HLA Class I alleles associated with HCV polymorphisms and response to antiviral therapy in HCV genotype 1-infected patients

Aims: The adaptive immune response against hepatitis C virus (HCV) is significantly shaped by the host's composition of HLA alleles. Thus, the HLA phenotype is a critical determinant of viral evolution during adaptive immune pressure. Potential associations of HLA class I alleles with polymorphisms of HCV immune escape variants are largely unknown. Methods: Direct sequence analysis of the genes encoding the HCV proteins E2, NS3 and NS5B in a cohort of 159 patients with chronic HCV genotype 1 infection who were treated with pegylated interferon-alfa 2b and ribavirin in a prospective controlled trial for 48 weeks was exhibited. HLA class I genotyping was performed by strand-specific reverse hybridization with the INNO-LiPA line probe assays for HLA-A and HLA-B and by strand-specific PCR-SSP. We analyzed each amino acid position of HCV proteins using an extension of Fisher's exact test for associations with HLA alleles. In addition, associations of specific HLA alleles with inflammatory activity, liver fibrosis, HCV RNA viral load and virologic treatment outcome were investigated. Results: Separate analyses of HCV subtype 1a and 1b isolates revealed substantially different patterns of HLA-restricted polymorphisms between subtypes. Only one polymorphism within NS5B (V2758x) was significantly associated with HLA B*15 in HCV genotype 1b infected patients (adjusted p=0,048). However, a number of HLA class I-restricted polymorphisms within novel putative HCV CD8+ T cell epitopes (genotype 1a: HLA-A*11 GTRTIASPK1086-1094 [NS3], HLA-B*07 WPAPQGARSL1111-1120 [NS3]; genotype 1b: HLA-A*24 HYAPRPCGI488-496 [E2], HLA-B*44 GENETDVLL530-538 [E2], HLA-B*15 RVFTEAMTRY2757-2766 [NS5B]) were observed with high predicted epitope binding scores assessed by the web-based software SYFPEITHI (>21). Most of the identified putative epitopes were overlapping with already otherwise published epitopes, indicating a high immunogenicity of the accordant HCV protein region. In addition, certain HLA class I alleles were associated with inflammatory activity, stage of liver fibrosis, and sustained virologic response to antiviral therapy. Conclusions: HLA class I restricted HCV sequence polymorphisms are rare. HCV polymorphisms identified within putative HCV CD8+ T cell epitopes in the present study differ in their genomic distribution between genotype 1a and 1b isolates, implying divergent adaptation to the host's immune pressure on the HCV subtype level.

Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.

We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.

Evolutionary patterns of MHC class II B in owls and their implications for the understanding of avian MHC evolution

Owing to its special mode of evolution and central role in the adaptive immune system, the major histocompatibility complex (MHC) has become the focus of diverse disciplines such as immunology, evolutionary ecology, and molecular evolution. MHC evolution has been studied extensively in diverse vertebrate lineages over the last few decades, and it has been suggested that birds differ from the established mammalian norm. Mammalian MHC genes evolve independently, and duplication history (i.e., orthology) can usually be traced back within lineages. In birds, this has been observed in only 3 pairs of closely related species. Here we report strong evidence for the persistence of orthology of MHC genes throughout an entire avian order. Phylogenetic reconstructions of MHC class II B genes in 14 species of owls trace back orthology over tens of thousands of years in exon 3. Moreover, exon 2 sequences from several species show closer relationships than sequences within species, resembling transspecies evolution typically observed in mammals. Thus, although previous studies suggested that long-term evolutionary dynamics of the avian MHC was characterized by high rates of concerted evolution, resulting in rapid masking of orthology, our results question the generality of this conclusion. The owl MHC thus opens new perspectives for a more comprehensive understanding of avian MHC evolution.

Impact of iron supplementation on substantial unexplained fatigue in iron deficient but not anaemic menstruated women

Aim: To determine the impact of iron therapy on the quality of life of non-anaemic iron-deficient women with substantial unexplained fatigue. Methods: Double blind randomised placebo controlled trial in 198 women aged 18 to 53 and having a ferritin level <50 ng/mL, assigned to either oral ferrous sulphate (80 mg/day of elemental iron daily; n = 102) or placebo (n = 96) for 12 weeks, by 44 general practices in France. Main outcome measures: Level of fatigue, depression and anxiety, measured by a 24-item self-administered questionnaire. Level of fatigue was also assessed with a visual analogue scale. Results: 171 (86.4%) women were eligible for efficacy analysis. Mean age, haemoglobin concentration, serum ferritin concentration, level of fatigue, depression, and anxiety were similar in both groups at baseline. Both groups were also similar for compliance and dropout rates. After 12 weeks, asthenia score decreased by −12.9 } 10.37 points (50.8%) in the iron group compared with -9.01 } 11.71 points (36.7%) in the placebo group (p = 0.02), whereas depression and anxiety scores, already low at inclusion, slightly decrease to the same extent in both groups. In an intention to treat analysis, by considering a responder to iron supplementation as having more than two points decrease on the fatigue 10-point visual analogue scale, iron group had 83,3% (85/102) responders vs. 69.8% (67/96) in the control group (p = 0.02). The number needed to treat to have a benefit was 7. Conclusion: Iron supplementation is an efficient inexpensive approach to manage unexplained fatigue in non-anaemic iron-deficient women.

T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions.

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

Intrauterine growth restriction and absent or reverse end-diastolic blood flow in umbilical artery (Doppler class II or III): A retrospective study of short- and long-term fetal morbidity and mortality.

