calculated from the standard curve.Cell viability was assayed as previously described . BrieflyCell lysate was prepared as previously described . Total pTaq DNA polymerase . PCR was performed with a hot start at 94 °C for 5 min and then with 30 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and elongation at 72 °C for 1.5 min on the ABI 7200 Thermal Cycler . The amplification products were then analyzed by gel electrophoresis in 2% agarose. For some experiments, CXCL1 mRNA level was analyzed by real-time PCR with the TaqMan gene expression assay system , using primers/probe sets Hs.708652 for human CXCL1 and Hs.520640 for human β-actin (as a control). PCRs were performed using a 7500 Real-Time PCR System . Relative gene expression was determined by the ΔΔCt method, where Ct was the threshold cycle. All experiments were performed in duplicate or triplicate.Oligonucleotide PCR primers targeting to human CXCL1 and β-actin were synthesized. The forward and reverse primers for CXCL1 were 5′-GCCCAAACCGAAGTCATAGCC-3′ and the forward and reverse primers for β-actin were 5′-ATCATGTTTGAGACCTTCAA-3′ and 5′-CATCTCTTGCTCGAAGTCCA-3′, respectively. Total RNA of A549 cells was extracted by Trizol reagents (Invitrogen Technologies) and reverse transcription reaction was performed by using Superscript III First-Strand Synthesis System (Invitrogen Technologies). Briefly, aliquots of 1–2 μg total RNA were incubated with random hexaprimers for 10 min at 65 °C and chilled on ice shortly. After primer annealing, RNA was reverse transcribed by the reverse transcriptase. Reactions were stopped and RNase H was added to remove RNA. Aliquots of transcribed cDNA were subjected to PCR in 25 μL of reaction mixture containing reaction buffer, dNTP, primers, and in vitro DNA Transfection Reagent  for 18 h in medium according to the manufacturer’s protocol. All DNAs were prepared using endotoxin-free plasmid preparation kits . All transient transfections included 0.375 μg of CXCL1 reporter construct and pSV-β-galactosidase control vector . Following transfection, cells were washed once with endotoxin-free medium and then allowed to grow for 16 h in complete medium containing antibiotics. CXCL1 reporter firefly luciferase values were obtained by analyzing 1 mL of purified cell extract according to standard instructions provided by the Luciferase Kit  in a Wallac Victor 3 1420 multilabel counter .The wild-type CXCL1 promoter fragment spanning nucleotides −1047 to −11 of the CXCL1 promoter cloned into pXP2 luciferase reporter plasmid was cloned. Briefly, the region was amplified from genomic DNA of A549 cells using the primers with linkers and restriction enzyme sites for cloning to the pGL3-luciferase reporter plasmid. The primers were 5′-GTGGAAGGTGCTT GCACACCAGG-3′ (forward) and 5′-GGAGCAGCAGTGCCACTCGCAGG-3′ (reverse). The accuracy of CXCL1 sequence was confirmed by DNA sequencing. Cells at approximately 80% confluence in 6-well culture plate  were transfected with 0.75 μg of total DNA, using PolyJet™ 5 cells/mL) and then assembled with the lower chamber. The coculture system was allowed to incubate at 37 °C for 16 h. All nonmigrant monocytes were removed from the upper face of the Transwell membrane with a cotton swab and migrated monocytes were fixed and stained with 0.5% toluidene blue in 4% paraformaldehyde. Migration was quantified by counting the number of stained cells per × 100 field  under a phase-contrast microscope  and photographed.Monocyte migration assay was performed using a modified Boyden chamber model . The lower chamber was seeded with/without A549 cells. After 90% of confluency, cells were filled with serum-free or VEGF-containing medium in the presence of vehicle, CXCL1 B/N Ab, CXCR2 inhibitor, TGF-β, or dexamethasone. The lower face of polycarbonate filters (Transwell insert) were coated with gelatin (20 μg/mL) for 30 min in the laminar flow hood. The upper chamber was loaded with human U937 monocytes (2.5 × 10t-test.Data were expressed as mean ± standard error of the mean (SEM). The means of two groups of data were compared using the unpaired, two-tailed Student’s In conclusion, in the present study we demonstrate that VEGF can induce CXCL1 mRNA and protein expression in A549 carcinoma epithelial cells through VEGFR, JNK and PI-3K-dependent pathway. Our results suggest that JNK is essential for CXCL1 synthesis, whereas PI-3K is for cellular CXCL1 release. The induction of CXCL1 release by VEGF in A549 cells functionally leads to the recruitment of monocytes toward themselves in the microenvironment. Lung cancer and/or cancer cells express different chemokines that chemokine receptor that modulate leukocyte infiltration within tumor microenvironment. Our results suggest the contribution of VEGF and elucidate its possible mechanism in causing CXCL1 release.


