DR Molecules Research Articles

Causal Involvement of Immune Cells in Chronic Obstructive Pulmonary Disease: A Mendelian Randomization Study.

The immune cells play a substantial role in the development and progression of chronic obstructive pulmonary disease (COPD). We aim to investigate the causal involvement of immune cells in COPD via a Mendelian randomization (MR) analysis. Published genome-wide association studies (GWAS) statistics on immune cells were analyzed, with genetic variants identified as instrumental variables (IVs). Inverse-variance weighting (IVW), weighted median, and MR-Egger regression methods were employed, along with simple mode and weighted mode adopted in the two-sample MR analysis. Sensitivity analysis was conducted to examine the heterogeneity, horizontal pleiotropy, and stability of the causal relationship. IVW results suggested that CCR2 on CD62L+ plasmacytoid dendritic cells (DC), CCR2 on plasmacytoid DC, CD11b on CD66b++ myeloid cells, CD19 on CD20- CD38- CD24+ memory B cell subset, CD25 on transitional B cells, and CD25++CD8br %CD8br T cells were risk factors for the development of COPD. Besides, CD127 on effector memory-like cytotoxic T lymphocytes lacking expression of co-stimulatory molecule 28 (CD28-EM CTLs) and HLA DR+ NK ACs expressing human leukocyte antigen DR molecules while being natural killer cells (%NK ACs) were protective factors for COPD. This study unveiled a causal relationship between immune cell phenotype and COPD. These findings offer new insights for the prevention and treatment of COPD using COPD-associated immune cells.

Ultrafast fragmentation of highly-excited doubly-ionized deoxyribose: role of the liquid water environment.

Ab initio molecular dynamics simulations are used to investigate the fragmentation dynamics following the double ionization of 2-deoxy-D-ribose (DR), a major component in the DNA chain. Different ionization scenarios are considered to provide a complete picture. First focusing on isolated DR2+, fragmentation patterns are determined for the ground electronic state, adding randomly distributed excitation energy to the nuclei. These patterns differ for the two isomers studied. To compare thermal and electronic excitation effects, Ehrenfest dynamics are also performed, allowing to remove the two electrons from selected molecular orbitals. Two intermediate-energy orbitals, localized on the carbon chain, were selected. The dissociation pattern corresponds to the most frequent pattern obtained when adding thermal excitation. On the contrary, targeting the four deepest orbitals, localized on the oxygen atoms, leads to selective ultrafast C-O and/or O-H bond dissociation. To probe the role of environment, a system consisting of a DR molecule embedded in liquid water is then studied. The two electrons are removed from either the DR or the water molecules directly linked to the sugar through hydrogen bonds. Although the dynamics onset is similar to that of isolated DR when removing the same deep orbitals localized on the sugar oxygen atoms, the subsequent fragmentation patterns differ. Sugar damage also occurs following the Coulomb explosion of neighboring H2O2+ molecules due to interaction with the emitted O or H atoms.

Reconditioned monocytes are immunomodulatory and regulate inflammatory environment in sepsis

Sepsis is caused by dysregulated immune response to severe infection and hyper inflammation plays a central role in worsening the disease. The immunomodulatory properties of mesenchymal stem cells (MSCs) have been evaluated as a therapeutic candidate for sepsis. Reconditioned monocytes (RM), generated from healthy human peripheral blood mononuclear cells (PBMCs) exhibit both macrophage and MSCs-like properties. RM were administered at different stages of sepsis in a mouse model. It reduced serum levels of IL6, MCP-1, IL-10, improved hypothermia, increased survival, and recovery from 0 to 66% when combined with antibiotics in the mouse model. The reduced human leucocyte antigen DR molecules expression on RM enables their co-culture with PBMCs of sepsis patients which resulted in reduced ROS production, and up-regulated TGF-β while down-regulating IL6, IL8, and IL-10 in-vitro. RM are potentially immunomodulatory, enhance survival in sepsis mouse model and modulate inflammatory behaviour of sepsis patient’s PBMCs.

Effective omalizumab treatment influenced eosinophil function in severe allergic asthmatics.

