, and hyperbilirubinemia is increased . Despite those common 3 types of diabetes, there are other specific types of diabetes including drugs or chemical-induced insulin deficiency, endocrinopathies, pancreatic destruction, and genetic defects. These unrelated forms of diabetes are included in the “Other Specific Types” and classified separately. The prevalence of T1D has been growing global by approximately 3-4% per year . Diabetes is an increasing health concern, worldwide. In 2000, around 171 million individuals worldwide were identified by diabetes; by 2011 the estimation was reported to be more than 366 million, and studies predicted that it will exceed 552 million by 2030 . According to the Diabetes Atlas (8th edition), Saudi Arabia is the 8th highest country for T1D in 20-year-old or younger patients, with a predicted around 35,000 affected children and adolescents . T1D is a polygenic disorder whose main locus is the human leukocytes antigen (HLA) region . Several studies pointed to the association between T1D and HLA, which were the first to report HLA associations with that type of diabetes currently considered T1D . Later, using advanced techniques, other studies defined both positive and negative HLA associations with T1D . The human leukocyte antigen (HLA) class II heterodimeric molecules consist of the alpha and beta chains and are encoded by numerous different alleles, which cause the high polymorphism characteristic of this locus. MHC class II molecules associated with T1D are encoded by three different loci are HLA-DR, HLA-DQ, and HLA-DP, which show high polymorphism. The polymorphism in these genes disturbs the ability to present antigens in the immune system, or even is made to detect which antigen is present in the population. HLA class II alleles are the main risk factors associated with several autoimmune diseases not only T1D . Numbers of viruses have high similarity with peptide sequences in the insulin-producing beta cells. So, the immune system could misguidedly destroy the beta cells instead of virus-infected cells. The antibodies against these intracellular beta-cell proteins exist in 75% of newly diagnosed T1D patients, although it is not clear if this is a reason or a consequence of the attacked beta cells . T1D affects between 0.5% and 1% of global population . This study aimed to determine the association between Human Leukocyte Antigens (HLA) Class II alleles and haplotypes with T1D in Saudi children by describing the frequency of T1D genetic susceptibility and resistance conferred by HLA*DRB1, *DQA1, *DQB1.
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DRB1*02/15 as the most commonly expressed DRB1 allele groups in Botswana, all occurring at frequencies above 20% in the study population. DQB1*06 was the most commonly ex- pressed allele group at the DQB1 locus, with 47.7% of the study population expressing it, followed by DQB1*03, DQB1*04, and DQB1*05 at 39.2%, 25.8%, and 21.5%, respec- tively. This is the first study to investigate the relative frequen- cies of HLA class II gene expression in Botswana, a country at the epicenter of the HIV/AIDS epidemic. This study addresses directly the problem of lack of information on generic HLA types in different populations, which is a significant obstacle in the design of an HLA-based HIV vaccine (51). Although the numbers in this study are small and may need to be extended to define the most common alleles within each allele group, this study provides evidence that by targeting the most com- monly encountered HLA allele groups within a specific region or population, it may be possible, at least in part, to overcome the problem of HLA polymorphism, which is likely to affect the widespread efficacy of HIV vaccines designed to elicit virus- specific T-cell responses. A particularly interesting finding from this study is that the population frequency of some com- monly expressed allele groups, such as DRB1*11 and DRB1*13, approximates that in other African populations (48). This suggests that by targeting these commonly expressed HLA class II alleles in HIV vaccine design strategies, it may be possible to overcome problems associated with genetic diver- sity within populations where HIV vaccines are urgently needed. Obviously, studies of HLA class II allele frequencies and distribution will need to be extended elsewhere in areas with high HIV prevalence and incidence rates, especially in sub-Saharan Africa, in order to define the extent of common- alities between populations. The significant differences found between our study population and well-studied populations in North America (such as in allele group specificities DRB1*11 and DRB1*13, which are those most commonly encountered in Botswana) suggest that disregarding local HLA gene fre- quencies in vaccine design may compromise the efficacy of a vaccine in the populations most likely to need it.