P < 0.01). No association was observed between donor gender and ΔHct, which was 3.02% following receipt of female blood versus 3.23% following receipt of male blood (P = 0.21). By multivariable analysis, recipient gender remained associated significantly with ΔHct (P < 0.01). In conclusion, recipient gender is independently associated with ΔHct following pRBCs transfusion. This association does not appear related to either demographic or anthropomorphic factors, raising the possibility of gender-related differences in recipient immune response to transfusion.The change in hematocrit (ΔHct) following packed red blood cell (pRBCs) transfusion is a clinically relevant measurement of transfusion efficacy that is influenced by post-transfusion hemolysis. Sexual dimorphism has been observed in critical illness and may be related to gender-specific differences in immune response. We investigated the relationship between both donor and recipient gender and ΔHct in an analysis of all pRBCs transfusions in our surgical intensive care unit (2006–2009). The relationship between both donor and recipient gender and ΔHct (% points) was assessed using both univariate and multivariable analysis. A total of 575 units of pRBCs were given to 342 patients; 289 (49.9%) donors were male. By univariate analysis, ΔHct was significantly greater for female as compared to male recipients (3.81% versus 2.82%, resp.,  Transfusion of critically ill patients with packed red blood cells (pRBCs) is commonplace; nearly all anemic intensive care unit (ICU) patients with a length of stay (LOS) ≥ 7 days receive at least one pRBCs transfusion , 2. DespResearch on the impact of gender on ICU outcomes has received recent attention and yielded conflicting results. Several studies have reported sexual dimorphism with respect to both overall and infection-related mortality among critically ill patient cohorts ranging from severe trauma , 13 to pEvidence of a possible immune-related sexual dimorphism with respect to outcomes following blood product transfusion has been reported by our group and others. Specifically, transfusion-related acute lung injury appears both more frequent and severe following transfusion of female blood as compared to male blood , 22. FurWe have investigated the change in hematocrit (ΔHct) following pRBCs transfusion as a clinically relevant potential surrogate marker of posttransfusion hemolysis. Specifically, based upon the associations between pRBCs storage time, accumulation of inflammatory mediators –29, and This study was a cross-sectional analysis of all leukocyte-reduced, pRBCs transfusions administered to critically ill patients from June 2006 through June 2009 at a 20-bed academic surgical ICU. Only single-unit transfusions that occurred while the patient was in the ICU were included; multiple unit transfusions as well as transfusions administered at other locations  were excluded.post) and the last pretransfusion value (Hctpre). The time from Hctpre determination to the blood transfusion , as well as the time from the blood transfusion to Hctpost determination  were also abstracted. In order to access for a potential effect of fluid equilibration over time, we also abstracted the second hematocrit determination following pRBCs transfusion . This data point was used to calculate ΔHct2 , defined as the difference between Hctpost2and Hctpre. Transfusion of additional blood products  along with the pRBCs transfusion was also recorded.The independent variables of interest were both donor and recipient gender. For the purposes of this analysis, gender refers to sex chromosomal status: XX for female and XY for male. The outcome variable was the ΔHct following pRBCs transfusion . ΔHct was calculated as the difference between the first posttransfusion value , recipient admission diagnosis (trauma versus other), the indication for transfusion , recipient admission weight (kg), recipient admission body mass index , recipient 24 hour fluid balance (L), time from surgical ICU admission to transfusion (days), Hctpre (%), and the age of transfused blood. Recipient ICU LOS (days) as well as mortality was also abstracted. The following covariates were hypothesized P < 0.05 being considered significant statistically, unless indicated otherwise. Continuous data are expressed as mean, range; categorical data are expressed as number, %. Correlations between predictor variables and ΔHct were assessed using Pearson's correlation coefficient (r2). The means of two continuous variables were