Omalizumab is an effective anti-immunoglobulin E (IgE) treatment for allergic asthma. Eosinophil plays a critical role in the pathogenesis of allergic airway inflammation. This study aimed to explore the influence of effective omalizumab treatment on circulating eosinophils. Allergic asthmatics enrolled in the study were treated with omalizumab for at least 16 weeks and exhibited a good or excellent response according to the global evaluation of treatment effectiveness (GETE) assessed by each patient and specialist physician. For eosinophil functional evaluation, peripheral blood eosinophils were separated; and examined the expression of human leukocyte antigen (HLA)-DR and co-stimulatory molecules cluster of differentiation (CD) 80, CD86 and CD40 by Flow Cytometry and serum were to measure the concentration of eotaxin-1 before and after 16 weeks of omalizumab treatment. Totally 32 allergic asthma patients who responded positively to omalizumab treatment were included. Omalizumab responders showed a significant decline in the expression of co-stimulatory molecules CD40, CD80, and CD86 on peripheral eosinophils and in serum eotaxin-1 concentration after treatment. Negative correlations (r=-0.61, P=0.048) were observed between the change in CD80+ eosinophils and the change in forced expiratory volume in the first second (FEV1)/forced vital capacity (FVC)% predicted and maximal expiratory flow (MEF) 25% after omalizumab treatment. Omalizumab improved FEV1/FVC% predicted (3.88, P=0.033), fractional exhaled nitric oxide (FeNO, -22.24, P=0.028), asthma control test (ACT, 4.22, P<0.001), mini asthma quality of life questionnaire (mini-AQLQ, -14.44, P=0.019), Leicester cough questionnaire (LCQ, 3.03, P=0.009) and visual analogue scale (VAS) for allergic symptoms (-13.00, P=0.001) in patients with severe allergic asthma statistically; reduced mini rhino-conjunctivitis quality of life questionnaire (mini-RQLQ, -8.50, P=0.047), and self-rating anxiety scale (SAS, -5.08, P=0.040) in allergic asthmatics with concomitant allergic rhinitis (AR) or anxiety, respectively. Our findings show a unique role of omalizumab in reducing co-stimulatory molecules expression on eosinophil and serum eotaxin-1 levels in severe allergic asthmatics accompanied by improvement of multiple clinical parameters of allergic diseases.

Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and β chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR β chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.

Circulating Placental Vesicles Carry HLA-DR in Pre-Eclampsia: A New Potential Marker of the Syndrome.

BackgroundPre-eclampsia (PE) is a common disorder of pregnancy that usually presents with hypertension and proteinuria. The clinical presentation arises from soluble factors released into the maternal circulation from the placenta owing to the stress of syncytiotrophoblast, consequence of defective placentation occurring in the first half of pregnancy. Reduced tolerance of the semiallogeneic fetus by the maternal immune system has been proposed as first trigger leading to poor placentation. We previously observed aberrant expression of human leukocyte antigen (HLA)-DR molecules in the syncytiotrophoblast of a subset of women with PE. Aim of this study was to investigate abnormal expression of circulating HLA-DR in syncytiotrophoblast-derived extracellular vesicles (STBEVs) in women with PE compared to normal pregnant women.Methodsperipheral venous blood was collected from 22 women with PE and 22 normal pregnant women. Circulating STBEVs were collected by ultra-centrifugation (120000 g) and analyzed for the expression of HLA-DR and placental alkaline phosphatase (PLAP), a specific marker of the placenta, by Western blot analysis and flow cytometry.Resultscirculating STBEVs positive for HLA-DR were observed in 64% of PE women while no HLA-DR positivity was detected in any of the controls (P<0.01).ConclusionsAberrant expression of HLA-DR in circulating STBEVs is specifically associated to PE. Further studies are required: a) to define the role of aberrant placental expression of HLA-DR molecules in the pathogenesis of PE; b) evaluate a possible application of detecting circulating HLA-DR positive STBEVs in the diagnosis and prediction of PE in the first and second trimester of pregnancy.