In view of the heterogeneity of the Arab population, which comprises people of distinct ethnic backgrounds and whose origins can be classified according to their area of habitation (North Africa, Arabian peninsula, and eastern Mediterra- nean), the present study was aimed at elucidating the diversity in HLA class II allele distribution among two distinct Arab communities, Bahrain and Lebanon. Bahrain is an island lo- cated in the Arabian Gulf whose inhabitants derive their origin from three major roots: Jaafari Arabs, Sunni Arabs, and Ira- nians (4). Lebanon is located in the eastern Mediterranean, and its population consists of Christians (Catholic, Maronite, Greek Orthodox, and Coptic) and Moslems (Sunni, Shiite, and Druze). This study provides basic information for further stud- ies of the MHC differences between Arabs of distinct origins * Corresponding author. Mailing address: Al-Jawhara Center for
Very few studies examined the prevalence of HLA class II allele and haplotype per se, and most examined a possible role for HLA class II in type 2 diabetes in relation to autoimmune markers and latent autoimmune diabetes in adults, genetic interaction between type 1 diabetes and type 2 diabetes, and association with complications of diabetes. This was exempli- fied by the findings that DRB1*0405 (7), DQB1*0201 and DQB1*0302 (32), DRB1*03/04-DQB1*0302 (15), and DRB1* 03-DQA1*0502-DQB1*0201 (DR3-DQ2) (8) were positively associated with type 2 diabetes in anti-glutamic acid decarbox- ylase (GAD)-positive type 2 diabetes subjects. In addition, increased frequency of HLA-DR3 and -DR4 was reported in islet cell autoantibody-positive patients with secondary failure to oral antidiabetic drugs (10, 12). Increased frequency of HLA-DRB1*1502 was also seen in anti-GAD-positive patients who remained well controlled on oral antidiabetic drugs (7). Furthermore, tumor necrosis factor alpha was associated with predisposition to subsequent insulin dependency in GAD-pos- itive HLA-DRB1*1502-DQB1*0602 (22) or DQB1*02 (19) type 2 diabetes patients, suggesting an interplay between HLA genotypes and other polymorphic markers in dictating the pro- gression of type 2 diabetes. Others failed to link HLA class II (DQA1 and DQB1) with type 2 diabetes (4, 5), and it would appear that the association between HLA class II and type 2 diabetes is racially and geographically restricted.
The biotin conjugation of sHLA was performed at Pure Protein laboratories at Oklahoma by chemical biotinylation. Briefly, a small-scale protocol was used starting with a 1 mg sHLA probe in PBS. The reaction was initiated by adding sodium bicarbonate buffer, pH 9.0 (final 0.1 M) and NHS-PEG12-Biotin at a final concentration of 393 µg/ml. The final mixture was incubation at room temperature for 2 hours at a total volume of 1 mL and a final sHLA concentration of 1 mg/ml. To complete the procedure, the reaction was terminated by adding Tris-HCl, pH 8.5 at a final concentration of 50 mM. An empirically determined molar ratio of 20:1 (Biotin-NHS : sHLA protein) was applied to all Class I molecules to assure that on average, not more than one biotin became attached to the sHLA protein to prevent disturbing the binding capability of the targeting antigen by excess biotinylation. The extent of biotin incorporation was determined using a HABA assay. To remove excess biotin, sHLA molecules were buffer-exchanged with PBS at pH 7.2 and concentrated using 10-kDa cut-off Macrosep centrifugal concentrators (Pall Filtron, Northborough, MA). The final product was filter-sterilized and stored at 4 °C until further use. The concentration of the purified biotinylated molecules was determined using the Micro BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL) using BGG as a standard.
