Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition.

Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process.

Tumor necrosis factor (TNF) alpha is a cytokine that has potent immune regulatory functions. To assess the potential role of this cytokine in the early development of autoimmunity, we investigated the effect of TNF on the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice, a spontaneous murine model for autoimmune, insulin-dependent type I diabetes. Treatment of newborn female NOD mice with TNF every other day for 3 wk, led to an earlier onset of disease (10 versus 15 wk of age in control mice) and 100% incidence before 20 wk of age (compared to 45% at 20 wk of age in control phosphate-buffered saline treated female mice). In contrast, administration of an anti-TNF monoclonal antibody, TN3.19.12, resulted in complete prevention of IDDM. In vitro proliferation assays demonstrated that mice treated with TNF developed an increased T cell response to a panel of beta cell autoantigens, whereas anti-TNF treatment resulted in unresponsiveness to the autoantigens. In addition, autoantibody responses to the panel of beta cell antigens paralleled the T cell responses. The effects mediated by TNF appear to be highly age dependent. Treatment of animals either from birth or from 2 wk of age had a similar effect. However, if treatment was initiated at 4 wk of age, TNF delayed disease onset. These data suggest that TNF has a critical role in the early development of autoimmunity towards beta-islet cells.

Dysrulation of T cell peripheral tolerance in type 1 diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which the insulin producing beta cells are destroyed. The breakdown of beta cell-specific self-tolerance by T cells involves a number of dysregulated events intrinsic and extrinsic to T cells. Herein, we review the key mechanisms that drive beta cell autoimmunity, with an emphasis on events that influence the expansion and differentiation of pathogenic T cells in the periphery.

Parameters that govern the regulation of immunoglobulin delta heavy-chain gene expression.

The mu and delta immunoglobulin heavy-chain genes comprise a complex transcriptional unit in which a single mRNA precursor gives rise to mu- and delta-specific transcripts. During the immature B-cell stage, posttranscriptional processing events involving alternate splicing and cleavage-polyadenylation site selection give rise to mu- but not delta-encoding transcripts. In terminally differentiated B cells, delta mRNA is not synthesized because of a transcription termination event occurring upstream of the delta-gene locus. In an attempt to gain insight into the respective contributions of alternate splicing and cleavage-polyadenylation in the control of delta mRNA synthesis, we have constructed a set of plasmids in which membrane mu (mu m)-delta intergenic sequences containing the mu m poly(A) site but differing in splicing capacity were inserted in between a VH and delta gene. The mu m-delta insertion vectors were transfected into a B lymphoma line representative of an immature stage, and proximal mu m poly(A) site usage and delta mRNA synthesis were assessed. To determine unequivocally whether the mu m-delta intergenic region can regulate termination, the insertion vectors were also transfected into a B myeloma line, and transcription through the region was measured. In immature B-cell transfectants, splicing site selection was found to have a key role in determining poly(A) site utilization and concomitant delta mRNA expression. Mature delta mRNA synthesis was blocked by an upstream cleavage-polyadenylation event only when the proximal poly(A) site was associated with appropriate splicing signals. Furthermore, in vitro transcription assays revealed that the mu m-delta intergenic region is sufficient to regulate transcription termination within a 1,2430-base-pair region containing the mu m poly(A) site in myeloma transfectants. The mu m-delta insertion vectors provide an excellent model system for studying the regulatory aspects of this transcription termination event.

Induction of GAD65-specific regulatory T-cells inhibits ongoing autoimmune diabetes in nonobese diabetic mice.

IDDM is a T-cell-mediated autoimmune disease in which the insulin-producing beta-cells are destroyed. The disease process is complex, involving the recognition of several beta-cell autoantigens. One of these, GAD65, appears to have a critical and not fully defined role in IDDM in humans and in the NOD mouse. We provide evidence that an ongoing diabetogenic response in NOD mice can be suppressed after intravenous administration of GAD65, but not by other beta-cell autoantigens. Furthermore, suppression of the diabetogenic response is mediated by the induction of GAD65-specific CD4+ regulatory T-cells. Finally, cytokine analysis indicates that these CD4+ regulatory T-cells have a T-helper 2 phenotype.

