Human Histocompatibility Leukocyte Antigen Research Articles

Development of mouse models with restricted HLA-B∗57:01 presentation for the study of flucloxacillin-driven T-cell activation and tolerance in liver injury

Flucloxacillin (FLX)-induced liver injury is immune-mediated and highly associated to HLA-B∗57:01 expression. Host factors leading to drug-induced liver injury are not yet well understood. Characterize invivo immune mechanisms determining the development of CD8+ T cells reactive to FLX in animals expressing the risk human leukocyte antigen (HLA) allotype. HLA-B∗57:01 transgenic mice (Tg) or Tg strains with H2-KbDb knockout (Tg/KO) or H2-KbDb/PD-1 double knockout (Tg/DKO) were treated with drug and/or anti-CD4 antibody. Drug-induced liver injury was evaluated on the basis of liver enzyme and histologic changes at day 10 of treatment. FLX-reactive CD8+ T cells were characterized invitro by release of effector molecules on drug restimulation, gene expression, and flow cytometry analysis, and functionality tested for hepatic cytotoxicity. CD8+ T-cell responses to FLX in Tg were dependent on both HLA and mouse major histocompatibility complex I presentation and invivo priming. Eliminating H2-KbDb in Tg/KO to allow exclusive presentation of FLX by HLA resulted in a less robust drug-specific CD8+T-cell response unless CD4+ cells, including regulatory T cells, were depleted. Treatment of Tg/KO with anti-CD4 antibody and FLX led to subclinical liver inflammation associated with an increase in PD1+CD8+ T cells in the lymphoid organs and liver. Impaired PD-1 expression in Tg/DKO led to liver histopathologic and transcriptional alterations but without hepatic enzyme elevations. Moreover, effector lymphocytes accumulated in the liver and showed FLX-dependent hepatic cytotoxicity invitro when tolerogenic liver cells were depleted. In our invivo models, FLX primes CD8+ T cells to recognize drug presented by HLA-B∗57:01 and murine major histocompatibility complex I. HLA-B∗57:01-dependent CD8+ T-cell reaction to FLX is limited by the presence of CD4+ cells, presumably regulatory T cells, and PD-1 expression. Tolerogenic hepatic cells limit clinical disease through PD-L1 or additional unexplored mechanisms.

Amino acid polymorphisms in human histocompatibility leukocyte antigen class II and proinsulin epitope have impacts on type 1 diabetes mellitus induced by immune-checkpoint inhibitors.

Immune-checkpoint inhibitors are effective in various advanced cancers. Type 1 diabetes mellitus induced by them (ICI-T1DM) is a serious complication requiring prompt insulin treatment, but the immunological mechanism behind it is unclear. We examined amino acid polymorphisms in human histocompatibility leukocyte antigen (HLA) molecules and investigated proinsulin epitope binding affinities to HLA molecules. Twelve patients with ICI-T1DM and 35 patients in a control group without ICI-T1DM were enrolled in the study. Allele and haplotype frequencies of HLA-DRB1*04:05, DQB1*04:01, and most importantly DPB1*05:01 were significantly increased in patients with ICI-T1DM. In addition, novel amino acid polymorphisms in HLA-DR (4 polymorphisms), in DQ (12 polymorphisms), and in DP molecules (9 polymorphisms) were identified. These amino acid polymorphisms might be associated with the development of ICI-T1DM. Moreover, novel human proinsulin epitope clusters in insulin A and B chains were discovered in silico and in vitro peptide binding assays to HLA-DP5. In conclusion, significant amino acid polymorphisms in HLA-class II molecules, and conformational alterations in the peptide-binding groove of the HLA-DP molecules were considered likely to influence the immunogenicity of proinsulin epitopes in ICI-T1DM. These amino acid polymorphisms and HLA-DP5 may be predictive genetic factors for ICI-T1DM.

