Cloning and characterization of a glutamate transporter cDNA from human brain and pancreas.

L-Glutamate is the major excitatory neurotransmitter in the brain. Sufficient removal from the synaptic cleft after neurotransmission by the L-glutamate transport system is essential to prevent excitotoxicity and neurotoxicity. We isolated mRNA from human brain and pancreatic islet cells and screened for sequences of high homology to a previously characterized rat brain glutamate transporter. An isolated sequence (GLTR) shows a 87.5% and a 92.5% sequence similarity at the nucleotide and amino acid level, respectively, with a rat brain specific L-glutamate transporter but only a 65% homology to the recently cloned human glutamate/aspartate transporter. The human mRNA is differentially expressed in brain and to a lesser degree in pancreas and in fetal liver. The gene encoding for the newly identified cDNA is located on chromosome 5.

Epidemiology and immunogenetic background of islet cell antibody--positive nondiabetic schoolchildren. Ulm-Frankfurt population study.

Islet cell antibodies (ICAs) were determined in a large cohort of white nondiabetic schoolchildren (n = 4287) from a homogenous population in southern Germany. The prevalence of ICA levels greater than or equal to 5 Juvenile Diabetes Foundation (JDF) U was 1.05% (95% confidence interval 0.8-1.4%). Analysis of HLA-DR beta and -DQ beta alleles revealed that the specificities found to be increased in insulin-dependent (type I) diabetic subjects with the same ethnic background were also associated with ICA positivity in the nondiabetic schoolchildren. HLA-DR3 (P less than 0.01) and -DR4 (P less than 0.01) phenotypes and absence of Asp residue (P less than 0.01) at codon 57 of the HLA-DQ beta-chain were significantly increased in ICA+ compared with control subjects. High levels of ICAs, which were categorized as either greater than or equal to 17 or greater than or equal to 30 JDF U, were found to be associated with amino acids other than Asp at position 57 of the HLA-DQ beta-chain. No association of ICA level was found for HLA-DR phenotypes.

T-cell-receptor repertoire in chronic plaque-stage psoriasis is restricted and lacks enrichment of superantigen-associated V beta regions.

Preferential usage of certain T-cell receptors by the lymphocytic infiltrate in psoriasis might indicate the involvement of an antigen in the pathogenesis of this disease. However, to date there are no data on the complete T-cell-receptor V alpha and V beta repertoire in psoriatic patients. We therefore compared the usage of T-cell-receptor variable regions in blood and skin of 10 patients with chronic plaque-stage psoriasis by means of semiquantitative polymerase chain reaction. Additionally, HLA class II alleles were analyzed by means of sequence-specific oligonucleotide typing. A considerable restriction of the T-cell-receptor repertoire was observed in the skin, where up to 20% of the variable regions present in the blood were not detectable. This was true for both alpha- and beta-chains. However, no interindividually constant pattern of T-cell-receptor restriction was deducible. Inconsistently, a certain preferential usage of some beta chains occurred within the cutaneous compartment. This report on the complete T-cell-receptor V alpha and V beta repertoire in psoriasis documents the restricted receptor repertoire of infiltrating T cells and a lack of enrichment of superantigen-associated V beta regions. Thus superantigens seem not to play a pathogenetically relevant role in chronic plaque-stage psoriasis.

Analysis of the alpha/beta T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging.

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the alpha/beta T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the alpha/beta T-cell repertoire in autoimmune and infectious diseases.

Non-productive human TCR beta chain genes represent V-D-J diversity before selection upon function: insight into biased usage of TCRBD and TCRBJ genes and diversity of CDR3 region length.

