Multi-locus analysis of HLA class II genes in DR2-positive IDDM haplotypes in Finland

Abstract: In this study we characterized the haplotypes found in IDDM patients that normally confer resistance to the disease in order to localize the polymorphisms relevant for the protection. We studied 15 DR2-positive subjects with IDDM for their DRB1, DRB5 and DQB1 genes using RFLP, polymerase chain reaction (PCR), oligonucleotide typing, and in some specific cases direct sequencing after allele-specific PCR. In addition we analyzed 39 DR2-positive, IDDM non-associated haplotypes representing those haplotypes that are not inherited to probands and hence are present only in healthy family members. The frequency of the DRB1*1501-DRB5*0101-DQB1*0602 haplotype was slightly decreased among diabetic patients (80% vs. 92%). In addition, two unconventional haplotypes DRB1*1501-DRB5*0101-DQB1*05031 and DRB1*1501-DRB5*0101-DQB1*0502 were found in patients with IDDM while all the control ones were conventional. The sequencing of the DQB1*0602 allele present in IDDM haplotypes showed no differences when compared to the controls. These results support the primary but not absolute role of DQ in the protection against IDDM. An additional role of factors centromeric to DQB1 gene was suggested by findings based on the biallelic TaqI RFLP polymorphism of the DQA2 gene. All DR2-DQB1*0602 IDDM haplotypes were associated with the 2.1-kb fragment while in the control group the 2.1-kb and 1.9-kb fragments were evenly distributed.     

Molecular oncogene markers and their significance in cutaneous malignant melanoma

Background: Oncogenes and other molecular tumor markers that predict tumor aggressiveness may allow individualization and optimization of surgical therapy of intermediate-thickness malignant melanoma. We examined the expression of selected markers, including the HLA-DR antigen, the heat shock protein-70 (HSP-70), and the c-myc oncogene in primary melanoma and regional nodes and related these findings to metastatic potential and survival. Methods: Forty patients with primary melanoma (1.5–4.0 mm) were studied, all of whom had prophylactic lymph node dissection and were followed for 18 months to 7 years. The primary tissue and nodes were examined using immunohistochemical techniques for the presence of HLA-DR antigen and HSP-70 protein and the expression of the c-myc oncogene. Results: Of 40 patients, there were 23 with lesions 1 to 2.9 mm thick and 17 with lesions 3 to 4 mm thick. Nodal metastases were present in 25 of the 40 patients who had elective node dissection. HLA-DR antibody stained the primary tumor in 10 patients (25%), but there was no correlation with survival in this group. HLA-DR antibody stained the stroma and cellular infiltrates surrounding the primary tumor in 28 of 40 patients; in this group there was a correlation of HLA-DR staining of the peritumoral stroma with improved survival overall. HLA-DR staining of the peritumoral stroma also influenced survival when patients were stratified by tumor thickness groups 1 to 2.9 mm and 3 to 4 mm and presence of nodal metastases. HSP-70 was demonstrated in the primary tumor in 25% of patients, who were also shown to have significantly improved survival when compared with those whose primary tumor did not stain with HSP-70. C-myc was expressed in the primary tumor in 25%, but showed no correlation with survival. None of these proteins correlated with or predicted the presence of nodal metastases. Conclusion: We conclude that the use of specific molecular-oncogene markers in intermediate-thickness primary melanoma may identify patients at high risk for conventional treatment failure and reduced survival who may profit from more aggressive surgery, adjuvant therapy, or both.Presented at the 48th Annual Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

中国内地人群类风湿关节炎与HLA-DRB1共同表位基因关联性的Meta分析

目的探讨中国内地人群类风湿关节炎(RA)与人类白细胞抗原(HLA)-DRB1共同表位(SE)基因的关联情况。方法检索已发表的有关中国内地人群RA和HLA-DRB1的文献,进行汇总分析分析。结果14项研究共纳入1118例RA患者和1301名正常对照,中国内地人群RA的关联基因有HLA-DR4[比值比(OR)=4.05,P<0.0001)]、DRB1*0401(OR=2.66,P=0.004)、DRB1*0404(OR=2.10,P=0.02)、DRB1*0405(OR=3.98,P<0.0001)、DRB1*0410(OR=3.36,P=0.008)、SE(OR=2.75,P<0.00001)。其中DRB1*0405是主要基因亚型。结论汇总分析分析不仅证实了DRB1*0405与中国内地人群RA的关联,而且发现了相对少见基因亚型DRB1*0401、DRB1*0404、DRB1*0410与RA的关联,样本大小对基因关联分析的结论有重要影响。     

结直肠癌HLA-DR表达与树突细胞浸润及其临床意义

目的:分析结直肠癌HLA-DR表达及癌组织中树突细胞(DC)的密度,探讨两者对结直肠癌患者临床分期及预后的影响。方法:47例结直肠癌石蜡标本,应用免疫组织化学SP法检测癌组织HLA-DR表达和S100阳性树突细胞的浸润密度,并与患者的临床分期和预后进行统计学分析。结果:结直肠癌HLA-DR表达率27.7%(13/47),表达率随临床分期增加而逐渐下降(P<0.05),但对预后无明显影响。S-100阳性DC丰富(≥10/HPF)的病例为44.7%(21/47),DC浸润密度在患者的临床分期和预后方面均有显著差异性(P<0.05)。HLA-DR表达与DC浸润程度密切相关(r=0.522;P<0.01)。结论:HLA-DR表达与DC浸润密度可反映了结直肠癌患者的抗肿瘤免疫状况,可能成为判断临床分期和预后的重要指标。     