LncRNA HCP5 acts as a ceRNA to regulate EZH2 by sponging miR‑138‑5p in cutaneous squamous cell carcinoma

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are essential for the progression of tumors, including cutaneous squamous cell carcinoma (CSCC). The present study aimed to examine the competing endogenous RNA (ceRNA) network in CSCC. Differentially expressed genes in CSCC were analyzed using the GSE66359 microarray data set, and the upstream miRNAs and lncRNAs were predicted using online database analysis (TargetScan 7.1, mirDIP 4.1, miRSearch V3.0, miRDB and RNA22 2.0) and were verified in clinical tissues. RNA pull-down and dual luciferase reporter gene assays were used to verify the targeting relationships among lncRNA human histocompatibility leukocyte antigen complex P5 (HCP5), miR-138-5p and enhancer of zeste homolog 2 (EZH2). Cell lines with a high and low HCP5 expression were screened, and a pcDNA-3.1-HCP5 overexpression vector, small interfering RNA against HCP5, miR-138-5p mimics and miR-138-5p inhibitors were transfected into the CSCC cells. Cell viability, invasion, migration, apoptotic rate and autophagy were evaluated. The effects of HCP5 on autophagy and apoptosis of CSCC cells were verified in vivo using Ki67 and TUNEL staining. EZH2 was demonstrated to be upregulated in CSCC cells. miR-138-5p target sequences were identified in HCP5 and EZH2. HCP5 was revealed to function as a putative ceRNA of miR-138-5p to positively regulate EZH2, and EZH2 was shown to regulate autophagy and apoptosis of CSCC cells through the STAT3/VEGFR2 pathway. HCP5 overexpression decreased miR-138-5p levels, increased EZH2 levels and promoted cell malignant behaviors and autophagy but decreased the apoptosis rate. These trends were opposite when HCP5 was silenced. In conclusion, HCP5 may competitively bind to miR-138-5p to regulate EZH2 in CSCC cells, promoting autophagy and reducing apoptosis through the STAT3/VEGFR2 pathway. This study may provide a new perspective for understanding the molecular mechanism and treatment of CSCC.

Down-regulation of HCP5 inhibits cell proliferation, migration, and invasion through regulating EPHA7 by competitively binding miR-101 in osteosarcoma.

Patients with osteosarcoma (OS) usually have poor overall survival because of frequent metastasis. Long non-coding RNAs (lncRNAs) have been reported to be associated with tumorigenesis and metastasis. In this study, we investigated the expression and roles of lncRNA human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in OS, aiming to provide a novel molecular mechanism for OS. HCP5 was up-regulated both in OS tissues and cell lines and high expression of HCP5 was associated to low survival in OS patients. Down-regulation of HCP5 inhibited cell proliferation, migration, and invasion, suggesting its carcinogenic role in OS. miR-101 was targeted by HCP5 and its expression was decreased in OS. The inhibitor of miR-101 reversed the impact of HCP5 down-regulation on cell proliferation, apoptosis, and metastasis in OS. Ephrin receptor 7 (EPHA7) was proved to be a target of miR-101 and had ability to recover the effects of miR-101 inhibitor in OS. In conclusion, lncRNA HCP5 knockdown suppressed cell proliferation, migration, and invasion, and induced apoptosis through depleting the expression of EPHA7 by binding to miR-101, providing a potential therapeutic strategy of HCP5 in OS.

Histocompatibility HLA class I in anterior uveitis patients with and without spondyloarthritis

Anterior uveitis (AU) and ankylosing spondylitis (AS) are associated with histocompatibility human leukocyte antigen (HLA)-B27. Previous genetic studies conducted in different populations have also demonstrated other genetic associations, including HLA, both general and individual ones for AU and AS. Objective: to investigate the association of HLA class I with AU depending on the presence or absence of spondyloarthritis (SpA). Patients and methods . The investigators used the data of HLA class I typing in the patients referred by ophthalmologists for examination to the V.A. Nasonova Research Institute of Rheumatology, as well as the previous databases of patients with AS. The investigation included retrospectively two groups of patients with AU: 1) 52 patients with a confirmed diagnosis of SpA (AU + SpA); 2) 96 patients who had other types of AU (idiopathic AU (n=52), viral uveitis (n=29), multiple sclerosis (n=2) toxoplasmosis (n=2), sarcoidosis (n=1), tuberculosis (n=1), chlamydiasis (n=3), Behcet's disease (n=2), juvenile chronic arthritis (n=3), and Fuchs' heterochromic cyclitis (n=1). A control group consisted of 150 healthy test donors. The distribution of HLA class I (A, B, and Cw) was analyzed when comparing the two groups of patients with AU and each control patient group. Results. HLA-B27 was detected in 96.1% of cases in the AU + SpA group, in 40.6% in the AU group, and in 7.3% in the controls. In HLA-27-positive patients, the risk (odds ratio (OR) for joint disease (AU + SpA) was 315.9 (95% confidence interval (CI), 61.9–2176.7); p<0.0000001; the risk for AU was 8.7 (95% CI, 3.9–19.4); p<0.000001. The HLA-C-locus antigens showed a high incidence of Cw2 antigen in patients with AU + SpA and in those with AU compared to the controls (64.0, 36.3, and 10.0%, respectively): AU + SpA versus the controls (p<0.000001), AU versus the controls (p<0.00001). In the two groups of patients with a high HLA-B27 frequency, this substantially higher rate of Cw2 antigen carriage was natural due to the non-equilibrium coupling phenomenon, including that for B27 and Cw2 antigens. The rate of Cw7 antigen was significantly lower in the AU + SpA group versus the controls: 12.8 and 38.7% (p=0.002). In the group of AU patients without SpA, the rate of this antigen did not differ significantly from that in the control and AU + SpA groups. Conclusion. The analysis of the distribution of HLA class I confirmed the association of B27 antigen with AU in the Russian population. There were no associations with other antigens other than Cw2. Cw7 antigen can play a protective role against SpA, since the frequency of this gene was not lower in AU patients compared to the controls.