The aim of the study was to assess the influence of constraints of V-D-J rearrangement on the nonrandom junctional diversity of productive T-cell receptor beta-chain genes in peripheral T-cells. Mature peripheral T lymphocytes are expected to display a biased repertoire of T cell receptors (TCRs), enriched for those that can recognize peptides presented by the major histocompatibility complex (MHC) molecules. Therefore, functional TCR rearrangements of peripheral T-cells are unsuitable to reveal the bias of the TCR repertoire, introduced by V-D-J rearrangement. To overcome this problem, we have studied nonfunctional TCR genes representing a repertoire of rearranged TCR gene sequences without any known post-rearrangement selection. Detailed molecular analysis of a database generated from more than 500 functional (TCRBV20S1) and nonfunctional (TCRBV10S1P and TCRBV19S1P) T-cell receptor genes from peripheral blood T-cells permitted a comparative analysis of recombination frequencies of each germline-encoded V, D, and J-segments, as well as exonucleolytic nibbling and addition of nucleotides in functional and nonfunctional transcripts. Our data demonstrate that V-D-J recombination generates a more diverse CDR3 length distribution than found among productive TCRBV genes, suggesting that selection constrains the CDR3 to an optimal junctional region length. Furthermore, the well established biased patterns of D- and J-usage in the rearranged TCRBV genes in human peripheral blood lymphocytes were also present in nonfuncrional transcripts. Therefore, V-D-J diversity is biased mainly by constraints of the rearrangement process rather than intrathymic T-cell selection and peripheral expansion of particular T-cell clones.

Rapid and simple subtyping of the HLA-DRB3 gene in Graves' disease by using temperature-gradient gel electrophoresis.

A HLA-DRB3-subtyping system that uses TGGE for analyzing DRB3 alleles was developed. The polymorphic second exon of the HLA-DRB3 gene was amplified from four homozygous typing cell lines, 223 healthy individuals, and 102 patients with Graves' disease by using the PCR. The PCR products were electrophoresed in a temperature gradient from 35 degrees C to 70 degrees C, and the resulting fragments were visualized by silver staining. Four DRB3 alleles (HLA-DRB3*0101, *0201, *0202, and *0301) were distinguished from one another by the migration of the corresponding homoduplex with the exception that DRB3*0201 was indistinguishable from DRB3*0202. The latter two alleles, however, were resolved by the artificial heteroduplexing approach. Arginine in position 74 of the DRB3 gene product (i.e., HLA-DRB3*0101) was significantly more frequent in Graves' patients than in controls. The relative risk conferred by the presence of the DRB3*0101 was 15.8 (p < or = 0.001). The presence of arginine in position 74 contributed to an etiologic fraction of 75% in our study population. The PCR-TGGE technique is a simple, nonisotopic method, which may be useful in rapid screening of large populations for HLA disease markers.

Opsonization with a trifunctional bispecific (alphaCD3 x alphaEpCAM) antibody results in efficient lysis in vitro and in vivo of EpCAM positive tumor cells by cytotoxic T lymphocytes.

Removab is a trifunctional bispecific antibody which can bridge CD3+ T cells and epithelial cell adhesion molecule positive (EpCAM+) tumor cells, and binds with its Fc fragment to antigen presenting cells. To explore a new approach for the treatment of patients with carcinoma of the upper aerodigestive tract, we investigated whether Removab can induce specific cellular responses to the EpCAM+ carcinoma cell line BHY. Particular emphasis was put on the opsonization of peripheral blood mononuclear cells (PBMN) with respect to clinical application. Tumor cells and allogeneic PBMN of healthy volunteers were incubated with or without Removab. In a third group, PBMN were opsonized with Removab and washed before incubation with tumor cells. Inverse microscopy, ELISPOT, flow cytometry analysis and cytotoxicity assays on the chorioallantois membrane (CAM) were performed. In comparison with PBMN alone, opsonization with Removab resulted in: a) activation of CD83+ antigen presenting cells, b) secretion of interferon gamma, and c) granzyme B mediated lysis of targeted BHY cells by EpCAM specific CD8+ T cells. The secretion of tumor necrosis factor alpha, interferon gamma and interleukin-2 by opsonized PBMN was significantly reduced after 24 h. Washed opsonized PBMN maintained their lytic activity against tumor cells as tested on the CAM. Removab is an appropriate agent for the therapeutic amplification of T cell responses against EpCAM+ tumor cells by opsonization of PBMN without putting patients at risk for severe adverse events caused by a cytokine storm.