Characterization of the HLA-Class-II (HLAcII)/Therapeutic Factor VIII (FVIII)-Derived Peptidome By FVIII Domain and HLAcII Isomer

Abstract Background: Hemophilia A (HA) is caused by F8 mutations and resultant plasma deficiencies in FVIII activity. While bleeding can be arrested by infusing therapeutic FVIII proteins (tFVIIIs), ~30% of severe HA patients will develop neutralizing tFVIII antibodies (inhibitors) and become resistant to treatment. For inhibitors to develop, foreign peptides must be liberated from a tFVIII and displayed on dendritic cells (DCs) in self HLAcII molecules. Little is known about the inhibitor risk attributable to distinct isomers of individual HLAcII repertoires and different tFVIII domains. Aims: Determine the contributions to FVIII immunogenicity risk of the distinct 1) HLAcII isomers DP, DQ and DR, and 2) tFVIII domains A1, A2, A3, B, C1 and C2. Methods: We analyzed HLAcII/tFVIII peptidomic data generated in DC-protein processing assays (PPAs) that used monocyte-derived DCs from 12 healthy donors to compare the immunogenicity of a full length (FL) and B-domain truncated (BDT) recombinant FVIII (FL- and BDT-FVIII) ± von Willebrand Factor (VWF) and a plasma derived (pd)FVIII + VWF. All tFVIIIs contain 3 small acidic-residue-rich peptides (a1, a2 and a3) that connect A1 to A2, A2 to B, and B to A3. To simplify our analysis, any LC-MS/MS individually sequenced peptide (ISP) with an N-terminal residue in either the (i) C-terminal portion of A1, A2 or B that extends into the adjacent a1, a2 or a3 region, respectively, was placed into that domain, or (ii) a1, a2 or a3 region, whether or not it extends into A3, C1 or C2, was placed into that domain. We then calculated the expected frequency of peptides from each FVIII domain (including, when appropriate, the adjacent acidic regions) based on residue number. Finally, we compared the observed and expected peptide proportions and performed a Z-test on the difference of proportions per domain. We performed this analysis for all ISP data in which the observed frequencies were obtained by a weighting scheme jointly taking into account contributions by tFVIII & by domain (All-data). We also performed this analysis by tFVIII. Results: For the All-data comparisons, we found that A3 had a higher observed than expected proportion (dP = 22%; p = 0.006). For the FL-FVIII comparisons, the B domain had a lower observed than expected proportion (dP = -12.1%; p We also approached these data from the HLAcII perspective. We found that our 12 donors expressed 12 distinct DR molecules based on their DRB1 alleles. After tallying the per allele density of FVIII residues (for all 2332 residues) falling in the DR-bound fraction of peptides, we constructed FVIII residue-by-allele matrices of dimensions 2332 x 12 per tFVIII and performed singular value decompositions that yielded their respective matrices of right singular vectors also known as principal components (PCs). Such PCs are said to span or generate a corresponding vector subspace. Using matrix analytic methods, we can compare the therapeutic-specific subspaces to see if such contrasts reflect differences in their associated density profiles. To compare two subspaces, we compute their projection onto their intersection subspace, and use this projection to compute a subspace distance. We test the null hypothesis of a subspace distance equal to 0 (for statistically similar subspaces) by a permutation test. As an example, we compared the FL-FVIII + VWF and pdFVIII + VWF subspaces, and found that they are significantly different (subspace distance (SD) = 0.96; p Conclusions: Our findings demonstrate that the distinct domains of tFVIIIs and different alleles of individual HLAcII repertoires differentially contribute to inhibitor risk. Download : Download high-res image (175KB) Download : Download full-size image Disclosures Hofmann: CSL Behring: Employment. Escobar: Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinh: Haplomics: Employment. Powell: CSL Behring: Employment. Howard: CSL Behring: Honoraria, Research Funding; Haplomics: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Experimentally and theoretically approaches for disperse red 60 dye adsorption on novel quaternary nanocomposites