compared using the student's t-test. The means of >2 groups were compared using ANOVA (F statistic); individual group differences were then compared using Tukey's test in the case of overall significance. It the case of multiple comparisons Bonferroni-corrected P-values were used and calculated as 0.05/k, where k is equal to the number of groups analyzed. Differences in proportions of categorical variables were compared using the chi-square test, except when expected cell counts were <10, in which case Fischer's exact test was used. Multivariable linear regression (sum of squares) was used to access for the independent contribution of gender to the variability in ΔHct. Variables correlated with either recipient gender or ΔHct (or both) at the P ≤ 0.10 level by univariate analysis were entered into the model using a forward selection method. Overall model significance was calculated using the F statistic, with the significance of individual variables calculated using the t statistic. This study was approved by the Colorado Multiple Institutional Review Board (Protocol no.10-0028).All statistical analyses were performed using SAS version 9.2 . The alpha error level was set at 0.05, with pre was 21.5 (range 8.2–32). The mean pRBCs storage time was 29.5 days, and 94.3% of pRBCs units were ≥14 days old. Donor gender was distributed equally among male (49.9%) and female (50.1%) donors.A total of 1,011 units of pRBCs were administered during the study period. Of these, 594 (56.9%) units were given to 342 patients as single-unit transfusions. Complete data were available for 575 (96.8%) units, which served as the final sample size. Sample demographics are shown in r2 = 0.23, P < 0.01) and Hctpre  were correlated most strongly with ΔHct. Additional significant correlations [in order of decreasing magnitude (|r2|)] were as follows: increasing recipient admission weight was associated with a decreased ΔHct , transfusion for ICU anemia as opposed to hemorrhage was associated with a decreased ΔHct , increasing age was associated with an increased ΔHct , increasing 24 hour fluid balance was associated with a decreased ΔHct , and increasing Tpre was associated with increased ΔHct . Admission diagnosis, recipient admission BMI, time from ICU admission to transfusion, Tpost, the age of blood, donor gender, and transfusion of additional blood products with the pRBCs unit were not correlated significantly with ΔHct.Correlations between independent variables and ΔHct are listed in P < 0.01), more likely to have an admission diagnosis of trauma (P < 0.01), more likely to be transfused for an indication of ICU anemia (P < 0.01), had a higher BMI (P < 0.01), and received older blood (P < 0.01).P < 0.01). However, donor gender did not impact ΔHct, which was 3.02% following receipt of female blood versus 3.23% following receipt of male blood (P = 0.21). In order to assess for any possible effect of fluid equilibration over time, these analyses were repeated using ΔHct2  instead of ΔHct. The mean ΔHct2 was 2.97%  and the mean Tpost2 was 14.1 hours (range 1–92). ΔHct2  remained significantly higher for female as compared to male recipients . Furthermore, donor gender was not associated with ΔHct2   .By univariate analysis, ΔHct was significantly greater for female recipients, as compared to male recipients , analysis of differences between individual pairs revealed only those with disparate recipient genders to be significant. By contrast, ΔHct was not significantly different when comparing pairs with disparate donor genders and identical recipient genders. Differences in ΔHct as well as adjusted P-values for individual pair comparisons are listed in The relationship between both donor and recipient gender and ΔHct was explored further by stratifying the sample into the 4 donor-recipient gender pairs listed in pre, recipient admission weight, Tpre, age of blood, and recipient 24 hour fluid balance. Of note, recipient BMI was not included in the model based on a high degree of colinearity with recipient weight . After controlling for these covariates, recipient gender remained significantly associated with ΔHct  . In additional to recipient gender, patient age, Hctpre, recipient admission weight, and Tpre were also associated independently with ΔHct.Results from multivariable logistic regression analysis are shown in  In this cross-sectional analysis of anemic, critically ill surgical patients, we observed an independent association between recipient gender and ΔHct following pRBCs transfusion. By