An EIA method on single donor solubilized HLA antigens for the identification of anti-HLA antibodies

An enzyme immunoassay (EIA) method based on solubilized human leucocyte antigens (HLA) derived from single donor platelets is described. The EIA results on these solubilized single donor HLA antigens (SDszHLA) correlated well with the complement dependent cytotoxicity (CDC) results on the lymphocytes of the same donors and also with the panel reactivity (PRA) in CDC. A concordancy rate of 78% was found for individual HLA specificities. The EIA+/CDC? (‘false positive’) discrepancies were more pronounced than EIA?/CDC+ (‘false negative’) discrepancies and varied for the different donors. To confirm discrepancies, our method was compared with a commercial PRA-STAT EIA method (based on secreted soluble HLA antigens). The same discrepancies between CDC and PRA-STAT EIA were found and are probably due to the higher and different sensitivity (e.g. non complement fixing antibodies) of EIA methods. A SDszHLA EIA method allows the identification of HLA specificities of HLA-antisera. The possibility of using individual and selected donors for the production of SDszHLA allows the directed search for well defined HLA specificities in order to confirm anti-HLA specificities found in other anti-HLA screening methods. An individualized HLA panel can be established with the support of blood banks that have HLA typed blood and platelet donors.     

Typing of HLA Class I by Polymerase Chain Reaction-Sequence Specific Oligonucleotide Primer (PCR-SSOP) Technique in Iranian Cord Blood Donors

Background: HLA compatibility between transplant donor and recipient is one of the major determinants of transplant outcome. Objective: To determine HLA class I by PCR- Sequence-Specific Oligonucleotide Probe (PCR-SSOP) in cord blood donors. Methods: Genomic DNA of 142 cord blood samples registered at the Cord Blood Bank of Iran at Hematology, Oncology, and Bone Marrow Transplantation Research Center, was prepared and HLA class I was determined by the PCR-SSOP. Results: A total of 284 HLA-A alleles was identified of which A*02 and A*24 were the most common. Among 284 HLA-B and HLA-C alleles, B*35, B*51, Cw*4 and Cw*12 were the most frequent alleles in the studied population. Conclusion: Amplification of HLA loci with PCR-SSOP has proved to be a reliable method for HLA-A, -B and -C genotyping.     

拉米夫定对慢性乙型肝炎患者可溶性HLA-1类分子含量的影响

目的:测定可溶性HLA-Ⅰ类分子的含量在慢性乙型肝炎治疗中的变化,并分析其意义。方法:将60例慢性乙型肝炎患者随机分为拉米夫定治疗组,甘利欣治疗组和拉米夫定加甘利欣联合治疗组每组均20例;血清可溶性HLA-Ⅰ类分子的含量用夹心ELISA方法测定。结果:治疗两个月,拉米夫定治疗组患者可溶性HIA-Ⅰ类分子的含量显著高于治疗前(P<0.01),甘利欣治疗组患者可溶性HLA-Ⅰ类分子的含量显著低于治疗前(P<0.05),联合治疗组患者可溶性HLA-Ⅰ类分子的含量与治疗前相比无显著变化;治疗4个月,3组患者可溶性HLA-Ⅰ类分子的含量均显著低于治疗前(P<0.01)。结论:可溶性HAL-Ⅰ类分子的含量反映了慢性乙型性肝炎治疗中患者免疫功能受到的调节。     

Stimulation of human neutrophils with sera containing HLA Class I alloantibody causes preferential degranulation of azurophilic granules and secretory vesicles

Background and Objectives The activation of neutrophils by human leukocyte antigen (HLA) Class I alloantibody is thought to be involved in transfusion‐related acute lung injury. Neutrophils contain various biological substances in four groups of granules, including secretory vesicles, azurophilic granules, specific granules and gelatinase granules. To characterize the activation of neutrophils by HLA Class I alloantibody, we investigated whether HLA Class I alloantibody could cause the degranulation of these groups of granules either coordinately or selectively. Materials and Methods Sera containing HLA‐A24 alloantibody were incubated with neutrophils in a washed whole blood system. CD11b expression (secretory vesicles) on neutrophils was analysed by flow cytometry, and the secretion of markers of each granule was determined by ELISA. Results The treatment of cross‐matching‐positive neutrophils with sera containing HLA‐A24 alloantibody caused the significant expression of CD11b, and the significant secretion of neutrophil elastase and myeloperoxidase, azurophilic granule markers and heparin‐binding protein (HBP), which is localized in secretory vesicles and azurophilic granules when compared with cross‐matching‐negative neutrophils. In contrast, no significant differences were observed in the secretion of lactoferrin, a marker of specific granules, and matrix methalloproteinase‐9, a marker of gelatinase granules between cross‐matching‐positive and cross‐matching‐negative cells upon stimulation with sera. CD11b expression and secretion of HBP by serum was partially inhibited by p38 mitogen‐activated protein (MAP)‐kinase inhibitors. Conclusion Neutrophils activated with sera containing HLA Class I alloantibody caused the preferential degranulation of azurophilic granules and secretory vesicles. This process was at least in part mediated by p38 MAP kinase‐involved signal transduction.     