A comprehensive study that combined both experimental and computational experiments was performed to evaluate the usage of organo-metal oxide nanocomposite for the elimination of disperse red 60 dye (DR) from aqueous solutions. Chitosan was modified by Schiff base to form nanoneedles chitosan-4-chloroacetophenone derivative. The derivatives were then impregnated with CeO2–CuO–Fe2O3 or CeO2–CuO–Al2O3 metal oxides to prepare a novel quarternary organo-metal oxide nanocomposite. The novel nanocomposite, chitosan-4-chloroacetophenone/CeO2–CuO–Fe2O3 (CF) and chitosan-4-chloroacetophenone/CeO2–CuO–Al2O3 (CA) are cheap and effective nano adsorbents that can be used for the uptake of DR from aqueous solution. The CF and CA nano-composites were characterized using different techniques. Moreover, the effect of adsorption parameters (initial DR concentration, time of contact, pH, temperature, and adsorbent mass) as well as CA and CF reusability tests were performed. Langmuir adsorption isotherm and pseudo-second-order kinetics models were best fitted with the adsorption process. The maximum amount of DR adsorbed was 100 mg/g on CF and CA at pH 2 and 4, respectively with a physical spontaneous, and exothermic adsorption process. Monte Carlo (MC) simulation studies indicated the adsorption of DR molecule on the CF and CA surfaces following a parallel mode in most of all studied configurations, confirming the strong interactions between the DR and surfaces atoms of CF and CA. The molecular structure analysis of DR dye adsorbed on the surface of CF and CA indicated that the adsorption process related to Van der Waals dispersion force. Consequently, this helps to trap DR dye molecules on the surface of CF and CA (i.e., physical adsorption), which supports our experimental results.

The role of posttranslational modifications in generating neo-epitopes that bind to rheumatoid arthritis-associated HLA-DR alleles and promote autoimmune T cell responses

While antibodies to citrullinated proteins have become a diagnostic hallmark in rheumatoid arthritis (RA), we still do not understand how the autoimmune T cell response is influenced by these citrullinated proteins. To investigate the role of citrullinated antigens in HLA-DR1- and DR4-restricted T cell responses, we utilized mouse models that express these MHC-II alleles to determine the relationship between citrullinated peptide affinity for these DR molecules and the ability of these peptides to induce a T cell response. Using a set of peptides from proteins thought to be targeted by the autoimmune T cell responses in RA, aggrecan, vimentin, fibrinogen, and type II collagen, we found that while citrullination can enhance the binding affinity for these DR alleles, it does not always do so, even when in the critical P4 position. Moreover, if peptide citrullination does enhance HLA-DR binding affinity, it does not necessarily predict the generation of a T cell response. Conversely, citrullinated peptides can stimulate T cells without changing the peptide binding affinity for HLA-DR1 or DR4. Furthermore, citrullination of an autoantigen, type II collagen, which enhances binding affinity to HLA-DR1 did not enhance the severity of autoimmune arthritis in HLA-DR1 transgenic mice. Additional analysis of clonal T cell populations stimulated by these peptides indicated cross recognition of citrullinated and wild type peptides can occur in some instances, while in others cases the citrullination generates a novel T cell epitope. Finally, cytokine profiles of the wild type and citrullinated peptide stimulated T cells unveiled a significant disconnect between proliferation and cytokine production. Altogether, these data demonstrate the lack of support for a simplified model with universal correlation between affinity for HLA-DR alleles, immunogenicity and arthritogenicity of citrullinated peptides. Additionally they highlight the complexity of both T cell receptor recognition of citrulline as well as its potential conformational effects on the peptide:HLA-DR complex as recognized by a self-reactive cell receptor.

Molecular mimicry between varicella, measles virus and Hsp60 in type 1 diabetes associated HLA-DR3/DR4 molecules

Background and aimsType 1 diabetes (T1D) is a multifactorial autoimmune disease that combines genetics and environmental factors. The aim of this study is to determine the environmental risk factors and to investigate how virals infections are risks factors for type 1 diabetics whom have HLA DR3/DR4 predisposition in our population. MethodsThis study includes 233 subjects, 145 diabetics and 88 controls from regions of the extreme western of Algeria. All the informations related to the disease were collected using predesigned questionnaire.Using in silico approach, we attempt to improve the understanding of this analytical result by molecular mimicry, which is associated with the breakdown of several autoimmune pathologies. ResultsThe statistical study showed that history of varicella and measles infection and T1D related inheritance and type 2 diabetes are risk factors for T1D in the population of Tlemcen.We have determined the homologous antigenic regions between the glycoprotein “gE” of the varicella virus, the “hemagglutinin” of measles and the human protein “HSP60” at the level of their sequence and 3D structure. These cross-reactive epitopes bind to MHC class II molecules (HLA DR3/DR4) that predispose to T1D but not to MHC class II molecules (HLA DR2) that protect against T1D.This epitopes induce Th2 cells but only “hemagglutinin” and “Hsp60” can activate Th1 differentiation. This indicates their potential to destroy pancreatic cells β. ConclusionOur study can allow us to adapt biological markers to genetically predisposed T1D and to establish a preventive strategy for healthy genetic predisposed individuals in Tlemcen population.