The level and the persistence of islet cell antibodies in healthy schoolchildren are associated with polymorphic residues of the HLA-DQ beta chain.

Cytoplasmic islet cell antibodies (ICA) were determined in a group of non-diabetic Caucasian schoolchildren (n = 4208). The prevalence rate for ICA positivity was 1.05 per cent (95 per cent confidence interval: 0.8-1.4 per cent). Analysis of HLA risk factors revealed that HLA-DRB1*03 (p less than 0.01), HLA-DRB1*04 (p less than 0.01) and HLA haplotypes with non-charged amino acids (non-Asp) at codon 57 of the HLA-DQ beta (p less than 0.01) chain were significantly increased when compared to controls. High levels of islet cell antibodies, i.e. Juvenile Diabetes Federation units (JDF units) equal to or greater than 30 JDF units were found to be associated with amino acids other than aspartic acid at codon 57 of the DQ beta chain molecule. Also the persistence of circulating ICA was found to be associated with non-Asp homozygosity of the proband (p less than 0.03).

Eradication of a dysfunctional HLA-haploidentical T cell system by a second HLA-identical BMT.

A 12-year-old boy treated for SCID at 1 month of age by HLA-haploidentical BMT developed a lymphoproliferative disease of unknown etiology at the age of 9 years characterized by sustained, marked elevation of circulating CD8+ donor T cells and by diffuse infiltration of the liver by CD8+ T cells. Because of progressive liver disease, the patient underwent a second BMT from a younger HLA-matched sister. This treatment induced an effective graft-versus-graft reaction and led to complete replacement of the HLA-nonidentical, dysfunctional T cell system, resolution of the hepatopathy and full reconstitution of T and B cell functions.

Expression of a glutamate transporter cDNA in human pancreatic islets.

A number of neuron-specific molecules have been found to be also expressed in pancreatic islets. Some of them might play a major role in the pathogenesis of an autoimmune reaction against the insulin-secreting beta-cells resulting in beta-cell destruction and the manifestation of insulin-dependent diabetes mellitus (IDDM). In ongoing studies our goal was to determine the role of other neuron-specific molecules in beta-cells function and in insulitis. We cloned a L-glutamate transporter from a highly purified fraction of human islet cells. The expression of that specific mRNA was marked in various regions of the brain; weak expression was detected in human islet cells, while no expression in any other tissue was observed. Sequence analysis revealed identity to the transcripts isolated from human brain. The cloned novel cDNA from human islets encodes a key molecule of the excitatory neurotransmission pathway. The techniques used provide a model for the cloning and expression of molecules expressed both in endocrine and neuronal tissue. With characterization of its structure the possible role of the L-glutamate transporter as autoantigen in IDDM is now under investigation.

T-cell receptor repertoire and function in umbilical cord blood lymphocytes from newborns of type 1 diabetic mothers.

Recent observations show that the development of the human fetal immune system is susceptible to conditions of the maternal immune system and vice versa. To investigate the impact of type 1 diabetes mellitus in pregnant women on the maturation of the immune system of their newborn infants, the umbilical cord blood (UCB) T-cell repertoire at birth was analysed for clonal or oligoclonal expansions and TCRBV gene usage. Quantitative PCR using TCRBV family-specific probes and the spectratyping method were used. The extent of oligoclonal expansions observed in cord blood was markedly lower than in peripheral blood of the diabetic mothers and healthy adults. Functional analysis revealed that UCB T cells from newborns of both type 1 diabetic and gestational diabetic mothers are mature and can be readily stimulated by superantigens and phytohemagglutinin in vitro. No significant differences of the clonal contingent and the TCRBV usage were found in newborns of type 1 diabetic mothers when compared to newborns of gestational diabetic mothers, independent from the presence or absence of islet autoantigens. Our findings indicate that the immune system of type 1 diabetic mothers has no major effects on the TCR repertoire of the newborn infants. In that respect, no evidence was found for superantigen-activated T cells, nor was chronic hyperglycaemia per se found to alter either T-cell repertoire or functional activity.