HLA class-I and -II antigens in chronic idiopathic autoimmune thrombocytopenia

Summary We studied MHC class-I and -II phenotypes in adult Caucasian patients with chronic idiopathic autoimmune þrombocytopenia (cAITP). Forty-five patients (median age 51 years, range 21–78 years) with a median disease duration of 7 years (range 2–26 years) were phenotyped for HLA-A, -B, -C by the standard lymphocytotoxicity test, HLA-DR and-DQ by restriction fragment-length polymorphism (RFLP), and-DP by oligonucleotide typing. Antiplatelet antibodies directed against glycoproteins Ib/IX and IIb/IIIa were determined by monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The comparison of antigen frequencies of the whole group of patients with healthy controls revealed no significant difference for any of the MHC class-I or class-II specificities (p>0.05). Patients were then divided into groups based on (a) their response to therapy, and (b) on whether they did or did not have detectable anti-platelet antibodies (n=16 versusn=29). All patients with a poor response to splenectomy carried the HLA-DPB1*0402 phenotype. The HLA-DPB1*1501 allele was found only among patients with detectable antiplatelet antibodies. These differences were not significant after correction for the number of tested antigens, however. Our data suggest that there is no association between MHC class-I/II alleles and adult cAITP or subgroups thereof.     

Distribution of HLA class I alleles differs in celiac disease patients according to age of onset

Celiac disease (CD) or gluten-sensitive enteropathy is strongly associated with HLA-DQ alleles; more than 95% of patients are DQB102. However, the uniform association with HLA-DQ alleles does not explain the clinical heterogeneity, especially the wide range in the age of onset of CD. We asked whether the age of onset of CD is also influenced by class I genes of the human MHC. We performed HLA typing in three groups of patients suffering from CD. The age of onset in the first group (N = 200) was before 15 years of age, in the second group (N = 62) between 15 and 40 years, in the third group (N = 59) after 40 years. We observed a statistically significant increase in the frequencies of HLA-B8 and Cw7 with increasing age of onset. In conclusion, we conclude that distinct alleles from the class I region of the human MHC might lead to late onset of CD. In particular, relatives of CD patients with the disease-prone HLA class I alleles HLA-B8 and Cw7 should be followed up carefully for late onset of CD.     

间日疟原虫抗原体外诱导间日疟患者PBMC凋亡的研究

目的分析间日疟原虫抗原(PvAg)诱导宿主外周血单个核细胞(PBMC)的凋亡水平方法采用流式细胞术结合应用膜联蛋白-V(Annexin-V),测定7例间日疟现症感染者PBMC经PvAg刺激后CD4+T细胞、CD8+T细胞及CD19+B细胞的凋亡率,并与空白对照组比较。结果经PvAg刺激后的间日疟现症感染者CD19+B细胞及CD4+T细胞凋亡率分别为7.26%和48.08%,与空白对照凋亡率4.08%和22.80%比较差异无统计学意义(P>0.05);CD8+T细胞凋亡率为55.16%,与对照凋亡率22.86%比较差异有统计学意义(P<0.05)。结论 PvAg可诱导间日疟现症感染者CD8+T细胞凋亡,从而可能参与介导间日疟原虫发生免疫逃逸。     

HLA Antigens Shed from the Surface of Synthetic or Naturally Occurred Platelet-Derived Microparticles During Storage of Platelet Concentrate

The demand for standard platelet concentrates (PCs) has continued to increase in the recent years. Infusible platelet membranes (IPM) prepared from new or outdated human platelets have been developed as an alternative to standard PCs, with the additional advantage of long shelf life and increased viral safety. Reduction of HLA antigens on the IPM has been assigned as one of the probable advantages of this product. In re-examining this issue, we studied the existence of HLA class I on the surface of IPM microparticles. In comparison we also surveyed HLA expression on the surface of the naturally occurred platelet-derived microparticles (nPMPs) during 7 days storage. Intended for producing IPM, PCs obtained from Iranian blood transfusion organization were lysed; virally inactivated with wet heat in the presence of a heat stabilizer and then sonicated. IPMs were separated using centrifugation and liquid-stored in 4°C. The expression of HLA class I antigens was surveyed using flow cytometry technique. HLA molecules were present on the microparticles. Shedding of HLA antigens was demonstrated from the surface of the both liquid-stored IPM and nPMPs during storage. Storage of IPM in 4°C was accompanied with significant reduction of HLA molecules. It seemed that achievement of HLA-free IPM could be impossible unless chloroquine treated platelets were used to prepare these microvesicles.     