A pilot study of preproinsulin peptides reactivity in Chinese patients with type 1 diabetes.

BackgroundThe aim of our study is to investigate whether preproinsulin (PPI) could trigger a proinflammatory CD4+ T cell response in Chinese patients with type 1 diabetes (T1D).MethodsPeripheral blood mononuclear cells were stimulated by a pool of 13 PPI peptides. Additional five PPI peptides previously proved to be antigenic in other cohorts of patients with T1D were also used. PPI reactive T cell responses were measured by interferon (IFN)‐γ ELISPOT assay.ResultsFifty‐one Chinese patients with T1D were enrolled in this study and 72.34% of them were positive for at least one islet autoantibody. The stimulation index (SI) value of IFN‐γ response to PPI peptide pool or peptides with dominant epitopes was below 3 in patients when SI≥3 was used as the positive cut‐off value. Two peptides (B9‐23 and C19‐A3) restricted to DQ8 or DR4 molecule failed to induce positive IFN‐γ response in patients with high‐risk HLA‐DQ8 or HLA‐DR4/DR9 alleles. RNA‐seq analysis of PPI specific CD4+ T cell lines further showed that most of the IFN‐γ associated genes remained unchanged.ConclusionsThis is the first report of CD4+ T cell epitope mapping of PPI in Chinese T1D. The lack of positive IFN‐γ response to PPI peptides indicates that PPI might not be the principal antigenic candidate for autoreactive CD4+ T cells in Chinese T1D. Therefore, the efficacy of PPI‐based immunotherapies in attenuating proinflammatory CD4+ T cell response requires further investigation.

Relevance of HLA-DQB1*02 Allele in the Genetic Predisposition of Children with Celiac Disease: Additional Cues from a Meta-Analysis

Background and Objectives: Celiac disease (CD) is a multifactorial immune-mediated disorder, triggered by the ingestion of gluten in genetically-predisposed subjects carrying MHC-DQ2 and -DQ8 heterodimers, which are encoded by four HLA-DQ allelic variants, overall. This meta-analysis aims at providing further epidemiological support to the predominant relevance of one specific allele, namely HLA-DQB1*02, in the predisposition and genetic risk of CD. Materials and Methods: We performed a search of MEDLINE/PubMed, Embase, Web of Science, and Scopus, retrieving all publications (case–control study, cross-sectional, and retrospective cohort study) on the association between HLA class II polymorphisms and first-degree relatives (FDRs) of children with CD. After a critical reading of the articles, two investigators independently performed data extraction according to the following inclusion criteria: HLA class II genes, any DQ and DR molecules, and CD diagnosed following the current clinical guidelines. A third participant was consulted for discussion to reach an agreement concerning discrepancies. Results: Our search strategy selected 14 studies as being eligible for inclusion, and those were submitted for data extraction and analysis. These studies were published between 1999 and 2016 and, collectively, enrolled 3063 FDRs. Positive and negative likelihood ratios (LR+ and LR−, respectively) for CD diagnosis, according to the presence of the HLA-DQ genotype coding a complete MHC-DQ2 and/or MHC-DQ8 molecules, were 1.449 (CI 1.279–1.642) and 0.187 (CI 0.096–0.362), respectively. If only the isolated presence of HLA-DQB1*02 allele is considered, the pooled estimation of LR+ was 1.659 (CI 1.302–2.155) and, importantly, the LR− still showed a very good discriminatory power of 0.195 (CI 0.068–0.558). Conclusions: Through our differential meta-analysis, comparing the presence of the genotype coding the full MHC-DQ2 and/or DQ8 molecules with the isolated presence of HLA-DQB1*02 allelic variant, we found that the LR− of the latter analysis maintained the same value. This observation, along with previous evidences, might be useful to consider potential cost-effective widened screening strategies for CD in children.