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In addition to stimulating antigen processing by T84 cells, g -IFN treatment has been observed to elicit a profound and relatively rapid decrease in TER in T84 monolayers without an immediate alteration in cell morphology or integrity of the monolayer (18). This alteration in barrier function after g-IFN treatment would limit the ability to selectively expose one sur- face of the cell to intact antigen and to independently study the polarity of antigen processing and presentation. Surprisingly, we observed that the T84 HLA-DRB1*0401 transfectants overexpressing CIITA were able to maintain a high TER (Fig. 5 C), despite showing many of the characteristics of transfec- tants treated with g -IFN with regards to class II antigen pro- cessing. Treatment of the T84 CIITA transfectants with g -IFN resulted in the characteristic decline in TER seen in the wild- type T84 cells (Fig. 5 C), suggesting that the directed overex- Figure 3. HLA-DR–restricted peptide pre- sentation occurs only at the basolateral surface of T84 cells. (Top) Schematic view of transwell antigen presentation assay. T84 cells are plated on upright transwell filters (3.0 m pore size), exposing the apical surface to the top chamber, or on upside- down filters that are subsequently inverted, exposing the basolateral surface to the top chamber. T cells are always added to the top chamber to maximize T cell-T84 con- tact. (Bottom) T84 HLA-DRB1*0401 transfectants plated in the apical or ba- solateral orientation were co-cultured with HSA-specific, HLA-DRB1*0401– restricted T cell hybridoma cells for 24 h in the absence or presence of 10 mM of the HSA peptide for which the hybridomas are specific. T84 transfectants expressing simi- lar levels of the HLA-DRB1*1101 (DR5) allele were used as a negative control. IL-2 production of the hybridomas was quanti- tated using the supernatant from the top chamber by [ 3 H]thymidine uptake (cpm) of
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The current study analyzed the distribution of HLA class II alleles in patients with anti-GBM disease and their potential significance. For HLA class II loci, HLA- DRB1 and -DPB1 encode relatively more variable gene products for HLA-DR and -DP molecules respectively, while both HLA-DQB1 and -DQA1 are variable in human population. Besides, previous studies have located some HLA-DRB1, -DQB1 and -DPB1 alleles with association with anti-GBM disease in Caucasian as well as Asian population [5,11,12]. But in Chinese patients, few studies have been done in this topic . Therefore, we choose to type HLA-DQB1, -DQA1 and -DPB1 loci in this study, on the basis of our previous study on HLA-DRB1 .
has a mutation at the 3′ splicing site of intron 1. Both mutations introduce a frame shift into the first third of the coding sequence, leading to the absence of a functional TAP1 subunit. Due to this deficiency, most HLA class I heavy chain/β2m complexes are retained in the ER and are unstable at 37°C. The complexes can be stabilized by binding with exogenous HLA-A–specific pep- tides. HLA-B appeared to be more unstable than HLA-A, probably as a result of the biochemical properties of these particular variants, since in TAP2- deficient patients we found HLA-B molecules to be more stable than HLA- A (13). On the other hand, the defi- ciency does not seem to affect the expression of CD1 antigen: it was pre- viously shown that Langerhans’ cells, in non-lesional skin, normally expressed this HLA class I–like molecule (6), in agreement with our previous observa- tion on TAP2-deficient dendritic cells (14).
alternative splicing pathway [8,33,36]. It is possible that serum HLA-II may be derived from similar processes. However, there is no supportive data for this assumption. Biochemical studies of sHLA-II in the synovial fluid of patients with rheumatoid arthritis revealed a preferential release of high-molecular-weight (1000 kDa) sHLA-II in the inflamed synovium, but not in the serum . In addition, attempts to induce production of similar mate- rial from a cell line expressing HLA-II on a cell surface have failed, indicating that release of sHLA-II is an active process. Of interest, sweat has been shown to possess pol- ymorphic structures identical to those of serum HLA-I. However, excretion of sHLA-I in sweat has been found to be in markedly lower quantities than in serum [11,38]. We reported the occurrence of 39 kDa sHLA-I in saliva as well as in serum during active Sjögren's disease and sys-
Before treatment CD4 + cell amount median in all patients was 155 cells/µl and HIV RNA viral load median - 55 thousand copies/ml. By studying the HLA class II haplotypes, it was concluded that the highest association with high immunological efficacy has haplotypes HLA-DRB1*/DQB1*/DQA1* 01:01/06:02-8/01:03, 01:01/03:01/01:02, 13:01/06:02-8/01:02, the incidence (gf = 0.36/0.09). After 12 weeks of treatment, CD4 + lymphocytes amount in the particular group has increased to 600-700 cells/µl, HIV RNA viral load has decreased to 5 thousand copies/ml. After 24-48 weeks of treatment - CD4 + lymphocytes amount has increased to 806-900 cells/µl (450 - 500 cells/µl), and HIV RNA viral load has decreased <400 copies/ml (decrease by 20 - 30 thousand copies/ml). These data suggest an efficiency of ART, as none of the patients in study groups with existing haplotypes has showed HIV clinical progression (the development of opportunistic infections) during the treatment. At the same time the association of low immunological efficacy has haplotypes: HLA- DRB1*/DQB1*/DQA1* 15:01/03:01/03:01, 17:01/05:01/02:01, 17:01/03:01/05:01, 07:01/03:01/02:01, 11:01/03:01/05:01,15:01/03:02/01:02, frequency (gf = 0.03/0.04/0.05). Patients with particular haplotype, treatment contribute to progressive CD4 + cells amount increase in the blood, and reduce the HIV viral RNA load in the study group of HIV/AIDS patients. After 12 weeks of treatment a tendency to increase in CD4 + cell amount was formed, but the increase was not large (50-100 cells/µl), HIV RNA amount decreased an average of 2000 copies / ml. Sufficiently high rates of HIV RNA were maintained after 24 - 48 weeks of treatment (55 thousand copies/ml).
