Pyrosequence-based typing of alleles of the HLA-DQB1 gene.

DNA typing of alleles of the highly polymorphic HLA-DQBI gene was performed by Pyrosequencing using purified DNA from the 11th International Histocompatibility Workshop human cell lines and samples from the Children's Hospital of Pittsburgh registry of diabetics and their first-degree relatives. Pyrosequencing was optimized for genotyping exon 2 of the HLA-DQB1 gene, but the procedure should be applicable to other HLA loci. The 47 HLA-DQB1 alleles were readily identifiable, as were the 1,128 potential allelic heterozygous combinations. The method required PCR conditions that specifically amplified DQB1 but not the pseudogene, DQB2. The new method of pyrosequence-based typing can be performed in 96- or 384-well format. The 61 polymorphic residues of DQB1 exon 2 were identified within four pyrosequencing reactions, obtained by a 70-nucleotide read length in each reaction, in about an hour's time. Allelic combinations of HLA-DQB1 most frequentlyfound in the population of diabetics and their immediate family members were analyzed and successfully compared to typing of the DQB1 alleles by sequence-specific oligonucleotide probe protocols. Pyrosequence-based typing is compatible with genotyping of allelic combinations expected from heterozygous individuals, resulting in nucleotide resolution of the highly polymorphic HLA system. Using pyrosequencing, more than 750 sample wells can be processed in a working day, resulting in the identification of more than 50,000 bases.

Progression to insulin-requiring diabetes in seronegative prediabetic subjects: the role of two HLA-DQ high-risk haplotypes.

AIMS/HYPOTHESIS: Most Caucasians with Type I (insulin-dependent) diabetes mellitus develop an autoimmune form of diabetes known as Type IA diabetes, based on the presence of humoral responses to islet autoantigens. Alleles at the HLA locus account for the strongest susceptibility to this form of diabetes, which requires insulin therapy. Because a number of patients who develop insulin-requiring diabetes are islet autoantibody negative, the HLA class II haplotypes, DQA1*0501-DQB1*0201 and DQA1*0301-DQB1*0302, were evaluated to assess whether they are an independent risk factor for progression to insulin requirement in first-degree relatives of Type I diabetic patients. METHODS: Both HLA-DQ genotyping and islet cell autoantibody assessment (insulin, GAD65, IA-2 autoantibodies and cytoplasmic islet cell antibodies) were evaluated prospectively in 74 relatives of Type I diabetic patients who developed diabetes treated with insulin (prediabetics) and in 426 control subjects who did not develop insulin-requiring diabetes. Based on the presence of DQA1*0501-DQB1*0201 and/or DQA1*0301-DQB1*0302, the number of HLA-DQ high-risk haplotypes was assigned as 0, 1 or 2. RESULTS: A higher prevalence of 2 HLA-DQ high-risk haplotypes was present in seronegative prediabetic subjects as compared to non-diabetic autoantibody negative first-degree relatives (33.3 % vs 10.1 % respectively; p < 0.05). Moreover, in seronegative relatives who developed insulin-requiring diabetes, the presence of 2 HLA-DQ high-risk haplotypes conferred an increased cumulative risk of developing insulin requirement of 27 % at 12.5 years of follow-up, compared to a risk of 6 % for non-diabetic relatives who were antibody-negative and had 0 or 1 HLA-DQ high-risk haplotypes (Log rank p = 0.01). CONCLUSION/INTERPRETATION: These data provide evidence for a phenotype, which is associated with the absence of conventional islet autoantibodies at initial screening, while usually remaining seronegative, and the presence of 2 HLA-DQ high-risk haplotypes with progression to clinical Type I diabetes after a prolonged follow-up. Given the fact that in humans the highest risk-conferring locus associated and linked to the disease is the HLA cluster, and that HLA-DQ molecules play a key role in the development of autoimmune diabetes, our observations imply that as yet unidentified immunologic abnormalities could well exist in seronegative relatives at risk of developing clinical diabetes and carrying 2 HLA-DQ high-risk haplotypes.