OR49 A pitfall of virtual crossmatching in pediatrics: Multiple solid phase reagents can show strong HLA antibodies that are not detectable in flow cytometry crossmatches

The usefulness of virtual crossmatching (VXM) is limited by the accuracy of current assays for HLA antibodies (Ab). We describe two cases of a specific false-positive pattern of class II Ab in toddler-age heart transplant (HTx) candidates. Subject 1 was a 13 month old female awaiting HTx for dilated cardiomyopathy, with no history of sensitizing events other than routine immunizations. Ab binding to DR4, DR7, and DR9 molecules in single antigen reagents from One Lambda (lot 11, 5,000–8,000 MFI) and Immucor (11,000–15,000 MFI) was suspect because it included a self-antigen (DRB1∗07:01). These specificities were also detectable in sera pretreated with DTT or fetal calf serum as well as with phenotype reagents (One Lambda and Immucor). Surrogate flow cytometry crossmatches (SFCXM) with DR4+ or DR7+ B cells treated with pronase were negative (positive without pronase). DR4, DR7, and DR9 specificities persisted during the 3 mo transplant waiting period. The patient underwent successful HTx with a DR4+ donor. The FCXM with donor B cells was negative (positive without pronase). Soon after transplant the strong DR4, DR7, DR9-signature was not detected. Nearly 2 years after transplant, no clinical or pathologic Ab-mediated or cellular rejection has occurred. Subject 2 (DR1,13) was listed for HTx due to dilated cardiomyopathy as an 18 month old male, without history of sensitizing events other than routine immunizations. His HLA Ab profile with single antigen reagents (One Lambda lot 12) showed a similar DR4, DR7, DR9 pattern (9,000–14,000 MFI) which was supported by data from phenotype reagents. SFCXM with DR4+ and DR7+ B cells were negative. This patient is still awaiting heart transplantation, and donors with these HLA types will not be excluded by VXM. DR4, DR7 and DR9 avoidance would exclude 50% of the American population, which may tragically slow the HTx waiting process for this patient population. We are skeptical of this particular pattern of antibodies, particularly in toddlers without previous HLA presensitizing events. These observations suggest that exclusive use of VXM without cell-based crossmatches to support clinical significance of the antibodies may unnecessarily limit donors. Investigation is underway to determine the cause for this discrepancy between the solid phase assays and cell-based crossmatches.

Roles of Hydration for Inducing Decomposition of 2-Deoxy-d-ribose by Ionization of Oxygen K-Shell Electrons.

To experimentally investigate the role of hydration in the initial process of the decomposition of 2-deoxy-d-ribose (dR), which is a major component of the DNA backbone, we used mass spectrometry to monitor the ions desorbing from hydrated dR films exposed to monochromatic soft X rays (560 eV). The X-ray photons preferentially ionize the K-shell electrons of the oxygen atoms in DNA. Hydrated dR samples were prepared under vacuum by exposing a cooled (∼150 K) dR film deposited on a Si substrate to water vapor. Using a quadrupole mass spectrometer, we observed the desorption of ions such as H+, CH x+, C2H x+, CH xO+, C3H x+ and C2H xO+ ( x = 1, 2, 3 and 4). In addition, the desorption of H2O+ or H3O+ was observed in the mass spectra of hydrated dR films. Except for H+, the yields of these ions decreased when one layer of water molecules was deposited onto the film. These ions are produced by C-C or C-O bond scission. This result suggests that the water molecules act as a quencher, suppressing Coulomb repulsion and thus the extensive molecular decomposition of dR. Ab initio molecular dynamics simulations were performed to rationalize the fragments observed in the experiments. The results of the dynamical process of a hydrated dR molecule after oxygen K-ionization revealed elongation of a C-O bond of dR and the O-H bonds of both dR and water molecules prior to the Auger process, resulting in the ejection of H+ ions. These results strongly suggest that the very early process contributes to reducing the dR fragmentation, producing the H3O+ and H+ detected from the hydrated dR films. These desorbed ions may be involved in the induction of other types of damage, such as oxidatively generated base lesions, concomitantly produced with a strand break when produced in DNA.