The role of cell adhesion molecules in the development of IDDM: implications for pathogenesis and therapy.

IDDM is a chronic inflammatory disease in which there is autoimmune-mediated organ-specific destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The migration of autoreactive lymphocytes and other leukocytes from the bloodstream into the target organ is of clear importance in the etiology of many organ-specific autoimmune/inflammatory disorders, including IDDM. In IDDM, this migration results in lymphocytic invasion of the islets (formation of insulitis) and subsequent destruction of beta-cells. Migration of lymphocytes from the bloodstream into tissues is a complex process involving sequential adhesion and activation events. This migration is controlled in part by selective expression and functional regulation of cell adhesion molecules (CAMs) on the surface of lymphocytes and vascular endothelial cells or in the extracellular matrix. Understanding the mechanisms that regulate lymphocyte migration to the pancreatic islets will lead to further understanding of the pathogenesis of IDDM. In this article, we summarize the recent advances regarding the function of CAMs in the development of IDDM in animal models and in humans and discuss the potential for developing CAM-based therapies for IDDM.

Prevention of diabetes in NOD mice by a mutated I-Ab transgene.

Susceptibility to the human autoimmune disease IDDM is strongly associated with those haplotypes of the major histocompatibility complex (MHC) carrying DQB1 alleles that do not encode aspartic acid at codon 57. Similarly, in a spontaneous animal model of this disease, the NOD mouse, the genes of the MHC play an important role in the development of diabetes. The DQB1 homolog in NOD mice, I-Ab(g7), encodes a histidine at codon 56 and a serine at codon 57, while all other known I-Ab alleles encode proline and aspartic acid, respectively, at these positions. We therefore mutated the NOD I-Ab allele to encode proline at position 56 and aspartic acid at position 57 and introduced this allele onto the NOD genetic background to study the effect of these substitutions on susceptibility to diabetes. No transgenic mice developed diabetes by 8 months of age, and transgenic mice had markedly reduced lymphocytic infiltration in the pancreas compared with nontransgenic littermates. Furthermore, splenocytes from transgenic mice failed to proliferate or secrete gamma-interferon in response to a panel of beta-cell autoantigens, although the mice did produce beta-cell specific antibodies. Interestingly, the proportion of IgG1 and IgE relative to IgG2a comprising these autoantibodies was much greater in transgenic mice compared with nontransgenic control mice. Finally, T-cells from transgenic mice inhibited the adoptive transfer of diabetes to irradiated recipients. This inhibition was partially reversed by treatment of the recipients with a combination of anti-interleukin (IL)-4 and anti-IL-10 monoclonal antibodies. Thus, a transgenic class II MHC allele encoding aspartic acid at B57 prevents diabetes, in part, by promoting the production of IL-4 and IL-10, which interfere with the effector phase of the diabetic process.

Isolation of nonobese diabetic mouse T-cells that recognize novel autoantigens involved in the early events of diabetes.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from chronic, cell-mediated, autoimmune islet damage. Our aim was to identify the earliest T-cell autoantigen in IDDM, reasoning that this antigen could be causally involved in the initiation of the disease. Identification of the earliest beta-cell-specific autoantigen is extremely important in allowing advances in prevention and treatment of initial events in the development of inflammatory insulitis that precedes beta-cell destruction and overt diabetes. Therefore, we analyzed the proliferative responses of peripheral T-cells from young, female nonobese diabetic (NOD) mice to extracts of pancreatic beta-cell lines. We were able to demonstrate that T-cells responsive to beta-cell antigens exist in the peripheral lymphoid tissue of these mice in the absence of deliberate priming before the manifestation of histologically detectable insulitis. T-cell lines and clones isolated from the peripheral lymphatic tissues of young, unimmunized, female NOD mice were also shown to react with extracts of beta-cells. Fractionation of the beta-cell extracts showed that these T-cell clones recognized multiple beta-cell-specific autoantigens but none of the previously reported putative autoantigens (glutamic acid decarboxylase [GAD]65, GAD67, Hsp65, insulin, ICA 69, carboxypeptidase-H, and peripherin). Thus, we can conclude that these responses are specific for novel beta-cell autoantigens. Finally, NOD T-cell proliferative responses were also seen to an extract of human islets suggesting potential shared antigenic determinants between human and mouse beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Report from the 1st International NOD Mouse T-Cell Workshop and the follow-up mini-workshop.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.