Immune related genetic polymorphisms and schizophrenia among the Chinese.

Genetic association studies were conducted among two independent cohorts of Chinese ethnicity. The samples consisted of cases and unrelated controls, ascertained from Guangzhou, China, and Singapore. The studies were prompted by our earlier report of an association between schizophrenia and HLA DQB1 alleles (HLA DQB1*0602 and HLA DQB1*0303) in the Singapore sample. Polymorphisms of HLA DQB1 and flanking markers on chromosome 6p21.3 were investigated in the first part of the study. A significant negative association with HLA DQB1*0402 was detected in the Guangzhou sample (Odds ratio, OR 0.26, 95% confidence intervals, CI 0.1, 0.6; p < 0.02, corrected for multiple comparisons). Additional analysis of the Guangzhou and Singapore samples revealed associations at three other anonymous markers flanking HLA DQB1. In the second part of the study, three polymorphisms at the Interleukin-1 gene cluster (IL-1, chromosome 2q13-q21) were investigated in both cohorts, since associations with schizophrenia have been reported in another sample. Persuasive evidence for an association at IL-1 was not detected in either sample. Our results suggest a susceptibility locus for schizophrenia in the HLA region among the Chinese, but further clarification is necessary.

The role of receptors in the maturation-dependent adenoviral transduction of myofibers.

One of the major hurdles facing the application of adenoviral gene transfer to skeletal muscle is the maturation-dependent transduction of muscle myofibers. It was recently proposed that the viral receptors (Coxsackie and adenovirus receptor (CAR) and the integrins alphavbeta3/beta5) play a major role in the poor adenoviral transduction of mature myofibers. Here we report the findings of morphological studies designed to determine experimentally the role of receptors in the adenoviral transduction of mature myofibers. First, we observed that the expression of both attachment and internalization receptors did not change significantly during muscle development. Second, when an extended tropism adenoviral vector (AdPK) that attaches to heparan sulfate proteoglycan (HSP) is used, a significant reduction of adenoviral transduction still occurs in mature myofibers despite HSP's high expression in mature skeletal muscle fibers. Third, when the adeno-associated virus (AAV) is used, which also utilizes HSP as a viral receptor, muscle fibers at different maturities can be highly transduced. Fourth, the pre-irradiation of the skeletal muscle of newborn mice to inactivate myoblasts dramatically decreased the transduction level of Ad and AdPK, but had no effect on AAV-mediated viral transduction of immature myofibers. These results taken together suggest that the viral receptor(s) is not a major determinant in maturation-dependent adenoviral transduction of myofibers.

Analysis of TCR Vbeta repertoire and cytokine gene expression in patients with idiopathic dilated cardiomyopathy.

Although the etiopathogenesis of idiopathic dilated cardiomyopathy (IDC) is still unclear, it is widely accepted that a complex interplay between viral infections and immune mechanisms is the basis of disease genesis. Previously, we showed that heart-infiltrating T cells of patients suffering from acute, fulminant Coxsackie virus B3+-IDC shared a preferential usage of three variable gene segments of the T cell receptor beta chain-(TCR-Vbeta) encoding families Vbeta3, 7 and 13.1. This indicated the possible presence of a superantigen-driven immune response. Here, we further investigated the IDC immunological scenario by analysing different phenotypes of heart-infiltrating cells: TCR repertoires, cytokine expression and presence of enterovirus-specific antigens. IDC patients who underwent heart transplantation at different times after the onset of heart failure were studied. A cardiac infiltrate of CD4+ and CD8+ T cells was present together with activated macrophages. Furthermore, the same Vbeta gene families, previously found to be skewed in hearts from fulminant cases of CVB3+-IDC, together with two additional Vbeta gene families, Vbeta1 and 5B, were increased. IL-1beta, IL-2, IL-6 and IFN-gamma were expressed in the myocardium while others, like IL-4 were not. In conclusion, an orchestrated complex of immune mechanisms seems to be the basis of IDC etiopathogenesis.

Protection of human islets from the effects of interleukin-1beta by adenoviral gene transfer of an Ikappa B repressor.