Echinococcus multilocularis metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococcosis patients.

Infection with the cestode Echinococcus multilocularis causes human alveolar echinococcosis (AE), a life-threatening disease affecting primarily the liver. Despite the severity of AE, clinical symptoms often develop only many years after infection, which suggests that E. multilocularis has developed mechanisms which depress anti-parasite immune response, thus favouring immune evasion. In this study we examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable E. multilocularis vesicles and E. multilocularis vesicle fluid antigen in vitro. After 48 h of co-culture, E. multilocularis metacestode culture supernatant and E. multilocularis vesicles depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-alpha and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-alpha was reduced in AE patients, which was accompanied by an increased number of CD4+ CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to E. multilocularis metacestode in AE patients. Instead the production of interferon (IFN)-gamma and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both regulatory and inflammatory Th1 and Th2 cytokines and chemokines. Such a mixed profile might be required for limiting parasite growth but also for reducing periparasitic tissue and organ damage in the host.

HLA-DR3 and variations of the T cell receptor beta gene in Graves' disease.

The association of Graves' disease with two allelic forms of the T cell receptor beta-chain gene (Bgl II restriction fragments 9.2/10.0 kb) was analysed in Caucasians suffering from Graves' disease (N = 54), randomly selected controls (N = 68), and HLA-DR3 homozygous typing control subjects (N = 12). While gene frequencies did not vary in the latter two groups, a significant reduction of 9.2 kb homozygosity was observed in patients with Graves' disease (11 vs 32%, chi 2 = 7.68, p less than 0.01, RR = 0.26). The excess of the 9.2/10.0 kb heterozygosity in patients, which is restricted to the DR3-positive individuals, did not reach significance. The increase in heterozygosity of genomic T cell receptor beta-chain polymorphisms may be attributed to the role the beta-chain plays in autoantigen recognition in Graves' disease.

TcR-alpha and TcR-beta dialellic RFLPs in insulin-dependent (type I) Caucasian diabetic patients.

We studied the association of a T-cell receptor (TcR) beta restriction fragment length polymorphism (RFLP) and a new TcR-alpha RFLP with insulin-dependent (Type I) diabetes mellitus. This study is part of our effort to find new non-HLA disease genes involved in this chronic organ-specific autoimmune disease. Distribution of a 9.2 kb and a 10.0 kb diallelic TcR beta 2 RFLP was not different in diabetics and controls. A new TcR-alpha RFLP, which gave a 2.7 kb Hind III restriction fragment (A2 allele) was found with a frequency of 0.78 in a population of 78 IDDM patients, compared to 0.68 in 68 control subjects (X2 = 3.62, p = 0.057). In 11 multiplex families studied, a high prevalence of the A2 allele was also observed, but cosegregation with the disease was not seen. Our data suggest that a TcR beta 2 RFLP is not associated with the disease, whereas a particular T-cell receptor alpha germline RFLP (A2 allele) is increased in Type I diabetics although formal proof of linkage is lacking. HLA typing reconfirmed that the HLA-DR4 specificity and the DQ allele HLA-DQw8 are primary risk markers in insulin-dependent (Type I) diabetes mellitus.

The X boxes from promoters of HLA class II B genes at different loci do not complete for nuclear protein-specific binding.