OBJECTIVE: Absent or reverse end-diastolic flow (Doppler II/III) in umbilical artery is correlated with poor perinatal outcome, particularly in intrauterine growth restricted (IUGR) fetuses. The optimal timing of delivery is still controversial. We studied the short- and long-term morbidity and mortality among these children associated with our defined management. STUDY DESIGN: Sixty-nine IUGR fetuses with umbilical Doppler II/III were divided into three groups; Group 1, severe early IUGR, no therapeutic intervention (n = 7); Group 2, fetuses with pathological biophysical profile, immediate delivery (n = 35); Group 3, fetuses for which expectant management had been decided (n = 27). RESULTS: In Group 1, stillbirth was observed after a mean delay of 6.3 days. Group 2 delivered at an average of 31.6 weeks and two died in the neonatal period (6%). In Group 3 after a mean delay of 8 days, average gestational age at delivery was 31.7 weeks; two intra uterine and four perinatal deaths were observed (22%). Long-term follow-up revealed no sequelae in 25/31 (81%) and 15/18 (83%), and major handicap occurred in 1 (3%) and 2 patients (11%), respectively, for Groups 2 and 3. CONCLUSION: Fetal mortality was observed in 22% of this high risk group. After a mean period of follow-up of 5 years, 82% of infants showed no sequelae. According to our management, IUGR associated with umbilical Doppler II or III does not show any benefit from an expectant management in term of long-term morbidity.

Specific binding of antigenic peptides to cell-associated MHC class I molecules.

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.

TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host.

It is well established that T cell-deficient nude and SCID mice can be reconstituted by i.v. injection of small numbers of purified peripheral CD4+ T cells; however, the requirements for expansion of the transferred T cells in such systems are not clear. We show here that blood and lymphoid organs of MHC class II-deficient mice (which selectively lack mature CD4+ T cells) cannot be reconstituted by transfer of purified splenic CD4+ T cells, whereas TCRalpha-deficient mice (which lack both CD4+ and CD8+ mature T cells) are readily reconstituted. The failure of CD4+ T cell reconstitution in MHC class II-deficient mice was not due to the presence of CD8+ T cells, since similar results were obtained in TCRalpha-MHC class II double-deficient mice. Consistent with most previous studies CD4+ T cells in reconstituted TCRalpha-deficient mice had a diverse TCR Vbeta repertoire and were predominantly of an activated/memory (CD44high) phenotype. Collectively our data demonstrate that the expansion of peripheral CD4+ T cells in a T cell-deficient host is dependent upon interactions of the TCR with MHC class II.

Decreased binding of peptides-MHC class I (pMHC) multimeric complexes to CD8 affects their binding avidity for the TCR but does not significantly impact on pMHC/TCR dissociation rate.

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.

Recognition judgments and the performance of the recognition heuristic depend on the size of the reference class

In a series of three experiments, participants made inferences about which one of a pair of two objects scored higher on a criterion. The first experiment was designed to contrast the prediction of Probabilistic Mental Model theory (Gigerenzer, Hoffrage, & Kleinbölting, 1991) concerning sampling procedure with the hard-easy effect. The experiment failed to support the theory's prediction that a particular pair of randomly sampled item sets would differ in percentage correct; but the observation that German participants performed practically as well on comparisons between U.S. cities (many of which they did not even recognize) than on comparisons between German cities (about which they knew much more) ultimately led to the formulation of the recognition heuristic. Experiment 2 was a second, this time successful, attempt to unconfound item difficulty and sampling procedure. In Experiment 3, participants' knowledge and recognition of each city was elicited, and how often this could be used to make an inference was manipulated. Choices were consistent with the recognition heuristic in about 80% of the cases when it discriminated and people had no additional knowledge about the recognized city (and in about 90% when they had such knowledge). The frequency with which the heuristic could be used affected the percentage correct, mean confidence, and overconfidence as predicted. The size of the reference class, which was also manipulated, modified these effects in meaningful and theoretically important ways.

Most residues on the floor of the antigen binding site of the class I MHC molecule H-2Kd influence peptide presentation.

A panel of 15 single alanine substitutions on the floor of the peptide binding groove of the murine class I histocompatibility molecule H-2Kd has been analyzed. All but two mutant molecules were expressed on the cell surface, and were tested for peptide binding and presentation to specific cytotoxic T lymphocytes. Eleven out of 13 mutant molecules appeared to be functionally altered. Five of the substituted residues were involved in the presentation of all peptides tested. Three participated in the presentation of certain peptides but not others. Three other residues participated in epitope formation through indirect interactions. Only two mutations had no detectable effect.

Combining molecular evolution and environmental genomics to unravel adaptive processes of MHC class IIB diversity in European minnows (Phoxinus phoxinus)

Host-pathogen interactions are a major evolutionary force promoting local adaptation. Genes of the major histocompatibility complex (MHC) represent unique candidates to investigate evolutionary processes driving local adaptation to parasite communities. The present study aimed at identifying the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of European minnows (Phoxinus phoxinus). To this end, we isolated and genotyped exon 2 of two MHCIIB gene duplicates (DAB1 and DAB3) and 1665 amplified fragment length polymorphism (AFLP) markers in nine populations, and characterized local bacterial communities by 16S rDNA barcoding using 454 amplicon sequencing. Both MHCIIB loci exhibited signs of historical balancing selection. Whereas genetic differentiation exceeded that of neutral markers at both loci, the populations' genetic diversities were positively correlated with local pathogen diversities only at DAB3. Overall, our results suggest pathogen-mediated local adaptation in European minnows at both MHCIIB loci. While at DAB1 selection appears to favor different alleles among populations, this is only partially the case in DAB3, which appears to be locally adapted to pathogen communities in terms of genetic diversity. These results provide new insights into the importance of host-pathogen interactions in driving local adaptation in the European minnow, and highlight that the importance of adaptive processes driving MHCIIB gene evolution may differ among duplicates within species, presumably as a consequence of alternative selective regimes or different genomic context.