Interleukin-1beta (IL-1beta) is a pro-inflammatory cytokine that inhibits beta cell function and promotes Fas-triggered apoptosis. IL-1beta is thought to act early in the initiation of the autoimmune destruction of pancreatic beta cells in type I diabetes. IL-1beta promotes beta cell impairment, in part, by activating NF-kappaB transcription factor-dependent signaling pathways. We have examined whether beta cells could be protected from the effects of IL-1beta by overexpressing an inhibitor of NF-kappaB activity, IkappaB, by adenoviral gene transfer to intact human islets in culture. Infection of islets with an adenoviral vector encoding a non-phosphorylatable, non-degradable variant of IkappaBalpha resulted in normal insulin responses to glucose in the presence of IL-1beta. Furthermore, nitric oxide production was prevented and, more importantly, Fas-triggered apoptosis was inhibited following IkappaBalpha gene transfer. These results suggest that blocking the NF-kappaB pathway might prevent cytokine-induced beta cell impairment as a means of facilitating islet transplantation.

Evidence for superantigen involvement in preeclampsia.

PROBLEM: Preeclampsia is the leading cause of maternal morbidity and premature fetal delivery in the United States, most likely involving the immune system in disease genesis. In this report, we tested the hypothesis that a superantigen phenomenon is an important factor in the pathogenesis of the disease. METHOD OF STUDY: A semi-quantitative polymerase chain reaction (PCR) was used to assess T-cell receptor (TCR) beta chain variable (Vbeta) regions as an indicator of T-cell expansion in both peripheral blood and basal plate of preeclamptic patients. All the subjects were also molecularly typed to identify their HLA-class II alleles. RESULTS: In peripheral blood of the majority of the patients, there was a high abundance of the Vbeta4 gene family, which was not observed in the control group. Polyclonality of this Vbeta gene family was confirmed by analysis of the Valpha chain and the complementary determining region 3 (CDR3). The majority of patients carried the Human Leukocyte Antigens (HLA)-DRB1*13 allele. CONCLUSION: We present evidence for the existence of a superantigen-like effect in at least a subset of patients with preeclampsia.

Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I.

Interleukin-1beta is a potent pro-inflammatory cytokine that has been shown to inhibit islet beta cell function as well as to activate Fas-mediated apoptosis in a nitric oxide-dependent manner. Furthermore, this cytokine is effective in recruiting lymphocytes that mediate beta cell destruction in IDDM onset. The insulin-like growth factor I (IGF-I) has been shown to block IL-1beta actions in vitro. We hypothesized that gene transfer of the insulin-like growth factor I to intact human islets could prevent IL-1beta-induced beta cell dysfunction and sensitization to Fas-triggered apoptosis activation. Intact human islets were infected with adenoviral vectors encoding IGF-I as well as beta-galactosidase and enhanced green fluorescent protein as controls. Adenoviral gene transfer of human IGF-I prevented IL-1beta-mediated nitric oxide production from human islets in vitro as well as the suppression of beta cell function as determined by glucose-stimulated insulin production. Moreover, IGF-I gene transfer prevented IL-1beta-induced, Fas-mediated apoptosis. These results suggest that locally produced IGF-I from cultured islets may be beneficial in maintaining beta cell function and promoting islet survival before and following islet transplantation as a potential therapy for type I diabetes.

Restricted TCR V beta gene expression and enterovirus infection in type I diabetes: a pilot study.