contrast, a relationship between donor gender and ΔHct was not observed. Specifically, ΔHct was higher for female, as compared to male recipients, irrespective of donor gender.pre and Tpost, would appear to influence ΔHct but were not associated strongly with either recipient gender or ΔHct in this analysis, nor did they significantly alter the observed relationship between these two variables in multivariable analysis. Furthermore, because pRBCs transfusion in effect entails adding a relatively fixed volume and concentration of erythrocytes to a variable recipient volume of distribution and Hct, variables such as Hctpre, recipient weight, 24 hour fluid balance, and recipient BMI are of relevance. Indeed, each of these variables was correlated with ΔHct but did not explain the association between recipient gender and ΔHct. Finally, male and female recipients may have differed with respect to the condition of the donor erythrocytes themselves. Specifically, we recently observed that older blood is less effective in raising the Hct [P < 0.01), this relatively small degree of difference in storage time was not found to significantly alter ΔHct in our previous work [Demonstration of sexual dimorphism with respect to recipient gender in this retrospective analysis raises the issues of both confounding and causality. In terms of the former, several covariates may confound the relationship between recipient gender and ΔHct. Chronologic factors, such as T the Hct . This poous work . FurtherThe immunomodulatory properties of pRBCs transfusions involve both donor and recipient qualities. Previous work has implicated transfusion of female blood in both antibody-mediated acute lung injury  and cancWe hypothesized originally a smaller ΔHct for female, as compared to male recipients based upon possible immune or sex hormone-mediated posttransfusion hemolysis. However, the opposite effect was observed. One possible explanation for this finding involves immunologic tolerance via microchimerism. Microchimerism entails proliferation of a relatively small (<5%) population of cells  from a distinct zygote and as a result of mixing of blood between the two organisms. Examples of circumstances leading to microchimerism include solid organ transplantation, pregnancy, and blood transfusion itself. One hypothesis for the observed findings thus involves a lesser degree of immune-mediated hemolysis of transfused pRBCs in female, as compared to male recipients, as a result of microchimerism-mediated immune tolerance. Although testing of this hypothesis is beyond the scope of our dataset, microchimerism and its relationship to immune tolerance represent an important development in the field of transfusion that may help to elucidate the sexual dimorphism observed in various outcomes of critically ill patients. Beyond microchimerism, immunologically relevant gender differences in sex hormone expression may contribute to the observed sexual dimorphism in ΔHct. Continued research is necessary to elucidate and potentially manipulate the observed gender-related disparity in the hopes of ultimately decreasing both patient exposure to and morbidity of allogeneic blood product transfusion.Out study is limited by a retrospective design. We were unable to capture additional variables that may have influenced ΔHct irrespective of recipient gender such as donor and recipient sex hormone concentrations, menopausal status, and obstetric and transfusion histories. Markers of hemolysis, such as haptoglobin, bilirubin, lactate dehydrogenase, and iron, as well as the presence of hemoglobinuria, were not measured routinely. Demonstration of an association between these markers and ΔHct would provide further evidence for the hemolysis hypothesis and is the focus of a current prospective study in our surgical ICU. Study strengths include a large and relatively uniform sample of critically ill surgical patients, rigorous collection of chronologic and demographic variables, and a low percentage of missing data.pre, and Tpre. The etiology of the observed sexual dimorphism is unknown but may involve gender-related differences in immune-mediated hemolysis of transfused erythrocytes; additional research at our institution is focused on exploring this hypothesis. Among this cohort of critically ill surgical patients, we observed a significant association between recipient gender and ΔHct following pRBCs transfusion, such that female recipients raised their hematocrit more than male recipients. This relationship was independent of either chronologic or anthropomorphic factors, differences in the age of transfused blood, and the gender of the donor. No relationship was observed between donor gender and ΔHct in any analysis. Additional factors that were associated independently with ΔHct included recipient age, recipient admission weight, Hct