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Cytotoxic T lymphocytes (CTL) target multiple epitopes in human immunodeficiency virus (HIV)-infected persons, and are thought to influence the viral set point. The extent to which HLA class I allele expression predicts the epitopes targeted has not been determined, nor have the relative contributions of responses restricted by different class I alleles within a given individual. In this study, we performed a detailed analysis of the CTL response to optimally defined CTL epitopes restricted by HLA class I A and B alleles in individuals who coexpressed HLA A2, A3, and B7. The eight HIV-1-infected subjects studied included two subjects with acute HIV infection, five subjects with chronic HIV infection, and one long-term nonprogressor. Responses were heterogeneous with respect to breadth and magnitude of CTL responses in individuals of the same HLA type. Of the 27 tested epitopes that are presented by A2, A3, and B7, 25 were targeted by at least one person. However, there was wide variation in the number of epitopes targeted, ranging from 2 to 17. The A2-restricted CTL response, which has been most extensively studied in infected persons, was found to be narrowly directed in most individuals, and in no cases was it the dominant contributor to the total HIV-1-specific CTL response. These results indicate that HLA type alone does not predict CTL responses and that numerous potential epitopes may not be targeted by CTL in a given individual. These data also provide a rationale for boosting both the breadth and the magnitude of HIV-1-specific CTL responses by immunotherapy in persons with chronic HIV-1 infection.
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Poliovirus (PV), a prototypical member of the Picornaviridae family, is a small, nonenveloped, positive-stranded RNA virus whose replication is limited to specific cells and tissues that express the PV receptor (PVR; CD155) (30, 40) on the cell surface. Most cells of the immune system, such as T and B lymphocytes, are not susceptible to PV infection (35, 41), as they do not express PVR. In contrast, Freistadt et al. demon- strated that human peripheral blood monocytes do express PVR (22). However, monocytes are not very permissive to PV infection; viral protein production could only be demonstrated at very low frequency in a subpopulation of monocytes (18, 21). Monocytes are precursor cells that are able to differentiate into macrophages or dendritic cells (DCs) (10–12, 36, 44). In response to pathogens, inflammatory cytokines, or necrotic cells, DCs and macrophages play central roles in the induction of immune responses. These antigen-presenting cells (APCs) acquire and process antigens, displaying them in the context of HLA class I and II molecules at the cell surface (9, 31, 43, 45). Subsequent interaction of the HLA-antigen complexes and costimulatory molecules on APCs with T cells in the presence of relevant secreted cytokines induces immune responses (26). DCs are critical APCs that, in contrast to macrophages, are unique in being able to induce primary immune responses from naive T cells to novel antigens in humans (6).