AIMS/HYPOTHESIS: High frequencies of T-cell receptor (TCR) V beta 7+ T cells were detected among the lymphocytes isolated from pancreatic islets of children at the onset of Type I (insulin-dependent) diabetes mellitus. We assessed whether a preferential expression of certain TCR V beta gene families could also be detected among the peripheral blood mononuclear cells from diabetic patients. METHODS: T-cell receptor repertoires were evaluated by using a semi-quantitative RT-PCR-based technique and confirmed by FACS analysis in peripheral blood mononuclear cells from diabetic patients before, at and after onset of the disease. These patients were also tested for exposure to enteroviruses by RT-PCR and by measuring titres of enterovirus-specific antibodies of the IgA, IgG, and IgM classes. RESULTS: T-cell receptor V beta 7 gene family values were higher in recently-diagnosed diabetic patients (10.5% +/- 3.7) than in age-matched non-diabetic control subjects (5.1% +/- 1.6) (p < 0.001). In a time-course analysis of people who developed diabetes during clinical monitoring (i.e., converters), T-cell receptor V beta 7 gene expression showed values consistently above 10% (p < 0.0005). Long-standing diabetic patients showed lower percentage of V beta 7 expression compared to values measured at disease onset. In the longitudinal study of the converters, multiple acute enterovirus infections were also detected. These infections appeared to be temporally related to increased percentage of V beta 7 gene transcripts. CONCLUSION/INTERPRETATION: The deviation in the T-cell receptor V beta repertoire among circulating T cells from diabetic patients seems to re-emphasize the importance of enterovirus infections in accelerating the progression of Type I diabetes.

Targeting autoimmune diabetes with gene therapy.

The autoimmune nature of insulin-dependent, or type 1, diabetes targets the beta-cells of the pancreas for destruction and results in a lifelong commitment to insulin replacement therapy. Although the number of formulations and dosing of insulin have become more sophisticated and more efficient in recent years, insulin therapy alone is unable to prevent nephropathy, retinopathy, or vascular and heart disease, which still occur in a large number of patients. Different approaches have been attempted to eliminate the requirement of exogenous insulin administration. Historically, these have included pancreatic and islet transplants, which were later combined with treatments intended to halt the destructive process directed against the islets. Despite significant advances made in all of these areas, each approach faces a hostile immunological response that frequently ends with the loss of the islets. Gene therapy-based approaches add a new dimension to the efforts aimed at specifically blocking the immunological attack against the islets in genetically at-risk individuals (autoimmunity) or the immunological response against transplanted allogeneic islets (rejection). This new technology may have an important role in the therapy and cure of type 1 diabetes.

Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1beta-induced beta-cell impairment and activation of islet cell apoptosis in vitro.

The beta-cells in the pancreatic islets of Langerhans are the targets of autoreactive T-cells and are destroyed in type 1 diabetes. Macrophage-derived interleukin-1beta (IL-1beta) is important in eliciting beta-cell dysfunction and initiating beta-cell damage in response to microenvironmental changes within islets. In particular, IL-1beta can impair glucose-stimulated insulin production in beta-cells in vitro and can sensitize them to Fas (CD95)/FasL-triggered apoptosis. In this report, we have examined the ability to block the detrimental effects of IL-1beta by genetically modifying islets by adenoviral gene transfer to express the IL-1 receptor antagonist protein. We demonstrate that adenoviral gene delivery of the cDNA encoding the interleukin-1 receptor antagonist protein (IL-1Ra) to cultured islets results in protection of human islets in vitro against IL-1beta-induced nitric oxide formation, impairment in glucose-stimulated insulin production, and Fas-triggered apoptosis activation. Our results further support the hypothesis that IL-1beta antagonism in in situ may prevent intra-islet proinsulitic inflammatory events and may allow for an in vivo gene therapy strategy to prevent insulitis and the consequent pathogenesis of diabetes.

Idiopathic dilated cardiomyopathy: a superantigen-driven autoimmune disease.