Potential molecular mimicry between the human endogenous retrovirus W family envelope proteins and myelin proteins in multiple sclerosis

Multiple sclerosis is an autoimmune disease caused by the destruction of the myelin sheath in the central nervous system. The major target molecules for the immune response are the myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein but the aetiology of the disease is as yet poorly understood. The HLA Class II allele DRB1*1501 in particular as well as DRB5*0101 and the expression of human endogenous retroviral envelope proteins have been linked to multiple sclerosis but the molecular mechanisms relating these remain to be elucidated. We hypothesised that cross-reactive peptide epitopes in retroviral envelope proteins and myelin proteins that can be presented by the two Class II DR molecules may play a role in initiating multiple sclerosis. Sequence homologies between retroviral envelope and myelin proteins and in silico predictions of peptides derived from them that are able to bind to the two Class II alleles were examined to test the hypothesis. The results support the hypothesis that molecular mimicry in peptide epitopes from envelope proteins of the HERV-W family of endogenous retroviruses and myelin proteins is possible and could potentially trigger multiple sclerosis. Mimicry between syncytin-1, a HERV-W envelope protein that is expressed during placentation, and myelin proteins may also explain the higher prevalence of multiple sclerosis in women. Experiments to test the ability of the identified peptide epitopes to activate TH cells are required to confirm the present findings.

The role of human leukocyte antigen DRB1-DQB1 haplotypes in the susceptibility to acquired idiopathic thrombotic thrombocytopenic purpura

The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying ADAMTS13-deficiency is caused by inhibitory autoantibodies against the protease. Human leukocyte antigens (HLA), responsible for antigen presentation, play an important role in the development of antibodies. The loci coding HLA DR and DQ molecules are inherited in linkage as haplotypes. The c.1858C>T polymorphism of the PTPN22 gene, which codes a protein tyrosine phosphatase important in lymphocyte activation, predisposes to a number of autoimmune diseases. We determined the HLA-DRB1-DQB1 haplotypes and the PTPN22 c.1858C>T genotypes in 75 patients with acquired idiopathic TTP and in healthy controls, in order to assess the role of these genetic factors and their interactions in the susceptibility to TTP. We found that the carrier frequencies of the DRB1∗11-DQB1∗03 and DRB1∗15-DQB1∗06 haplotypes were higher, while those of the DRB1∗07-DQB1∗02 and DRB1∗13-DQB1∗06 haplotypes were lower in TTP patients. There was no difference in the overall frequency of the PTPN22 c.1858T allele between TTP patients and controls. In conclusion, we identified four HLA-DRB1-DQB1 haplotypes associated with an increased (DRB1∗11-DQB1∗03 and DRB1∗15-DQB1∗06) or a decreased (DRB1∗07-DQB1∗02 and DRB1∗13-DQB1∗06) susceptibility to acquired idiopathic TTP.

A Comparative Analysis of the Peptide Repertoires of HLA–DR Molecules Differentially Associated With Rheumatoid Arthritis

To evaluate similarity of the peptide repertoires bound to HLA-DR molecules that are differentially associated with rheumatoid arthritis (RA), and to define structural features of the shared peptides. Peptide pools bound to HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*10:01 (RA associated) and those bound to HLA-DRB1*15:01 (non-RA-associated) were purified and analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS) and LC-ion-trap MS. Peptide pools from each allotype were compared in terms of size, protein origin, composition, and affinity (both theoretical and experimental with some peptides). Finally, 1 peptide sequenced from DR1, DR4, and DR10, but not from DR15, was modeled in complex with all 4 HLA-DRB1 molecules and HLA-DRB5*01:01. A total of 6,309 masses and 962 unique peptide sequences were compared. DR10 shared 29 peptides with DR1, 9 with DR4, and 1 with DR15; DR1 shared 6 peptides with DR4 and 9 with DR15; and DR4 and DR15 shared 4 peptides. The direct identification of peptide ligands indicated that DR1 and DR10 were the most similar molecules regarding the peptides that they could share. The peptides common to these molecules contained a high proportion of Leu at P4 and basic residues at P8 binding core positions. The degree of overlap between peptide repertoires associated with different HLA-DR molecules is low. The repertoires associated with DR1 and DR10 have the highest similarity among the molecules analyzed (∼10% overlap). Among the peptides shared between DR1 and DR10, a high proportion contained Leu(4) and basic residues at the P8 position of the binding core.