The role of environmental factors in insulin-dependent diabetes mellitus: an unresolved issue.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by the destruction of the insulin-secreting beta cells found in the islets of Langerhans. Reduced beta-cell mass results in overt diabetes, requiring lifelong exogenous insulin administration and the possibility of numerous sequelae. Incidence and development of IDDM depend upon a variety of genetic and nongenetic factors. Environmental factors such as chemicals, diet, and infection are suspected to influence the development of disease. This review describes the work performed to date to elucidate the role of these environmental factors in IDDM.

The receptor tyrosine kinase MerTK regulates dendritic cell production of BAFF.

The MerTK receptor tyrosine kinase is an important negative regulator of dendritic cell function and is required to prevent B cell autoimmunity in vivo. It is not currently known however, if any causal relationship exists between these two aspects of MerTK function. We sought to determine if dendritic cells (DC) from mice lacking MerTK (mertk(- / - ) mice) have characteristics that may aid in the development of B cell autoimmunity. Specifically, we found that mertk(- / - ) mice contain an elevated number of splenic DC, and this population contains an elevated proportion of cells secreting the critical B cell pro-survival factor, B cell activating factor (BAFF). Elevated numbers of BAFF-secreting cells were also detected among mertk(- / - ) bone marrow-derived dendritic cell (BMDC) populations. This was observed in both resting BMDC, and BMDC stimulated with lipopolysaccharide (LPS) or treated with exogenous apoptotic cells. We also found that DC in general have a pro-survival effect on resting B cells in co-culture. However, despite containing more BAFF-secreting cells, mertk(- / - ) BMDC were not superior to C57BL/6 or baff-deficient BMDC at promoting B cell survival. Furthermore, using decoy receptors, we show that DC may promote B cell survival and autoimmunity through a BAFF-and a proliferation-inducing ligand-independent mechanism.

Plasmid DNAs encoding insulin and glutamic acid decarboxylase 65 have distinct effects on the progression of autoimmune diabetes in nonobese diabetic mice.

We previously demonstrated that administration of plasmid DNAs (pDNAs) encoding IL-4 and a fragment of glutamic acid decarboxylase 65 (GAD65) fused to IgGFc induces GAD65-specific Th2 cells and prevents insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To assess the general applicability of pDNA vaccination to mediate Ag-specific immune deviation, we examined the immunotherapeutic efficacy of recombinants encoding murine insulin A and B chains fused to IgGFc. Insulin was chosen based on studies demonstrating that administration of insulin or insulin B chain by a variety of strategies prevents IDDM in NOD mice. Surprisingly, young NOD mice receiving i.m. injections of pDNA encoding insulin B chain-IgGFc with or without IL-4 exhibited an accelerated progression of insulitis and developed early diabetes. Exacerbation of IDDM correlated with an increased frequency of IFN-gamma-secreting CD4(+) and CD8(+) T cells in response to insulin B chain-specific peptides compared with untreated mice. In contrast, treatment with pDNAs encoding insulin A chain-IgGFc and IL-4 elicited a low frequency of IL-4-secreting Th cells and had no effect on the progression of IDDM. Vaccination with pDNAs encoding GAD65-IgGFc and IL-4, however, prevented IDDM. These results demonstrate that insulin- and GAD65-specific T cell reactivity induced by pDNA vaccination has distinct effects on the progression of IDDM.

Systemic antigen in the treatment of T-cell-mediated autoimmune diseases.

Systemic injection of antigen is one of the approaches that reproducibly induces effective antigen-specific hyporesponsiveness. Here, Roland Liblau and colleagues discuss the cellular and molecular bases of such tolerance, review the current use of this therapeutic strategy in experimental organ-specific autoimmune diseases and analyse what steps are necessary to make this approach suitable for clinical use.

Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent.

Peptide-based immunotherapy is one strategy by which to selectively suppress the T cell-mediated destruction of beta cells and treat insulin-dependent diabetes mellitus (IDDM). Here, we investigated whether a panel of T cell epitopes derived from the beta cell autoantigen glutamic acid decarboxylase 65 (GAD65) differ in their capacity to induce Th2 cell function in nonobese diabetic (NOD) mice and in turn prevent overt IDDM at different preclinical stages of disease development. The panel consists of GAD65-specific peptides spanning aa 217-236 (p217), 247-265 (p247), 290-309 (p290), and 524-543 (p524). Our studies revealed that all of the peptides effectively prevented insulitis and diabetes when administered to NOD mice before the onset of insulitis. In contrast, only a mixture of p217 and p290 prevented progression of insulitis and overt IDDM in NOD mice exhibiting extensive beta cell autoimmunity. Immunization with the GAD65-specific peptides did not block IDDM development in NOD mice deficient in IL-4 expression. These findings demonstrate that GAD65-specific peptide immunotherapy effectively suppresses progression to overt IDDM, requires the production of IL-4, and is dependent on the epitope targeted and the extent of preexisting beta cell autoimmunity in the recipient.

Assessment of antigen-specific receptor function of surface immunoglobulin M and D with identical hapten specificity.

Monoclonal B-cell lines expressing antigen-specific surface IgM or IgD were established by transferring the genes encoding immunoglobulin heavy (mu or delta) and light (kappa) chains specific for the hapten 2,4,6-trinitrophenyl (TNP) into a B-cell lymphoma line. Two IgMTNP and two IgDTNP transformants were selected on the basis of similar levels of anti-TNP idiotype expression by flow microfluorimetric analysis. The IgMTNP and IgDTNP transformants were compared in quantitative assays for their ability to bind TNP-carrier and present TNP-carrier to carrier-specific T cells. Our results indicate that IgMTNP and IgDTNP transformants have an equal capacity to bind and present specific antigen. Thus surface IgM and IgD, when present in equivalent amounts, function similarly as antigen receptors.

Functional differences between immunoglobulins M and D expressed on the surface of an immature B-cell line.

Crosslinked IgM molecules expressed on the surface of immature B cells mediate responses that inhibit further development, in contrast to the activational and proliferative events that follow crosslinking of the mu heavy chain in mature B cells. Concomitant with this change in IgM signaling capacity is the appearance of surface IgD, which has been proposed to modulate the response elicited by the mu heavy chain. In an attempt to gain insight into the mechanism(s) by which surface IgM is able to generate such disparate responses, delta heavy chain gene transfectants of the murine B-cell lymphoma line WEHI-231 were established. WEHI-231 cells resemble phenotypically immature B cells, in addition to being highly susceptible to the growth-inhibitory effect of surface IgM cross-linking. Endogenous mu and exogenous delta heavy chains expressed on the surface of the transfectants were compared for their role in cell proliferation and on gene expression. Our results indicate that the growth-inhibitory response is associated only with the mu heavy chain and that surface IgD does not mediate such a response. Furthermore, in contrast to IgM, IgD molecules appear to have an inductive effect on the expression of Myc and the endogenous mu and exogenous delta Ig heavy chain genes but not on the expression of the housekeeping gene encoding beta 2-microglobulin. These findings suggest that IgM and IgD are functionally distinct when expressed on the surface of an immature B cell.

An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells.