Clinical significance of HLA DRB103-DRB104 in type 1 autoimmune hepatitis.

BACKGROUND: HLA DRB1*03-DRB1*04 combines both susceptibility factors for type-1 autoimmune hepatitis. AIMS: Determine whether this phenotype is a risk factor for autoimmune hepatitis in white North American patients, assess its associations with clinical features and treatment outcome, and determine whether alleles within this phenotype affect prognosis. METHODS: One hundred and ninety-eight patients with type 1 autoimmune hepatitis and 102 normal adults were evaluated. HLA typing was performed by DNA-based techniques. RESULTS: Twenty-eight patients had HLA DRB1*03-DRB1*04, and the frequency was higher than in normal subjects (14% vs 4%, OR 4.0%, 95% CI 1.4-11.8, P = 0.01). Patients with DRB1*03-DRB1*04 relapsed less frequently than patients with DRB1*03 (1.3 +/- 0.3 vs 2.1 +/- 0.2, P = 0.04), but they otherwise had outcomes similar to patients with other phenotypes. Patients with DRB1*03-DRB1*04 who had 3-4 alleles encoding lysine at position DRbeta71 within the class II molecule of the major histocompatibility complex developed cirrhosis more commonly (75% vs 9%, P = 0.05) and had a higher frequency of hepatic-related death or liver transplantation (40% vs 0%, P = 0.04) than patients with fewer alleles. CONCLUSIONS: HLA DRB1*03-DRB1*04 is a risk factor for type-1 autoimmune hepatitis, and its impact on outcome relates to the diversity of DRB1*04 alleles that encode a critical motif.     

The nature of peptides presented by an HLA class I low expression allele

Background

The functional integrity of human leukocyte antigen low expression variants is a prerequisite for considering them as essential in the matching process of hematopoietic stem cell donors and recipients to diminish the risk of serious complications such as graft-versus-host disease or graft rejection. The HLA-A*3014L variant has a disulfide bridge missing in the α2 domain which could affect peptide binding and presentation to T cells.

Design and Methods

HLA-A*3014L and HLA-A*3001 were expressed as truncated variants and peptides were eluted and subjected to pool sequencing by Edman degradation as well as to single-peptide sequencing by mass spectrometry. Quantitative analysis of binding peptides presented in vivo was performed by a flow cytometric peptide-binding assay using HLA-A*3001 and HLA-A*3014L-expressing B-LCLs.

Results

The truncated HLA-A*3014L protein was secreted in the supernatant and it was possible to elute and sequence peptides. Sequence analysis of these eluted peptides revealed no relevant differences to the peptide motif of HLA-A*3001, indicating that the Cys164Ser substitution does not substantially alter the spectrum of presented peptides. Strong binding of one of the shared in vivo identified HLA-A*3001/3014L ligands was confirmed in the peptide-binding assay.

Conclusions

This study is the first to demonstrate that HLA low expression variants are able to present peptides and, thus, can be considered as functionally active. When comparing peptide motifs, it is likely that HLA-A*3014L and HLA-A*3001 represent a permissive mismatch with low allogenicity in hematopoietic stem cell transplantation. These results indicate that surface expression, as well as peptide-binding data of HLA variants with similar disulfide bridge variations (e.g. HLA-A*3211Q) need to be considered as functionally active in an allogeneic hematopoietic stem cell transplantation setting as long as the opposite has not been shown. Otherwise a relevant but not considered HLA mismatch could result in a severe allogeneic T-cell response and graft-versus-host disease.     

