Analysis of Autoantibodies to T-Cell Receptors among HIV-Infected Individuals: Epitope Analysis and Time Course

Individuals seropositive for human immunodeficiency virus type 1 (HIV) express elevated levels of autoantibodies (AAbs) directed against recombinant T-cell receptors (TCRs) and synthetic peptide epitopes duplicating β chain markers. We performed longitudinal studies of anti-TCR AAbs in HIV-1-infected individuals, making comparisons with uninfected sera and sera from other individuals infected with a nonviral agent. We determined levels of autoantibodies by titration using enzyme-linked immunosorbent assay (ELISA) and developed a means for characterizing “autoantibody CDR recognition spectrotypes” for individual sera. Antibody levels against certain defined synthetic epitopes were substantially elevated in HIV-infected subjects relative to reactivities by control groups. Individual sera showed relatively high AAb levels to a subset of CDR1 peptide epitopes. Two patients who subsequently developed AIDS showed particular reactivity to Vβ2.1, 8.1, 10.1, and 22.1 epitopes. Our results show that production AAbs to TCR Vβ epitopes is a general consequence of HIV infection. The response is individual but shows some restriction and shifts in AAb subpopulations often occur with time.     

HLA-DR3- and HLA-DR7-Restricted T-Cell Hyporesponsiveness to Gluten Antigen: A Clue to the Aetiology of Coeliac Disease?

Coeliac disease (CD) is strongly associated with the human class-II HLA determinants HLA-DR3 and -DR7. We investigated the relative frequency of gluten-reactive T cells from DR3- or DR7-positive. CD patients and healthy controls who were heterozygous at the DR locus. We found a consistently and significantly lower frequency of gluten-reactive T cells when the antigen was presented by monocytes in conjunction with DR3 or DR7 than in conjunction with the other DR determinant of the T-cell donor. In contrast, the frequency of reactive T cells in these donors to other antigens was not reduced in conjunction with DR3 or DR7. These results indicate a specific immunoregulatory function associated with class-II HLA molecules. The reduced frequency of gluten-reactive T cells in association with HLA-DR3 or -DR7 may be directly involved in the development of CD.     

Identification of a Gliadin T-Cell Epitope in Coeliac Disease: General Importance of Gliadin Deamidation for Intestinal T-Cell Recognition

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified γ-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.     

Nearly Identical T-Cell Receptor V-Gene Usage at Birth in Two Cohorts of Distinctly Different Ethnic Origin: Influence of Environment in the Final Maturation in the Adult

In this study we report on functional characteristics of pustule as well as blood polymorphonuclear neutrophils (PMN) in a patient suffering from relapsing bullous staphyloderma. Large numbers of viable PMN from newly formed pustules as well as from the peripheral blood were investigated. During the course of disease chemotactic migration, enzyme degranulation, superoxide-anion generation and leukotriene B4 production were determined simultaneously. The results revealed C5a- and NAP-1/IL-8-specific dysfunction of pustule PMN as compared with blood PMN. In contrast, FMLP-elicited functional activities of pustule PMN were only slightly affected. Our findings provide evidence that in inflamed tissue invading PMN are regulated by in situ generated mediators. C5a produced by staph, aureus-induced activation of the alternative pathway of the complement cascade represents a predominant regulatory factor in situ. Furthermore, the results substantiate previous observations concerning different modulation of C5a and f-met-peptide receptors on human PMN.     

T-Cell Receptor Diversity Expressed by CD4+ T Cells Activated by Primary Allogeneic HLA-DR Stimulation: Estimation of the Degree of CDR3 Diversity

In order to analyse the diversity of T-cell receptors (TCRs) expressed by the T-cell population activated by allogeneic HLA-DR stimulation, TCRβ cDNA was synthesized from mRNA of human CD4⁺ T cells that had been stimulated in a primary mixed lymphocyte reaction (MLR). The TCRβ cDNA was amplified by the polymerase chain reaction (PCR), subjected to bacterial cloning, and sequenced from Vβ through Jβ. Twenty-six different Vβ genes and 10 different Jβ segments were detected among 56 randomly selected cDNA clones. Occurrences of Vβ17.1 and Jβ1.5 were higher than those found in the CD4⁺ T-cell population activated with a CD3-specific antibody. A total of 53 different CDR3 sequences, two of them occurring more than once, were detected among the 56 cDNA clones. In order to estimate the degree of CDR3 diversity, amino acid similarity in the CDR3 region of the cDNA was calculated and compared with those of the anti-CD3-activated T-cell sequences as well as those of various published T-cell clone sequences, each directed to either alloantigens or single antigenic peptides. It was found that the similarity score among CDR3 sequences obtained from the MLR (56.4 ± 10.3) was comparable to those of anti-CD3-activated T cells (55.7 ± 10.7) and those of T-cell clones directed toward alloantigens (range, 48.4 ± 12.4−59.4 ± 13.1), but significantly smaller than those of T-cell clones directed toward single antigenic peptides such as those derived from myelin basic protein (75.6 ± 17.9) and cytochrome c (76.9 ± 20.5). These results provide quantitative proof that TCRs of T cells activated by primary allogeneic HLA-DR stimulation have a larger diversity than those recognizing single antigenic peptides.     