In Malaysia, Azizah et al.  reported significant asso- ciation of HLA-DR2, -DQB1 ∗ 0501, and -DQB1 ∗ 0601 with SLE in Malays. A significant positive association of DR2 and DQB1 ∗ 0501 with renal involvement and DR8 with alopecia in Malays was also described in their study. For the investi- gation of the role of HLA genes in autoantibody expression, they found significant association of DQB1 ∗ 0601 with anti- Sm/RNP, DR2 with anti-Ro/La, and DR2, DRB1 ∗ 0501 and ∗ 0601 with anti-dsDNA. The same group of researchers also carried out similar study on Chinese population and suggested that DQB1 ∗ 0102, DQB1 ∗ 0501, ∗ 0601, and DPB1 ∗ 0901 were significantly associated with SLE . Clinically, a strong association of DR2 and DQA1 ∗ 0301 with renal involvement and DQA1 ∗ 0102 with alopecia was re- ported. In contrast to Malays, DQA1 ∗ 0102 and DQA1 ∗ 0301 were observed to be strongly associated with anti-Ro/La and anti-dsDNA, respectively, in Chinese. Earlier on, Doherty et al.  reported that HLA-DRw15 and DQw1 were observed to be significantly associated with SLE among Southern Chinese in Malaysia and most prevalent in patients with lupus nephritis and cutaneous manifestation.
autoantigen presentation by HLA class II expressing thyroid follicular cells thus only occurs in Graves' disease, suggesting that HLA class II expression on thyroid follicular cells is an essential feature, but by itself not sufficient for the induction of autoimmunity. Additional factors, the possible nature of which […]
Allergic bronchopulmonary aspergillosis (ABPA) is a hy- persensitivity lung disease characterized by Aspergillus fu- migatus (Af) colonization, IgE and IgG anti-Af antibodies, pulmonary infiltrates, bronchiectasis, and pulmonary fibro- sis. Little is known regarding T cell responses and their role in the pathogenesis of ABPA. To examine T cell reactivity to Af antigens, T cell clones (TCC) specific to the Asp f 1 antigen, an 18-kD protein of Af, were established from the peripheral blood of three ABPA patients. The majority of TCC isolated from ABPA patients, and specific for the Asp f 1 allergen of Af, are IL-4 producing CD4 1 cells of the Th2 phenotype. Further analysis in this study revealed that the majority of TCC reacted to mainly two epitopes of Asp f 1, while the remaining TCC reacted to three additional “mi- nor” epitopes. Blocking studies using monoclonal antibod- ies specific for class II HLA-D region gene products showed that most TCC, 19/21, were restricted by HLA-DR mole- cules, and the remaining two clones by HLA-DP molecules. The use of a panel of HLA-matched and mismatched EBV- transformed B cells as antigen presenting cells revealed that the HLA-DR restriction was mediated exclusively by either the HLA-DR2 or HLA-DR5 alleles. Genotyping of DRB1 gene products showed that class II presentation for most clones was not restricted to a single allele, representing DRB1 gene products of either HLA-DR2 or DR5. These studies offer in- sight into the cellular and molecular determinants which contribute to the immunopathophysiology of ABPA. ( J. Clin. Invest. 1996. 97:2324–2331.) Key words: allergic bron- chopulmonary aspergillosis • T cell clones • HLA-restriction • epitope mapping • cytokines
studies from Australia , Turkey , and Spain  demonstrating that a higher frequency of the HLA DRB1*15 allele could be associated with OCBs. Australian researchers in their cohort trial demonstrated that the presence of OCBs might also be related to the HLA DRB1*03 and *04 alleles . We failed to confirm such a relation in the present study. An association be- tween OCBs and the HLA DRB1*15 allele suggests that immunological changes affecting the prognosis of the disease might be regulated by genetic factors, i.e., sup- ports an idea of genetic origin of MS. Our findings also suggest that the presence of OCB is associated with the HLA DRB1 genotype and that different genotypes are linked to the different rates of intrathecal immunoglobu- lin synthesis, in turn reflecting the levels of B cell activ- ity in the CNS .
Assessment of the viral set points for individuals identified during PHI. Longitudinal viral-load data were collected from all study subjects. Two infec- tious-disease physicians independently determined the average viral set points for all untreated subjects. Only subjects with untreated HIV-1 infection and multiple (four or more) available viral loads 6 months after first presentation were included in the analysis. Subjects were excluded (i) if they had ever received antiretroviral therapy during the first 6 months of infection, (ii) if results for less than four independent viral-load measurements were available during the viral set point period, and (iii) if one of the two infectious-disease physicians con- cluded that no stable viral set point was reached. Viral set points were defined following the algorithm previously described by Fellay et al. (10). Viral set points were obtained for 110 out of 428 study subjects. The main reasons for the absence of a viral set point in the remaining 318 study subjects were as follows: treatment during acute infection (n ⫽ 242), loss to follow-up (n ⫽ 38), treatment before the establishment of a viral set point (n ⫽ 26), or lack of a stable viral set point (n ⫽ 12). The 110 subjects, for whom a viral set point was obtained were representative of the cohort of 428 subjects in terms of gender, race, and HLA distribution.