RETRACTED: Role of HCP5-miR-139-RUNX1 Feedback Loop in Regulating Malignant Behavior of Glioma Cells

Aberrant expression of long noncoding RNAs has recently been reported in tumorigenesis and plays a pivotal role in regulating malignant behavior of cancers. In this study, we confirmed that the long noncoding RNAs human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) was up-regulated in glioma tissues as well as in U87 and U251 cells. Knockdown of HCP5 inhibited the malignant biological behavior of glioma cells by reducing proliferation, migration and invasion, and inducing apoptosis. HCP5 regulated the malignant behavior of glioma cells by binding to microRNA-139, which functions as a tumor suppressor. Moreover, knockdown of HCP5 down-regulated Runt-related transcription factor 1, a direct and functional downstream target of microRNA-139 that is involved in microRNA-139-mediated tumor-suppressive effects in glioma cells. Runt-related transcription factor 1 increased promoter activities and upregulated expression of the oncogenic gene astrocyte elevated gene-1 (AEG-1). Runt-related transcription factor 1 also increased the promoter activities and expression of HCP5, which showed a positive feedback loop in regulating the malignant behavior of glioma cells. In conclusion, this study demonstrated that the HCP5-microRNA-139- Runt-related transcription factor 1 feedback loop plays a pivotal role in regulating the malignant behavior of glioma cells, which may provide a potential therapeutic strategy for treating glioma.

Elimination of Latently HIV-infected Cells from Antiretroviral Therapy-suppressed Subjects by Engineered Immune-mobilizing T-cell Receptors

Persistence of human immunodeficiency virus (HIV) in a latent state in long-lived CD4+ T-cells is a major barrier to eradication. Latency-reversing agents that induce direct or immune-mediated cell death upon reactivation of HIV are a possible solution. However, clearance of reactivated cells may require immunotherapeutic agents that are fine-tuned to detect viral antigens when expressed at low levels. We tested the antiviral efficacy of immune-mobilizing monoclonal T-cell receptors against viruses (ImmTAVs), bispecific molecules that redirect CD8+ T-cells to kill HIV-infected CD4+ T-cells. T-cell receptors specific for an immunodominant Gag epitope, SL9, and its escape variants were engineered to achieve supraphysiological affinity and fused to a humanised CD3-specific single chain antibody fragment. Ex vivo polyclonal CD8+ T-cells were efficiently redirected by immune-mobilising monoclonal T-cell receptors against viruses to eliminate CD4+ T-cells from human histocompatibility leukocyte antigen (HLA)-A*0201-positive antiretroviral therapy-treated patients after reactivation of inducible HIV in vitro. The efficiency of infected cell elimination correlated with HIV Gag expression. Immune-mobilising monoclonal T-cell receptors against viruses have potential as a therapy to facilitate clearance of reactivated HIV reservoir cells.

Neuroinvasive West Nile Infection Elicits Elevated and Atypically Polarized T Cell Responses That Promote a Pathogenic Outcome.

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.

Prediction and prevention of type 1 diabetes: update on success of prediction and struggles at prevention.