Comparison of Outcomes of Allogeneic Hematopoietic Cell Transplantation for Multiple Myeloma Using Three Different Conditioning Regimens

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapy for patients with multiple myeloma, as it provides a graft-versus-myeloma effect alongside a myeloma-free graft. Although reduced-intensity conditioning regimens decrease nonrelapse mortality (NRM), there is a paucity of data with regard to the ideal conditioning regimen in myeloma. We conducted a retrospective comparison of 3 different preparative regimens used for allo-HCT for multiple myeloma at our institution in recent clinical trials: busulfan/fludarabine (BuFlu), fludarabine/melphalan 100 mg/m² (FM100), and fludarabine/melphalan 140 mg/m² (FM140). NRM, progression-free survival (PFS) at 3 years, and overall survival (OS) at 3 years were the primary endpoints. Secondary endpoints included time to engraftment, and the incidence of grades II through IV acute graft-versus-host disease and chronic graft-versus-host disease. A total of 73 patients received allo-HCT with these regimens. NRM at 3 years was seen in 3 (21%), 5 (28%), and 6 (24%) patients in the BuFlu, FM100, and FM140 groups, respectively. Three-year PFS in the BuFlu, FM100, and FM140 groups was 16% (hazard ratio [HR], 1.2; 95% confidence interval [CI], 0.6 to 2.1), 26% (HR, 0.6; 95% CI, 0.3 to 1.2), and 11% (reference), respectively. Three-year OS in the BuFlu, FM100, and FM140 groups was 39% (HR, 1.1; 95% CI, 0.5 to 2.2), 43% (HR, 0.7; 95% CI, 0.3 to 1.4), and 32% (reference), respectively. High-risk cytogenetics and relapsed disease prior to allo-HCT were found to be independent predictors of inferior OS on multivariate analysis, with a HR of 2.1 (P = .02) and 2.6 (P = .004), respectively. In contrast, the preparative regimen did not emerge as a predictor of PFS or OS. Durable clinical remission can be achieved in 11% to 25% of patients with multiple myeloma with the use of allo-HCT without any significant difference in the safety or efficacy of the conditioning regimen. High-risk cytogenetics and relapsed disease prior to transplant were associated with inferior PFS and OS.     

Assay for the Two Different Types of Lymphocyte Complement Receptors

Abstract. This paper describes the assay system for two different types of lymphocyte complement receptors, the immune adherence receptor (C3b receptor) and the C3d receptor.     

Blueprints of Signaling Interactions between Pattern Recognition Receptors: Implications for the Design of Vaccine Adjuvants

Innate immunity activation largely depends on recognition of microorganism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other mediators. Moreover, PRR engagement in antigen-presenting cells initiates the activation of adaptive immunity. Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity, synergistic effects between two or more PRRs are necessary. No systematic analysis of the interaction between the major PRR pathways were performed to date. In this study, a systematical analysis of the interactions between PRR signaling pathways was performed. PBMCs derived from 10 healthy volunteers were stimulated with either a single PRR ligand or a combination of two PRR ligands. Known ligands for the major PRR families were used: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RigI-helicases. After 24 h of incubation, production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-10 was measured in supernatants by enzyme-linked immunosorbent assay (ELISA). The consistency of the PRR interactions (both inhibitory and synergistic) between the various individuals was assessed. A number of PRR-dependent signaling interactions were found to be consistent, both between individuals and with regard to multiple cytokines. The combinations of TLR2 and NOD2, TLR5 and NOD2, TLR5 and TLR3, and TLR5 and TLR9 acted as synergistic combinations. Surprisingly, inhibitory interactions between TLR4 and TLR2, TLR4 and Dectin-1, and TLR2 and TLR9 as well as TLR3 and TLR2 were observed. These consistent signaling interactions between PRR combinations may represent promising targets for immunomodulation and vaccine adjuvant development.     