Association between NDO-LID and PGL-1 for leprosy and class I and II human leukocyte antigen alleles in an indigenous community in Southwest Amazon

The frequencies of the Human leukocyte antigen (HLA) alleles in the Puyanawa indigenous reserve population and their association with the NDO-LID and ELISA PGL-1 rapid serological test was assessed. This was a cross-sectional study with an epidemiological clinical design conducted in two indigenous communities in the state of Acre, Brazil. Blood was collected in a tube with EDTA to identify HLA alleles and perform serological tests. DNA was obtained using the salting out procedure. The LabType™ technique (One-Lambda-USA) was used for HLA class I (loci A*, B* and C*) and II (loci DRB1*, DQA1* and DQB1*) typing. Allele frequency was obtained by direct count, and the chi-square test was used to assess the association with the NDO-LID and PGL-1 tests. The most frequent alleles in the two communities were: HLA-A*02:01, HLA-B*40:02, HLA-DRB1*16:02, HLA-DQA1*05:05 and HLA-DQB1*03:01. The allele HLA-C*04:01 was the most common in the Barão community, and the allele HLA-C*07:01 in Ipiranga. Among individuals who presented seropositivity to the NDO-LID test, the association with alleles HLA-A*02 (43.18% vs 24.8%, p = 0.03, OR = 2.35) and HLA-B*53 (6.83% vs 0.0%, p = 0.03, OR = 8.95) was observed in the Barão community. HLA-B*15 was associated with non-seroconversion to the NDO-LID test in Ipiranga. In both communities, HLA-B*40 and HLA-C*03 were associated with positive serological response to ELISA PGL-1. The HLA class I and II alleles most frequently found in this study have already been described among Terena indigenous groups, and HLA class I contributes to seroconversion to NDO-LID and PGL-1 tests in inhabitants of the Barão and Ipiranga communities.     

血清饥饿法用于细胞周期同步化的方法学研究

目的探讨血清饥饿法进行细胞周期同步化实验的影响因素。方法用无血清和低血清浓度培养基饥饿Anip973和AGZY83-a两种人肺腺癌细胞系，分别在培养48h、72h和5d时收集各组细胞，用流式细胞分析仪分析细胞周期各时相细胞百分数。结果Anip973细胞系在含O．2％FBS的RPMI1640培养基中培养5d得到理想结果，G0-G1期细胞百分比高达84．19％。AGZY83-a在含O．2％FBS的RPMI1640培养基中培养48h也获得较满意的结果，G0-G1期细胞百分比达61．30％。结论细胞类型、血清浓度和饥饿时间是血清饥饿法进行细胞周期同步化实验成功与否的影响因素。可应用血清饥饿法使不同细胞系同步于G0-G1期。     

恩替卡韦治疗慢性乙型肝炎过程中可溶性HLA-I类分子的变化及意义

目的测定可溶性HLA-I类分子的含量在慢性乙型肝炎治疗中的变化,并分析其意义。方法慢性乙型肝炎患者随机分为恩替卡韦治疗组、甘利欣治疗组和恩替卡韦加甘利欣联合治疗组;血清可溶性HLA-I类分子的含量用夹心ELISA方法测定。结果治疗3个月,恩替卡韦治疗组患者可溶性HLA-I类分子的含量显著高于治疗前（P〈0.01）,甘利欣治疗组患者可溶性HLA-I类分子的含量显著低于治疗前（P〈0.05）,联合治疗组患者可溶性HLA-I类分子的含量与治疗前相比无显著变化;治疗6个月,三组患者可溶性HLA-I类分子的含量均显著低于治疗前（P〈0.05）。结论可溶性HLA-I类分子的含量可反映慢性乙型肝炎治疗中患者免疫功能的调节。     

Peptides Derived from a Soluble Molecule of the Human Leukocyte Antigen (HLA) Class I Cause Apoptosis in Gastric Cancer Cell Lines

We have reported that the levels of the soluble molecule of the human leukocyte antigen class I (sHLA-I) in patients with advanced gastric cancer were significantly lower than those in patients with cancer in the early stages. However, the effect of sHLA-I on gastric cancer cells has not been elucidated. Using human gastric cancer cell lines, MKN28, MKN45, and MKN74, we evaluated the effects of sHLA-I on cell growth, DNA synthesis, and apoptosis induction. Three types of synthesized peptides derived from HLA-I were also examined for their capacity to induce apoptosis. sHLA-I and a synthesized peptide, nos. 220–232 of the α3 domain of HLA-B7, caused cell growth inhibition by inducing apoptosis in human gastric cancer cells. This peptide also inhibited the in vivo growth of cancer dissemination caused by an intraperitoneal injection of MKN45 into severe combined immunodeficient mice. In conclusion, sHLA-I and the peptides derived from HLA-I cause apoptosis in human gastric cancer cell lines.     