Conventionally, the assessments of endothelial function and arterial stiffness require different sets of equipment, making the inclusion of both tests impractical for clinical and epidemiological studies. Pulse wave analysis (PWA) provides useful information regarding the mechanical properties of the arterial tree and can also be used to assess endothelial function. PWA is a simple, valid, reliable, and inexpensive technique, offering great clinical and epidemiological potential. The current paper will outline how to measure arterial stiffness and endothelial function using this technique and include discussion of validity and reliability. Cardiovascular disease (CVD), the leading cause of mortality in the Western world , has a vConventionally, assessments of endothelial function and arterial stiffness require different sets of equipment, making the inclusion of both tests impractical for clinical and epidemiological studies. Pulse wave analysis (PWA) is a simple and noninvasive technique that has been widely used in epidemiological  and inteArterial stiffness is a general term that collectively describes distensiblility, compliance, and elastic modulus of the arterial vascular system. These properties are not homogenous along the arterial tree and muscular and elastic vessels differ. Arterial stiffness can be measured systemically, regionally, or locally. Local measurements provide important physiological information and are more quantitative and sensitive than systemic indices. However, these measurements give no indication of how the artery of interest interacts with central function  as part of an integrative system. Regional arterial stiffness is measured at arterial sites of major physiologic importance such as the aorta where the arterial buffering function is principally expressed, or a particular limb. Systemic arterial stiffness affects the global buffering properties of the arterial system, just as arterial blood pressure can be considered as a global value of hemodynamic load, systemic arterial stiffness reflects the overall opposition of large arteries to the pulsatile effects of ventricular ejection.in vivo assessment of arterial stiffness. These methodologies fall into three broad groups: (1) relating change in area of an artery to distending pressure, that is, local arterial stiffness, (2) measuring pulse wave velocity, that is, regional arterial stiffness, and (3) pulse wave analysis, that is, systemic arterial stiffness. Ultrasound and magnetic resonance imaging (MRI) are capable of measuring local arterial stiffness [systemic arterial stiffness using PWA.A number of methodologies have been applied to the tiffness –23 as wetiffness –30, but tiffness –34, tonotiffness , 35–37, tiffness , 39, andtiffness –43 devicA number of commercial devices are available to automate PWA assessments, including Compilor (tonometric device) , 45, SphTr). Tr represents the time from the onset of the ejected pulse waveform to the onset of the reflected wave and reflects aortic pulse wave velocity [systemic arterial stiffness [velocity . AP is tvelocity . AIx is tiffness , 69. Alttiffness , suggesttiffness  and is itiffness  and heartiffness , 74, wittiffness . AIx shoUsing the SphygmoCor to illustrate the procedure , an alternative index to AIx, has been reported in several studies [Tr [Any valid technique utilised for the measurement of physiological variables must be reproducible . A high  studies , 91, 94. studies , 94. ICC studies , and 0.4dies [Tr , 91, 94.dies [Tr , 91, 93,Tr. Since AIx is strongly influenced by heart rate, both AIx and AP should be corrected for this confounding factor [The PWA technique is particularly suitable for incorporation into clinical trials. Strengths of this technique include simplicity of assessment, relatively short training requirements for investigators, low time commitment for subjects, noninvasiveness, portability, and cost-effectiveness. Both AIx and AP confer similar reproducibility and have been reported as more reliable than g factor . FurtherFunctionally, the endothelium is a large autocrine, paracrine, and endocrine organ that plays a key role in vascular homeostasis . EndotheEstablished