HLA-DR、DQ基因多态性与遵义地区汉族流行性出血热相关性的研究

目的：探讨HIA-DR、DQ基因多态性与遵义地区汉族流行性出血热（EHF）的关联性。方法：采用群体研究方法，应用聚合酶链反应-序列特异性引物（PCR-SSP）技术对100例流行性出血热患者（患者组）和100例健康对照者（健康对照组）进行HIA-DR、DQ基因分型，比较其等位基因频率（GF），并计算其相对危险度（RR）。结果：EHF患者组HLA-DRB1＊16基因频率较对照组明显增高（Pat=3．58，χ^2=4．916，P=0．0266〈0．05）；患者组HIA-DQ各等位基因频率与对照组比较差异无显著性。结论：研究提示在遵义地区汉族人群中，HLA-DRB1＊16基因与EHF呈正相关，HLA-DRB1＊16基因可能是EHF的易感基因。     

Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma

We have previously reported that human T cell receptors (TcR) selected in the class II-restricted (HLA-DRB1*1302) response to a tetanus toxin peptide (tt830-843) frequently used the Vβ2 germ-line segment which paired with several Vα segments and that the putative CDR3 of both α and β chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 α^?β^?, by expressing the human α and β variable regions joined to the mouse α and β constant regions, respectively. The chimeric TcR, expressing the same Vβ germ-line segment (Vβ2), two expressing Vα21.1, twoVα17.1 and one Vα8.1 were shown to have the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ-line segments but distinct CDR3 showed large differences in their capacity to react with the ligand. Second, interchanging the α and β chains from tt830-843/DR1302-specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ-line Vα17.1 could functionally replace Vα21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, Vα replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity. Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site-directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti-tt830-843 TcR may have a similar orientation with respect to the peptide/DR complex. The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.     

特异性寡核苷酸探针检测胰岛素依赖型糖尿病病人HLA－DR基因频率

为了探讨ＨＬＡ－ＤＲ基因在胰岛素依赖型糖尿病（ＩＤＤＭ）发病机制中的作用，利用聚合酶链反应技术及３０个特异性寡核苷酸探针检测３２例中国北方汉族ＩＤＤＭ病人及２５５名正常中国北方汉族人的ＨＬＡ－ＤＲ基因频率的分布。结果ＩＤＤＭ组ＤＲ３和ＤＲ４频率分别为２５．０％和４０．６％，明显高于正常组（８．６％和１８．６％），ＤＲ３和ＤＲ４的相对危险率分别为３．５ｌ和３．１０。提示ＤＲ３、ＤＲ，与ＩＤＤＭ易感性呈正相关。     

Distribution of HLA-DQA1 and -DQB1 alleles and DQA1-DQB1 genotypes among Senegalese patients with insulin-dependent diabetes mellitus

Transracial analysis is one method for distinguishing primary associations between insulin-dependent diabetes mellitus (IDDM) and HLA II alleles from those related to linkage disequilibrium. Black people have different DR-DQ relationships from other races and are a useful group to investigate HLA-D regions associated with IDDM. In this study, we compared the frequencies of HLA-DQA1 and DQB1 alleles in Senegalese IDDM and control subjects. DQA1*0301 was positively associated with insulin-dependent diabetes mellitus (p<10^-9, OR 5.21), as were DQB1*0201 and *0302 (p<10^-7 OR=3.55, p<10^-3 OR=3.20, respectively). The positive associations with DQA1*0301, DQB1*0201 and DQB1*0302 are consistent with all racial groups investigated. However, taken together, the data in Senegalese population show that susceptibility and resistance to IDDM are associated both with particular haplotypes and DQA1-DQB1 heterodimers.     

A T cell epitope-based vaccine protects against chlamydial infection in HLA-DR4 transgenic mice

Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4⁺ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4⁺ T cells in host defense against Chlamydia. In this study, we identify CD4⁺ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4⁺ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4⁺ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4⁺ clones could be detected in the mouse spleen following Chlamydia muridarum and a human Chlamydia trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4⁺ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4⁺ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.     

Limitations of an ocular surface inflammatory biomarker in impression cytology specimens

Context: A number of ocular conditions, such as dry eye, are associated with inflammation on the surface of the eye leading to irritation and ocular pain. Many drugs such as chemotherapeutics, beta blockers, angiotensin-converting enzymes and so forth also cause dry eye but currently there are no validated ocular surface biomarkers available.

Objective: We evaluated sample stability, assay sensitivity, reproducibility and overall performance of impression cytology (IC) utilizing the cellular surface biomarker human leukocyte antigen DR-1 (HLA-DR) as an ocular surface inflammatory biomarker by flow cytometry in a fit-for-purpose validation study. Additionally, subjects classified as normal or having various degrees of dry eye were evaluated to determine if HLA-DR could demonstrate a clear separation between normal and dry eye samples.

Results: The assay demonstrated high dynamic range detecting a broad range of fluorescent intensities in healthy donors. Additionally, inter, intra and stability assay results demonstrated strong concordance and low variability. Overall CV% for both assays were less than 25% for all measured parameters. However, high variability was observed for donor samples assayed beyond day 10 post IC sample collection (4.2–110.8 CV%).

Discussion: HLA-DR expression demonstrated a progressive increase in patients with mild to severe levels of dry eye disease providing sufficient evidence it is sensitive enough to monitor inflammatory effects of dry eye when coupled with additional biomarkers and/or methodologies such as cytokine analysis or ICAM-1. This biomarker can be used to monitor ocular surface disorders in patients and to evaluate potential treatment options during drug development. Although our results demonstrate this methodology is reproducible for routine evaluation, limitations around sample integrity exist.

Conclusion: The ocular cell surface inflammatory biomarker, HLA-DR coupled with impression cytology is a simple non-invasive robust, specific and reproducible assay that can be utilized to measure inflammatory infiltrates on the surface of the eye in IC samples less than 10-days old.