Type 1 diabetes mellitus (T1DM) is the archetypal example of a T cell-mediated autoimmune disease characterized by selective destruction of pancreatic β cells. The pathogenic equation for T1DM presents a complex interrelation of genetic and environmental factors, most of which have yet to be identified. On the basis of observed familial aggregation of T1DM, it is certain that there is a decided heritable genetic susceptibility for developing T1DM. The well-known association of T1DM with certain human histocompatibility leukocyte antigen (HLA) alleles of the major histocompatibility complex (MHC) was a major step toward understanding the role of inheritance in T1DM. Type 1 diabetes is a polygenic disease with a small number of genes having large effects (e.g., HLA) and a large number of genes having small effects. Risk of T1DM progression is conferred by specific HLA DR/DQ alleles [e.g., DRB1*03-DQB1*0201 (DR3/DQ2) or DRB1*04-DQB1*0302 (DR4/DQ8)]. In addition, the HLA allele DQB1*0602 is associated with dominant protection from T1DM in multiple populations. A concordance rate lower than 100% between monozygotic twins indicates a potential involvement of environmental factors on disease development. The detection of at least two islet autoantibodies in the blood is virtually pre-diagnostic for T1DM. The majority of children who carry these biomarkers, regardless of whether they have an a priori family history of the disease, will develop insulin-requiring diabetes. Facilitating pre-diagnosis is the timing of seroconversion which is most pronounced in the first 2 yr of life. Unfortunately the significant progress in improving prediction of T1DM has not yet been paralleled by safe and efficacious intervention strategies aimed at preventing the disease. Herein we summarize the chequered history of prediction and prevention of T1DM, describing successes and failures alike, and thereafter examine future trends in the exciting, partially explored field of T1DM prevention.

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

COST-EFFECTIVENESS OF HUMAN LEUKOCYTE ANTIGEN MATCHING IN PENETRATING KERATOPLASTY.

Background: The matching of favorable human leukocyte antigen (HLA) combinations is rarely performed in penetrating keratoplasty procedures for primary prophylaxis of immune reactions. However, clinical studies suggest that the incidence of graft rejection decreases substantially when patients receive favorably matched grafts. Objective: The aim of this study was to assess the cost-effectiveness of HLA matching for patients undergoing penetrating keratoplasty in everyday clinical practice. Methods: In the absence of a randomized controlled clinical trial, we used administrative data from the Freiburg University Eye Hospital in Germany. Our study population consisted of all patients who underwent their first keratoplasty between 11/2003 and 01/2010 and for whom information on HLA histocompatibility was available. We used propensity score matching to estimate a causal effect of favorable HLA matching, parametric survival regression techniques to predict graft survival and expert opinion to model incremental cost for HLA matching. Because the availability of favorable HLA histocompatibility ultimately depends on the patients' HLA phenotype, we modeled the incremental cost-effectiveness ratio (ICER) as a function of the probability that a patient will receive a favorably matched HLA, and used expert opinion to set a point estimate. Results: We predicted that corneal grafts with favorable HLA matching were associated with improved rejection-free graft survival time (more than 1,000 days). We estimated the incremental cost of HLA matching at EUR 1,200 and the ICER at EUR 4.62 per additional day of graft survival. Conclusions: The ICER of HLA matching is acceptable, given the high cost of alternative treatment and the shortage of corneal donors in Germany.

Detection of respiratory syncytial virus fusion protein variants between 2009 and 2012 in China

Respiratory syncytial virus (RSV) causes respiratory tract infection, particularly acute lower respiratory tract infection (ALRTI), in early childhood. The RSV fusion protein (F protein) is an important surface protein, and it is the target of both cytotoxic T lymphocytes (CTL) and neutralizing antibodies; thus, it may be useful as a candidate for vaccine research. This study investigated the genetic diversity of the RSV F protein. To this end, a total of 1800 nasopharyngeal aspirates from hospitalized children with ALRTI were collected for virus isolation between June 2009 and March 2012. There were 333 RSV-positive cases (277 cases of RSV A, 55 of RSV B, and 1 with both RSV A and RSV B), accounting for 18.5 % of the total cases. Next, 130 clinical strains (107 of RSV A, 23 of RSV B) were selected for F gene sequencing. Phylogenetic analysis revealed that the F gene sequence is highly conserved, with significant amino acid changes at residues 16, 25, 45, 102, 122, 124, 209, and 447. Mutations in human histocompatibility leukocyte antigen (HLA)-restricted CTL epitopes were also observed. Variations in RSV A F protein at the palivizumab binding site 276 (N→S) increased between 2009 and 2012 and became predominant. Western blot analysis and microneutralization data showed a substitution at residue 276 (N→S) in RSV A that did not cause resistance to palivizumab. In conclusion, the RSV F gene is geographically and temporally conserved, but limited genetic variations were still observed. These data could be helpful for the development of vaccines against RSV infection.