methodologies for evaluating peripheral endothelial function include strain-gauge venous occlusion forearm plethysmography , 105, ulβ2 agonist and endothelium-dependent vasodilator, in part reduces wave reflection by activation of the L-arginine-NO pathway.Endothelial function can be assessed noninvasively by evaluating the effects of inhaled salbutamol on the AIx –8. AIx iμg inhaled salbutamol. The maximal decrease in AIx from baseline following a salbutamol challenge is used as an index of endothelial function. Endothelium-independent function can also be assessed using PWA by measurement of the reduction in AIx following sublingual GTN administration.The test takes approximately 35 minutes to complete; following at least 10 minutes supine rest, baseline PWA is recorded as described above, and PWA recordings are then repeated after 5, 10, 15, and 20 minutes after the administration of 400 Wilkinson et al.  and Haywd = 0.9 ± 2%). Wilkinson et al. [Studies examining the reproducibility of salbutamol-mediated effects on AIx are limited , 8, 82. n et al.  also assn et al. . RelativSalbutamol-mediated effects on AIx can be used to estimate endothelial function. This technique offers a number of advantages, namely simplicity of assessment, relatively short training requirements for investigators, low time requirement, portability, cost-effectiveness, ease of use, good reliability indicated by preliminary studies, and the salbutamol challenge isolating the NO pathway. However, more clinical studies are required to further validate this test. Also, while NO is unarguably an important molecule governing endothelial function, the endothelium can release additional complimentary or compensatory molecules , most noPWA is a simple technique capable of assessing systemic arterial stiffness and endothelial function. This test is particularly suitable for clinical and epidemiological studies. Further study is required to determine: (1) sample size requirements, and (2) whether baseline AIx  and AIx following salbutamol inhalation confer additive prognostic value.


The Italian Ministry of Health and the Ministry of Labour and Social Policies have supported a three-year research project titled “Development of disability evaluation protocols based on the biopsychosocial model of the International Classification of Functioning, Disability and Health – ICF”. The Project has been led by the Italian WHO Collaborating Centre for the Family of International Classifications - Friuli Venezia Giulia Regional Health Directorate, in collaboration with the Italian National Institute of Statistics “ISTAT”, the Rehabilitation Research Institute “Medea”, the National Neurological Institute “Carlo Besta”, and the National Agency for Labour Policies “Italia Lavoro SpA”. The research focus was on defining criteria, methodological principles, instruments and data analysis perspectives oriented to the following main objectives:a) To introduce into the Italian legislation the concept of disability as defined by the UN Convention for the Rights of Persons with Disability;b) To develop a common technical framework for the evaluation of disability, thus overcoming the current fragmentation in the Italian welfare system;c) To test the capability of ICF to serve as a reference descriptive language supporting such disability evaluation framework, as well as providing a frame for the electronic socio-health record of people with disability.As an integral and crucial part of the discussion of the results achieved, an International seminar was organized in Rome on 19-20 April 2010, aimed to compare different perspectives on the topics and problems addressed by the project.This volume contains the main papers presented at the workshop and provides an interesting overview of the processes of implementation of ICF in different areas and different social and cultural contexts. We believe that the work presented here can give a contribution to the international debate and suggest innovative insights and research perspectives.A special thanks is due to the colleagues in the Ministry of Health and the Ministry of Labour and Social Affairs, Guido Ditta, Fiammetta Landoni, Raffaele Tangorra, Romolo De Camillis, Federica Francescone, who have supported and encouraged the development of the project and made the realization of the seminar possible, and to Professor Luigi Tesio of the University of Milan who, with enthusiasm and competence, accepted to be the editor of this special issue of BMC Journal of Public Health.