Soluble HLA Class I antigens in sera of patients with chronic hepatitis

Soluble HLA Class I antigens in sera (serum-HLA Class I, s-HLA Class I) of patients with chronic hepatitis (CH) were measured with an enzyme-linked double determinant immunoassay (EDDIA). The mean titers of s-HLA Class I antigens of patients with CPH (mean±standard deviation, 2.22±1.60), CAH2A (2.24±1.65) or CAH2B (2.73±1.46) were significantly higher than that of normal subjects (0.36±0.27) (P< 0.01). The titer of s-HLA Class I correlated significantly with the level of serum glutamic pyruvic transaminase (s-GPT) (r=0.73), and weakly with serum level of β₂-microglobulin (r=0.43). In patients with chronic hepatitis type B (CH-B) treated with human lymphoblastoid interferon alpha (IFNα), the titer of s-HLA Class I antigens increased. The increased level of s-HLA Class I antigens in the clinical course of chronic hepatitis may be caused by their release from necrotizing hepatocytes which have acquired the expression of HLA Class I antigens on the cell-surface membrane during viral infection.     

应用PCR-SSOP技术进行大样本脐血HLA基因分型的探讨

目的：探讨一种高效、快速、准确、省力并能批量检测HLA基因分型的方法。方法：采用聚合酶链反应一序列特异性寡核苷酸探针(PCR-SSOP)反向杂交技术对天津地区汉族健康新生儿脐血HLA-A、B、DR位点进行中低分辨基因分型。结果：共检出72种HLA-Ⅰ、Ⅱ类特异性抗原，其中A位点抗原检出19种；B位点抗原检出40种；DR位点抗原检出13种。结论：PCR-SSOP是一种结果相对准确稳定、操作简便快速、适用于脐血库等进行大样本分型检测HLA多态性的分子生物学技术。     

Genetic heterogeneity of autoimmune diabetes: age of presentation in adults is influenced by HLA DRB1 and DQB1 genotypes (UKPDS 43)

Aims/hypothesis. Juvenile-onset, insulin-dependent diabetes is associated with islet cell antibodies and with specific “high-risk” HLA-DRB1 and HLA-DQB1 genotypes. Patients with Type II (non-insulin-dependent) diabetes mellitus can have islet-related antibodies, but the genotypic associations at different ages of onset have not been evaluated. Our aim was to determine (i) the prevalence of DRB1 and DQB1 genotypes in patients at diagnosis of Type II diabetes at different ages from 25 to 65 years compared with the general population, and (ii) whether the presence of islet cell antibodies (ICA) or glutamic acid decarboxylase antibodies (GADA) or both by age is associated with different DRB1 and DQB1 genotypes. Methods. The antibodies to islet cells and those to glutamic acid decarboxylase were measured in 1712 white Caucasian diabetic subjects at diagnosis of diabetes and they were genotyped for HLA DRB1 ^* 03 and DRB1 ^* 04 and the high-risk DRB1 ^* 04-DQB1 ^* 0302 haplotype. To assess over-representation of high-risk alleles for Type I (insulin-dependent) diabetes mellitus, the prevalence of high-risk alleles in diabetic patients was expressed relative to the prevalence of low-risk alleles, non-DR3/non-DR4, that provided a reference denominator in both the diabetic patients and in 200 non-diabetic control subjects. The prevalence of ICA or GADA or both in patients with different HLA genotypes was assessed in those diagnosed in four age groups, 25–34 years, 35–44 years, 45–54 years and 55–65 years. Results. In Type II diabetic patients presenting at ages 25–34, 35–44 and 45–54 years, there was an increased prevalence of DR3/DR4 compared with the general population with approximately 6.5-fold, 2.9-fold, 2.1-fold over-representation, respectively (p < 0.0001, < 0.01, < 0.05) but this was not found in those aged 55–65 years old. In the group aged 25–34 years, 32 % of patients with ICA or GADA or both had DRB1 ^* 03/DRB1 ^* 04-DQB1 ^* 0302 compared with 10 % in those aged 55–65 years and expected 3 % prevalence. Conversely, only 14 % of those aged 25–34 years with antibodies had non-DR3/non-DR4, compared with 35 % in those aged 55–65 years. There was thus pronounced age heterogeneity in DRB1 and DQB1 predisposition to Type II diabetes. The antibodies displaced DRB1 or DQB1 genotypes in the multivariate model for requiring insulin therapy by 6 years of follow-up. Conclusion/hypothesis. The age of presentation of Type I diabetes in adulthood was in part dependent on the DRB1/DQB1 genotype. Islet cell antibodies and glutamic acid decarboxylase antibodies were strongly associated with DRB1 ^* 03/DRB1 ^* 04-DQB1 ^* 0302 in early adulthood but showed little relation with specific HLA genotypes after the age of 55 years. [Diabetologia (1999) 42: 608–616] Received: 27 April 1998 and in final revised form: 20 January 1999