Vaccines and Autoimmunity

Vaccines have eradicated or controlled many infectious diseases, saving each year millions of lives and quality of life of many other millions of people. In spite of the success of vaccines over the last two centuries, parents (and also some health care workers) gloss over the devastating consequences of diseases, which are now avoided thanks to vaccines, and direct their attention to possible negative effects of immunization. Three immunological objections are raised: vaccines cause antigenic overload, natural immunity is safer and better than vaccine-induced immunity, and vaccines induce autoimmunity. The last point is examined in this review. Theoretically, vaccines could trigger autoimmunity by means of cytokine production, anti-idiotypic network, expression of human histocompatibility leukocyte antigens, modification of surface antigens and induction of novel antigens, molecular mimicry, bystander activation, epitope spreading, and polyclonal activation of B cells. There is strong evidence that none of these mechanisms is really effective in causing autoimmune diseases. Vaccines are not a source of autoimmune diseases. By contrast, absolute evidence exists that infectious agents can trigger autoimmune mechanisms and that they do cause autoimmune diseases.

Strategy for the creation of clinical grade hESC line banks that HLA‐match a target population

Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.

Silencing HLA Class I Expression in Human Corneas to Decrease the Risk of Graft Rejection after Keratoplasty

The high variability of the human histocompatibility leukocyte antigen (HLA) complex, and of the minor histocompatibility antigens significantly contributes to elicit an immune response after allogeneic cornea transplantation, and may lead to the development of acute or chronic rejection. In addition, often inevitable second cornea transplantation is always associated with an increased risk of graft rejection. This project aims to permanently reduce HLA expression on cornea transplants to decrease the risk of graft rejection after keratoplasty. This method represents a new therapeutic concept and may contribute to prevent graft rejection. For this purpose, lentiviral vectors encoding short hairpin RNAs (shRNA) targeting β2-microglobulin were used to induce the downregulation of HLA class I expression. Levels of β2-microglobulin mRNA or cell surface HLA class I expression were determined by Real-Time PCR and flow cytometry, respectively. Cell viability tests using 7-Aminoactinomycin (7-AAD) and Propidium iodide (PI) staining were performed after transduction. Light and fluorescence microscopic analysis were carried out to evaluate the integrity of the corneal endothelium. A mean of 92%±7% of the total cornea cells were transduced. In comparison to the non-engineered cornea, the delivery of the β2-microglobulin-specific shRNA caused a reduction by up to 95% in β2-microglobulin transcript levels as detected at day 5 and day 15 after cornea transduction. Furthermore, the reduction of β2-microglobulin levels induced a decrease in the cell surface expression of HLA class I proteins by up to 90%. Microscopic analysis of the cornea endothelium showed that this cell layer was not affected by the transduction procedure or the silencing effect. The integrity of the HLA class I silenced endothelium and the absence of a positive signal upon 7-AAD/PI staining indicated the absence of off target effects that might impair the endothelial function or viability. This data demonstrates the feasibility of silencing HLA class I expression in the original 3D tissue-structure by RNAi-mediated nucleic acid targeting. As in vivo data in an allogeneic rat model showed a superior survival of RT1-silenced cells, silencing HLA expression has the potential to overcome alloimmunization and tissue rejection caused by HLA mismatches in allogeneic keratoplasty.