BACKGROUND: Many cases of idiopathic dilated cardiomyopathy (IDC) result from an inflammatory myocarditis. The specific immunological mechanisms are not yet defined. Various autoimmune diseases are associated with superantigen-triggered immune responses, resulting in massive T-cell activation and tissue damage. We studied 3 cases in a search for evidence that such a phenomenon is also implicated in IDC. METHODS AND RESULTS: Myocardial, lymph node, and thymic tissue samples were obtained from IDC patients who were undergoing heart transplantation. Infiltrating immune-cell phenotypes and gene expression of T-cell receptor (TCR) alpha- and beta-chain variable (Valpha and Vbeta) regions were analyzed by immunostaining and polymerase chain reaction. Similar technical approaches were used to assay the tissues for the presence of coxsackievirus B (CVB). In all the specimens analyzed, an overexpression of the TCR Vbeta3, Vbeta7, and Vbeta13.1 gene families was detected among the infiltrating T cells. These tissues were also found to be CVB3-positive. In vitro exposure of peripheral blood mononuclear cells to lysates of cells infected with CVB3 was capable of stimulating expansion of the same TCR Vbeta families. The TCR Valpha repertoire was never found to be skewed. CONCLUSIONS: A superantigen-mediated immune response is involved in human heart disease. CVB3 may directly or indirectly trigger this response, suggesting a possible mechanistic link between CVB infection and myocarditis development progressing to IDC.

Flt-3 ligand increases microchimerism but can prevent the therapeutic effect of donor bone marrow in transiently immunosuppressed cardiac allograft recipients.

C3H (H2k) mice received 50 x 10(6) B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 microg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 microg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.

Lack of association between schizophrenia and HLA DQB1 alleles in a Swedish sample.

Three studies have reported a negative genetic association between schizophrenia and HLA DQB1*0602, an allele of the human leucocyte antigen (HLA) DQB1*06 gene. In a sample of ethnic all homogeneous Caucasians living in Sweden, the frequency of HLA DQB1 alleles in patients with schizophrenia (n = 124) was compared with that in a control group (n = 85). No significant differences were found. Together with previous investigations, the present study indicates that the reported genetic association of DQB1*0602 with schizophrenia may be limited to non-Caucasians.

Impact of Flt-3 ligand on donor-derived antigen presenting cells and alloimmune reactivity in heart graft recipients given adjuvant donor bone marrow.

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II+ cells in the lymphoid tissues of noncytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2k) mice received a nonvascularized B10 (H2b) heart allograft in the dorsal ear pinna, followed by an i.v. infusion of 50 x 10(6) donor BM cells. They were given FL (10 microg/day i.p., x7 days), tacrolimus (2mg/kg/day i.p., x13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IAb+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II+ cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days). The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cells against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus +/- FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedly increased in conventionally immunosuppressed organ allograft recipients given donor BM + a potent haematopoietic and DC-growth promoting cytokine. Although withdrawal of systemic immunosuppression appears to allow exhibition of the potential allostimulatory activity of these donor APC leading to rejection, the model provides a useful basis for further evaluation of the persistence and manipulation of donor haematopoietic cells and in particular, donor-derived APC, on the outcome of organ transplantation.

Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation.

Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.

Double-labeled fluorescent probes for 5' nuclease assays: purification and performance evaluation.

An inexpensive method for the purification and evaluation of user-synthesized or crude commercially prepared double-labeled fluorescent probes is presented. These probes exhibit the characteristics required for use in 5'-nuclease assays, including efficient reporter dye quenching, target specificity and susceptibility to cleavage by Taq DNA polymerase during PCR amplification. The method is suitable for research laboratories that wish to develop 5' nuclease assays for the detection of PCR-amplified target sequences to eliminate the requirement for agarose gels and to advance throughput.

Negative association of schizophrenia with HLA DQB1*0602: evidence from a second African-American cohort.

The authors attempted a replication of an earlier study of African-Americans, in which they detected a negative association of schizophrenia with HLA DQB1*0602. Patients with schizophrenia (n = 75, DSM-III-R criteria) and screened adult controls of African-American ethnicity (n = 66) were genotyped with respect to HLA DQB1*0602 using a combination of two polymerase chain reaction (PCR) based assays: amplification with sequence specific primers and a dot blot assay. A significant negative association with HLA DQB1 was not noted overall, but was present among women (female patients vs. female controls: odds ratio, OR = 0.42, 95% confidence intervals, CI = 0.32, 0.55). Reanalysis of the earlier study also revealed a gender related association. When the present and earlier samples from both genders were combined, the association persisted (OR 0.48; 95% CI: 0.12, 0.52). The present findings support and association between schizophrenia and the HLA DQB1 gene locus among African-Americans.