Susceptibility to the human autoimmune disease insulin-dependent diabetes mellitus is strongly associated with particular haplotypes of the major histocompatibility complex (MHC). Similarly, in a spontaneous animal model of this disease, the nonobese diabetic (NOD) mouse, the genes of the MHC play an important role in the development of diabetes. We have produced transgenic NOD mice that express the class II MHC molecule I-Ad in addition to the endogenous I-Ag7 molecules in order to study the role of these molecules in the disease process. Although the inflammatory lesions within the islets of Langerhans in the pancreas appear similar in transgenic and nontransgenic animals, transgenic mice develop diabetes with greatly diminished frequency compared to their nontransgenic littermates (10% of transgenic females by 30 weeks of age compared to 45% of nontransgenic females). Furthermore, adoptive transfer experiments show that T cells present in the transgenic mice are able to interfere with the diabetogenic process caused by T cells from nontransgenic mice. Thus, the mechanism by which I-Ad molecules protect mice from diabetes includes selecting in the thymus and/or inducing in the periphery T cells capable of inhibiting diabetes development.

Administering glutamic acid decarboxylase to NOD mice prevents diabetes.

Type 1 diabetes is the result of an ongoing autoimmune response to specific proteins expressed by the insulin producing beta cells. Recently, a number of beta cell autoantigens have been identified. However, their role in mediating the diabetogenic response is not known. Here we assess the relative importance of a panel of beta cell autoantigens in the disease process. The approach was to inhibit T cell proliferation to a given autoantigen by either i.t. or i.v. injections, and then determine the effect this had on the diabetogenic response. We show that administering murine glutamic acid decarboxylase (GAD) to 3-week-old NOD females can reduce the frequency of insulitis and prevent the onset of diabetes. In contrast, carboxypeptidase H or peripherin do not induce a similar protective effect, suggesting that GAD has a critical role in the diabetogenic response. These results also suggest that GAD may provide a useful target for antigen-specific immunotherapy.

The analysis with monoclonal antibodies of the heterogeneity of Ia glycoproteins on chronic lymphocytic leukemia cells.

The accessible Ia molecules on the surface of chronic lymphocytic leukemia (CLL) cells were quantitated in the cellular radioimmunoassay with saturating concentrations of monoclonal antibodies. Monoclonal antibody 21w4, like DA/2 antibody, recognizes monomorphic determinants of human Ia antigens. The amount of 21w4 or DA/2 bound to CLL cells derived from eight patients (varying from 2.6 to 13.9 x 10(5) molecules/cell) appears to be the maximum observed with the antibodies studied. Two other antibodies, 18d5 and 21r5, although also directed at nonpolymorphic Ia determinants, bind differentially to CLL cells, with the ratios of 21r5/21w4 and 18d5/21w4 varying from 0.08 to 0.90. Sequential immmunoprecipitation studies have established that the four epitopes 18d5, 21r5, 21w4, and DA/2 were present on the same molecules. All Ia molecules express 21w4 and DA/2 epitopes, whereas only certain subsets of Ia molecules carry accessible 21r5 or 18d5 epitopes. Competitive binding studies showed that the epitopes recognized by the four monoclonal antibodies were different. Monoclonal antibodies 21r5 and 21w4 did not inhibit each other's binding. Furthermore, binding of 21w4 to CLL cells potentiated the binding of 125I-21r5 IgG to the same cells, suggesting that binding of 21w4 antibody induces a conformational change in the molecule that renders 21r5 epitopes more accessible.

Prevention of diabetes and insulitis by neonatal intrathymic islet administration in NOD mice.

The murine model of human insulin dependent diabetes mellitus (IDDM), the non-obese diabetic (NOD) mouse, develops a T cell-dependent destruction of pancreatic islets. While the target antigens are unknown, there is clearly a lack of tolerance to them. Neonatal intrathymic (i.t.) antigen injection has been successfully employed to prevent insulitis in BB rats but previous i.t. islet antigen studies in NOD mice were done on older mice. We have injected syngeneic islets into the thymus of NOD mice at birth and found that diabetes and insulitis can be completely prevented by this procedure. The effect is islet antigen-specific since other T cell responses, including autoimmune salivary infiltration, ard unaffected. Furthermore, contrary to previous studies, cyclophosphamide administration was unable to induce diabetes in treated mice which suggests that deletion or anergy might be the mechanism by which neonatal intrathymic islet injection protects from disease. However, anti-islet antigen antibodies were still present in these mice which suggests that the mechanism of disease protection may be more complex.