Retrospective Analysis of Clinical Performance of an Estonian Speech Recognition System for Radiology: Effects of Different Acoustic and Language Models

The aim of this study was to analyze retrospectively the influence of different acoustic and language models in order to determine the most important effects to the clinical performance of an Estonian language-based non-commercial radiology-oriented automatic speech recognition (ASR) system. An ASR system was developed for Estonian language in radiology domain by utilizing open-source software components (Kaldi toolkit, Thrax). The ASR system was trained with the real radiology text reports and dictations collected during development phases. The final version of the ASR system was tested by 11 radiologists who dictated 219 reports in total, in spontaneous manner in a real clinical environment. The audio files collected in the final phase were used to measure the performance of different versions of the ASR system retrospectively. ASR system versions were evaluated by word error rate (WER) for each speaker and modality and by WER difference for the first and the last version of the ASR system. Total average WER for the final version throughout all material was improved from 18.4% of the first version (v1) to 5.8% of the last (v8) version which corresponds to relative improvement of 68.5%. WER improvement was strongly related to modality and radiologist. In summary, the performance of the final ASR system version was close to optimal, delivering similar results to all modalities and being independent on user, the complexity of the radiology reports, user experience, and speech characteristics.     

A family with an apparent HLA-DR triplet: evidence for exchange of functional HLA-DR beta-genes between different haplotypes

Serological and protein analysis showed that HLA-DR1 and DR2 short (s) segregated together as one haplotype, resulting in a HLA-DR triplet as found in a family study. Both DR1 and DR2 molecules were coordinately expressed and were shown to function as restriction elements in antigen presentation assays. This unique HLA-DR1, 2s haplotype was further studied by Southern blot analysis. Based upon well-known restriction fragment length polymorphisms for the involved gene sets, i.e. DR1 and DR2 along with the DR type from the other haplotype, the genes as identified by restriction fragment length polymorphism of the triplet could be established. Pseudogenes, which are included in the previously described gene sets of HLA-DR1 and DR2 are apparently lacking in the triplet. We therefore postulate that during an unequal crossing-over event the DR beta-pseudogene of DR2 could be exchanged by a functional DR1 beta-gene.     

Human T-Cell Hybrids Secreting Lymphokines: Effect of Different Parent Cell Clones of the Human T-Cell Acute Lymphoblastoid Leukaemia Line CCRF-CEM

We have cloned a number of cell lines from the human T-lymphocyte acute lymphoblastoid leukaemia (ALL) line CCFR-CEM, and attempted to construct functionally active human T-lymphocyte hybrids with them. Functional hybrids were generated using only one particular clone, 3H6. The activities found in the supernatants of two of these hybrids, DB1G7 and DB2D10, are described. Supernatant from DB1G7 was found to suppress strongly the migration of normal human peripheral blood mononuclear cells, while that from DB2D10 was shown to inhibit the proliferative response of human T lymphocytes to both phytohaemagglutinin and concanavalin A. There was no cross-reactivity between the two supernatants, confirming the usefulness of the human T-lymphocyte hybrid technique in dissecting human T-lymphocyte function. The successful use of 3H6 is contrasted with the failure of another clone, 2H2, to permit the production of functional hybrids. Problems relating to the use of CCRF-CEM and its clones as parent cell lines in the production of human T-lymphocyte hybrids are discussed.     

Comparison of Outcomes after Two Standards-of-Care Reduced-Intensity Conditioning Regimens and Two Different Graft Sources for Allogeneic Stem Cell Transplantation in Adults with Hematologic Diseases: A Single-Center Analysis

Recent advances in allogeneic stem cell transplantation (allo-HSCT) have included the advent of reduced-intensity conditioning (RIC) regimens to decrease the toxicity of myeloablative allo-SCT and the use of double umbilical cord blood (dUCB) units as a graft source in adults lacking a suitable donor. The FB2A2 regimen (fludarabine 30 mg/kg/day for 5-6 days + i.v. busulfan 3.6 mg/kg/day for 2 days + rabbit antithymocyte globulin 2.5 mg/kg/day for 2 days) supported by peripheral blood stem cells (PBSCs) and the TCF regimen (fludarabine 200 mg/m² for 5 days + cyclophosphamide 50 mg/kg for 1 day + low-dose [2 Gy] total body irradiation) supported by dUCB units are currently the most widely used RIC regimens in many centers and could be considered standard of care in adults eligible for an RIC allo-SCT. Here we compared, retrospectively, the outcomes of adults patients who received the FB2A2-PBSC RIC regimen (n = 52; median age, 59 years; median follow-up, 19 months) and those who received the dUCB-TCF RIC regimen (n = 39; median age, 56 years; median follow-up, 20 months) for allo-SCT between January 2007 and November 2010. There were no significant between-group differences in patient and disease characteristics. Cumulative incidences of engraftment, acute grade II-IV and chronic graft-versus-host disease were similar in the 2 groups. The median time to platelet recovery, incidence of early death (before day +100), and 2-year nonrelapse mortality were significantly higher in the dUCB-TCF group (38 days versus 0 days [P <.0001]; 20.5% versus 4% [P = .05], and 26.5% versus 6% [P = .02], respectively). The groups did not differ in terms of 2-year overall survival (62% for FB2A2-PBSC versus 61% for dUCB-TCF), disease-free survival (59% versus 50.5%), or relapse incidence (35.5% versus 23%). In multivariate analysis, the presence of a lymphoid disorder was associated with a significantly higher 2-year overall survival (hazard ratio, 0.42; 95% confidence interval, 0.20-0.87; P = .02), whereas patients receiving a FB2A2-PBSC allo-SCT had a significantly lower 2-year nonrelapse mortality (hazard ratio, 0.24; 95% confidence interval, 0.1-0.7; P = .01). There were no factors associated with higher 2-year disease-free survival or lower relapse incidence. This study suggests that the dUCB-TCF regimen provides a valid alternative in adults lacking a suitable donor and eligible for RIC allo-SCT. Prospective and randomized studies are warranted to establish the definitive role of dUCB RIC allo-SCT in adults. In addition, strategies for decreasing nonrelapse mortality after dUCB RIC allo-SCT are urgently needed.     