Expression of Env and HLA class II protein incorporation. One possible mechanism for HLA class II protein incorpora- tion is that the packaging of Env might cause the placement of HLA class II proteins onto the virion. To test this hypothesis, the presence of HLA class II proteins on virions with and without Env was examined. Since microvesicles containing HLA class II proteins have been shown to contaminate even purified H9 and peripheral blood mononuclear cell (PBMC) virion preparations (4, 14), simply examining virus stocks for HLA class II proteins would detect protein from both virions and microvesicles. Therefore, the presence of HLA class II proteins on the virions was examined by a whole-virus immu- noprecipitation with HLA class II antiserum (i.e., without detergent lysis of the particle), followed by detection of pre- cipitated virions by immunoblotting for Gag. To produce Env- deficient virions from HLA class II-expressing cells, these cells were infected with VSV-G pseudotypes of wild-type or Env mutant viruses produced by cotransfection of 293T human kidney cells with a proviral DNA construct (pNL4-3  or its mutants) and a VSV-G expression plasmid, pCMVHg (6), by using a calcium phosphate mammalian cell transfection kit (5 Prime-3 Prime, Inc., Boulder, Colo.). These pseudotyped stocks were then used to infect HLA class II-expressing cells, either H9 cells or phytohemagglutinin-stimulated PBMCs, in the presence of 2 g of Polybrene per ml. The inoculating virus was removed by washing at 4 h postinfection, and virus-con- taining clarified supernatants were harvested 48 to 72 h postin- fection.
HLA-DQB1 alleles were determined by a PCR sequence- specific priming (SSP) technique using AllSet Gold SSP low resolution kit according to the specifications of the manufacturer (Invitrogen, USA). PCR products were an- alyzed on a 2.0 % agarose gel stained with ethidium bromide (0.5 g/ml) for specific amplification patterns and alleles were assigned with SSP UniMatch (v5.1) soft- ware. Genotyping was performed at the Human Genetics Unit, Faculty of Medicine, University of Colombo. Exons 2 to 3 of HLA-A and HLA-B genes and Exons 2 to 4 of HLA-DRB1 gene were sequenced in a subset of 140 each of patients and controls, on an Ion Proton next gener- ation sequencing platform at a commercial sequencing laboratory . The HLA alleles were analyzed and assigned at the same facility, by aligning against refer- ence sequences of IMGT/HLA database and using an in house protocol adapted from published methods .
Expression of MHC class II also has been studied in NSCLC. In many NSCLC cell lines, expression of MHC class II had been detected. Using immuno ﬂ uorescent HLA-DR, -DQ, -DP expression had been detected in A549, SKMES1, CALU6, A427 cell lines. 45 MHC class II could be induced by IFN- γ in TKB-3 and TKB-4 cell lines and the induced MHC class II expressed on cell surface instead of cytoplasm. 46 MHC class II expression in adenocarcinoma, squamous cell carcinoma and large cell carcinoma was detected in surgery samples by immu- nohistochemistry (IHC). It demonstrated that MHC class II expressed on both TILs and lung cancer cells. 46 Another study showed high expression of MHC class II on tumor cells was associated with high MHC class II expression on TILs, and expression of MHC class II on adenocarcinoma cells was higher than non-adenocarcinoma cells. 14 As for prognosis, MHC class II expression on TILs was asso- ciated with a better recurrence-free survival (RFS) and overall survival (OS). 14 Ohri et al analyzed 40 NSCLC patients ’ specimens by IHC and found non-macrophage expression of HLA-DR in tumor islets was signi ﬁ cantly elevated in patients who had better prognoses. 47 HLA-DPB1 expression detected in biopsy tissue also had positive correlation with good prognosis in lung cancer patients. 48