DENV-specific CD4 T-cells dominantly recognize capsid-derived epitopes and display a cytotoxic phenotype

Abstract While DENV-specific CD8+ T cell responses have been extensively studied, the breadth and specificity of CD4+ T cell responses remains to be defined. Here we map CD4 T cell responses in individuals previously exposed to dengue virus. To identify HLA class II candidates, we utilized a panel of algorithms for sixteen common HLA DR molecules representative of the main DR super types. These efforts led to the identification of 365 epitopes derived from all 10 DENV proteins. In contrast to CD8 T cell targets, the highest number of epitopes was associated with the structural capsid protein (C), followed by nonstructural NS3, NS2A, NS5 and envelope proteins (E). Phenotyping of the responding CD4+ T cell subset revealed a DENV specific T cell subset, specifically expanded in donors carrying an allele associated with protection from severe disease and which is absent in DENV negative donors. This subset of DENV specific CD4 T cells is associated with markers of cytotoxic T cells such as Granzyme B, Perforin, CD107a, T bet, Eomes and co-expression of CD8α. We are currently establishing specific gene expression signatures associated with these characteristic phenotypes, as a function of different HLA restricting alleles. Detailed knowledge of phenotype and function of DENV-specific T cells will aid in the establishment of correlates of protection against severe disease.

Artificial antigen-presenting cells expressing HLA class II molecules as an effective tool for amplifying human specific memory CD4(+) T cells.

Owing to their multiple immune functions, CD4(+) T cells are of major interest for immunotherapy in chronic viral infections and cancer, as well as for severe autoimmune diseases and transplantation. Therefore, standardized methods allowing rapid generation of a large number of CD4(+) T cells for adoptive immunotherapy are still awaited. We constructed stable artificial antigen-presenting cells (AAPCs) derived from mouse fibroblasts. They were genetically modified to express human leukocyte antigen (HLA)-DR molecules and the human accessory molecules B7.1, Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3). AAPCs expressing HLA-DR1, HLA-DR15 or HLA-DR51 molecules and loaded with peptides derived from influenza hemagglutinin (HA), myelin basic protein (MBP) or factor VIII, respectively, activated specific CD4(+) T-cell clones more effectively than Epstein-Barr virus (EBV)-transformed B cells. We also showed that AAPCs were able to take up and process whole Ag proteins, and present epitopes to specific T cells. In primary cultures, AAPCs loaded with HA peptide allowed generation of specific Th1 lymphocytes from healthy donors as demonstrated by tetramer and intracellular cytokine staining. Although AAPCs were less effective than autologous peripheral blood mononuclear cells (PBMCs) to stimulate CD4(+) T cells in primary culture, AAPCs were more potent to reactivate and expand memory Th1 cells in a strictly Ag-dependent manner. As the availability of autologous APCs is limited, the AAPC system represents a stable and reliable tool to achieve clinically relevant numbers of CD4(+) T cells for adoptive immunotherapy. For fundamental research in immunology, AAPCs are also useful to decipher mechanisms involved in the development of human CD4 T-cell responses.

Evaluation of Thermal Stability of Organic Electro-Optic Device by Using Thermally Stimulated Current.

Thermally stimulated current (TSC) measurement was employed to study the thermal stability of electro-optic (EO) polymers, i.e., guest/host polymer DR1/PMMA and side-chain polymer PMMA-co-DR1. Here the isothermal relaxation test showed that the relaxation time τ (85 °C) of side-chain polymer PMMA-co-DR1 is longer than that of guest/host polymer DR1/PMMA. TSC peaks appeared symmetrically in proportion to the poling electric field Ep, indicating that DR1 molecules make a dominant contribution to dipolar depolarization. Thermal sampling (TS) method showed that the activation energy of the DR1/PMMA is around 1 eV, while that of the PMMA-co-DR1 is distributed >1 eV. Results suggested that side-chain polymer is preferable to the guest/host polymer in the thermal stability. TSC measurement is helpful as a conventional method for studying the life time of EO polymers in terms of dipolar motion.