Cytochemical Characterization and Localization of Oligomannosidic Oligosaccharide Receptors on the Normal Human Spermatozoa Using Fluorescent Lectins: Comparison of Different Fixation Procedures

ABSTRACT: Similar localizations of the Con A and wheat germ lectin receptors were obtained by using fluorescent lectins, in nonfixed spermatozoa and in spermatozoa fixed with formaldehyde and methanol, showing that in samples with the same previous treatment, worked out by the same operator, in which enough determinations have been performed to eliminate individual variations, the different procedures of fixation produced similar results. The localizations obtained with fluorescent lectins confirm previous results, produced with the peroxidase technique, indicating that the lectins interact with oligomannosidic oligosaccharide receptors situated mainly in the equatorial segment of the acrosome and postnu-clear cap. They also indicate the presence of similar receptors that were not detected previously on the neck and intermediate segment. The larger size of the lectin-peroxidase-diaminebenzidine reagent compared to that of the fluorescent lectins suggests that the new receptors are semicriptic and were not detected by steric effects in the first case, but were able to interact with lower volume, fluorescent probes. It is suggested that these oligomannosidic chains could be recognition signals for the elimination of incompetent sperm during their passage through the female reproductive track. Also these oligosaccharides and its possible metabolic variations could be involved in the interaction between the acrosome-reacted spermatozoa with the zone pellucida.     

3种 DNA 甲基化定量分析方法的比较

目的：比较特定基因区域 DNA 甲基化检测方法的优缺点。方法采用焦磷酸测序、克隆测序和DNA 甲基化芯片等3种 DNA 甲基化检测方法,对7例唐氏综合征和5例正常对照样本的 PRDM8内含子7 CpG 岛中的部分序列进行甲基化检测。从准确度、重复性和精确度(分辨率)等方面来比较3种方法,探讨哪种方法更适合特定基因的 DNA 甲基化水平定量检测。结果①焦磷酸测序可在单碱基水平定量检测样本的甲基化水平,结果重复性好,准确度高,而且能发现样本中 CpG 位点甲基化水平变化的规律;②克隆测序也可在单碱基水平定量检测样本的甲基化水平,但结果重复性较差,无法发现 CpG 位点甲基化水平变化的规律;③甲基化芯片可通过所测区域的两个探针来判断样本间甲基化水平的差异,但无法在单碱基水平定量检测样本的甲基化水平;④上述3种方法检测结果之间存在一定的差异,但是样本间甲基化水平总体趋势基本一致。此外,唐氏综合征样本的甲基化水平高于正常对照样本。结论焦磷酸测序准确度高、重复性好、费用较低、操作较为简单快捷,更为适合在单碱基水平检测特定基因区域的 DNA 甲基化水平。     

Lymphocyte Recirculation: the Need for Site-Specific Receptors to Dictate T-Lymphocyte Localization into Different Tissue Sites

This paper addresses the need for receptors involved in recirculation of T lymphocytes both during development and during an immune response or inflammation. Some known receptors present on T lymphocytes which are important in maintaining normal T-cell localization and function, i.e. the integrins, LECAMs, LPAM-1 and H-CAM (CD44) are considered in terms of the specificity which they confer on lymphocyte recirculation. A clear understanding of lymphocyte trafficking patterns and of the receptors involved may provide, for example, novel therapies for treatment of malignant cancer, alleviation of damage caused by lymphocytes during inflammatory responses, and targeting of cells to sites of infection.