The Forensic Genetics Laboratory of the University of Barcelona: The evolution of paternity testing cases over the past 35 years

Biological paternity research in Catalonia (north-eastern Spain) began in 1976 when Dr. Castillo, Dr. Gelabert and Dr. Huguet studied the first case at the Department of Forensic and Legal Medicine of the University of Barcelona in cooperation with the Haemostasis and Hemotherapy Service and the Blood Bank of the Hospital Clínic in Barcelona. In the knowledge that each human is genetically unique as a result of genetic polymorphism, blood group antigen typing and other immunological techniques including human histocompatibility leukocyte antigen (HLA) typing became widely used for paternity testing and forensic science application until the end of the 1970s. However, biological tests were not accepted by the courts in Spain until 1981 when the Civil Code was reformed. With the support of Dr. Carracedo from the University of Santiago, in 1982 Dr. Huguet and Dr. Gené, who are still actively researching, set up the Haemogenetic Forensic Laboratory at the University of Barcelona and this area of the Legal Medicine Unit was thus consolidated. It was the first Forensic Genetics Laboratory in Catalonia. The aim of this study is to conduct a retrospective analysis of paternity testing cases over the past 35 years. We report on the social and legal development of the cases analysed, grouped into different periods of time. The results on paternity and non-paternity claims were studied included exclusion and non-exclusion rates. Biomathematical performance values were reported for each methodology stage (number of exclusions, average paternity probability values, population index, etc.).

HLA mismatches and hematopoietic cell transplantation: structural simulations assess the impact of changes in pep-tide binding specificity on transplant outcome

The success of hematopoietic cell transplantation from an unrelated donor depends in part on the degree of Human Histocompatibility Leukocyte Antigen (HLA) matching between donor and patient. We present a structure-based analysis of HLA mismatching, focusing on individual amino acid mismatches and their effect on peptide binding specificity. Using molecular modeling simulations of HLA-peptide interactions, we find evidence that amino acid mismatches predicted to perturb peptide binding specificity are associated with higher risk of mortality in a large and diverse dataset of patient-donor pairs assembled by the International Histocompatibility Working Group in Hematopoietic Cell Transplantation consortium. This analysis may represent a first step toward sequence-based prediction of relative risk for HLA allele mismatches.

On the estimation of the necessary inventory for hellenic public cord blood banks using simulation

Allogeneic hematopoietic stem cell transplantation is a relative new treatment method for a wide range of diseases including hematopoietic malignancies, immunodeficiency disorders, metabolic diseases and genetic disorders. Umbilical Cord Blood (UCB) is a modern and effective source of hematopoietic stem cells with increasing usage throughout the world. Thus, the establishment and operation of public UCB banks has emerged as a common practice all over the world. In Greece, the development of such a UCB bank network is still in its infancy. This paper presents the development of a methodology for estimating the required inventory level of UCB units in Greece (bank size), in order to ensure an adequate probability that a Greek patient finds a Human Leukocyte Antigen (HLA)-compatible unit/donor (with a match at least for 4 out of 6 HLA histocompatibility antigens). To this end, we have adopted a Monte Carlo simulation approach. The available histocompatibility data include detailed genotypes of 8,500 Greeks. These data were fed into the system simulation model to emulate a UCB bank operation. The results of the simulation process reveal that an inventory of 10,000 cryopreserved UCB units could ensure a 4 out of 6 HLA matching for a Greek patient with a probability exceeding 95%. The determination of the bank size is a strategic, pivotal decision; thus, the proposed methodology provides the input for the National Healthcare System to decide on developing or not an UCB Bank (along with its designing parameters).

The clinical significance of gene polymorphism in children's primary nephrotic syndrome

The pathogenesis of children's primary nephrotic syndrome has not been completely clear at present.In the past few years,The polymorphism of these genes,such as angiotensin-converting enzyme gene,apolipoprotein E gene,platelet-activating factor acetylhydrolase gene,human histocompatibility leukocyte antigen gene,glucocorticoid receptor gene and cytokine gene,ect have been discovered that were significantly correlated with susceptibility,pathological progress,steroid response,disease recurrence and prognosis of primary nephrotic syndrome.This article reviewed the research progress of PNS and gene polymorphism. Key words: Children; Nephrotic syndrome; Gene; Polymorphism(Genetics)

Helper T Cell Epitope-Mapping Reveals MHC-Peptide Binding Affinities That Correlate with T Helper Cell Responses to Pneumococcal Surface Protein A

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA) is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA) would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC) class II. We also characterized spleen- and cervical lymph node (CLN)-derived helper T lymphocyte (HTL) cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA199–246) consistently caused the greatest IFN-γ, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4+ T cells isolated from S. pneumonia strain EF3030-challeged F1 (B6×BALB/c) mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA199–246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th) cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.