Expression of HLA class II genes is coordinately regulated by cis-acting elements present in their promoter regions immediately upstream from the 5' end of their transcription start sites. Trans-acting factors from the nuclear proteins of the cell are able to positively or negatively regulate transcription of these genes by binding to highly conserved sequences, called boxes. After cloning the promoter regions of all the transcribed class II B genes present in the cell line Priess, we were able to identify certain protein-box complexes and to determine the affinity of these proteins for their respective boxes by comparing promoter boxes of each gene to those of the other genes. Different nuclear proteins seemed to bind to the X boxes of the different class II B genes tested. In the case of the Y box-protein complexes, the various Y boxes competed with similar affinities. The protein(s) which specifically bound to the DRB1-CCAAT box also bound to DPB1-CCAAT box, but completely failed to bind the homologous box from DQB1. Further, CCAAT box-specific protein(s) did not bind to the Y box of the same gene, excluding the possibility that these proteins just recognize the reverse CCAAT box (ATTGG) present within Y.

Aspartic acid at position 57 of the HLA-DQ beta chain is protective against future development of insulin-dependent (type 1) diabetes mellitus.

Insulin-dependent (Type I) diabetes mellitus is a chronic autoimmune disease. From studies in discordant twins and multiplex families a long prediabetic period has been reported. In a population-based program started in 1983, fifteen individuals at possible risk for future Type I diabetes were followed for up to 74 months. Two individuals (13%) developed Type I diabetes. These probands were characterized by the presence of high-level cytoplasmic islet cell antibodies (ICA), complement-fixing ICA, and an impaired first-phase insulin response after intravenous glucose load. Both were homozygous for a high-risk immunogenetic marker of Type I diabetes, i.e., non-Asp at codon 57 of the HLA-DQ beta chain. In all other subjects studied, the immunogenetic marker that confers "dominant resistance", aspartic acid at codon 57, was found. On the basis of our data we conclude that a combination of assays which determine ICA, first-phase insulin release, and HLA-DQB1 polymorphisms will identify individuals with a high probability of developing Type I diabetes at the population level. Conversely, HLA haplotypes positive for aspartic acid seem to confer resistance to the disease.

Pro-inflammatory (IL-1beta, IL-18) cytokines and IL-8 chemokine release by PBMC in response to Echinococcus multilocularis metacestode vesicles.

In humans infected with Echinococcus multilocularis, larval metacestodes will develop, proliferate and progressively infiltrate the surrounding host tissues by exogenous budding of parasitic microvesicles or cell lines which detach from the original tumour and thus become transported through blood or lymph vessels into other organs. Cellular effector mechanisms constitute the most effective means to restrict parasite persistence and proliferation, and here we demonstrate that E. multilocularis vesicle antigens will induce pro-inflammatory, regulatory and chemokine release by PBMC from patients. The pro-inflammatory cytokines IL-1beta and IL-18 were reduced in echinococcosis patients, regulatory IL-10 was similar, but parasite vesicle-induced IL-8 was dominant and clearly elevated in patients. Such selective and opposite dynamics of inflammatory cytokines and chemokine release may prevent overwhelming and pathogenic inflammation, and constitute an appropriate response for attraction of effector cells into the periparasitic tissues with the capacity to limit E. multilocularis metacestode proliferation and dissemination.

The HLA-DQ beta non-Asp-57 allele: a predictor of future insulin-dependent diabetes mellitus in patients with autoimmune Addison's disease.

HLA-DR specificities in 72 Addison's (AD) patients and 808 local controls were compared. We confirmed earlier reports that the HLA-DR3 specificity is significantly increased in AD patients. In our study a relative risk of 3.4 chi 2 = 22.5; pc = 0.01) for the disease was calculated. Analysis of HLA-DQB1 alleles in DR4+ Addison's patients with diabetes mellitus (N = 6) and without IDDM (14 of 18 individuals tested) revealed that the HLA-DQw8 allele (DQB1*0302) was significantly increased in AD patients with IDDM (chi 2 = 13.5; p = 0.001); conversely, a clustering of the HLA-DQw7 allele was detected in DR4+ Addison's patients without IDDM. We thus conclude that particular polymorphic alleles corresponding to non-charged amino acids at position 57 of the HLA-DQ beta-chain [non-Asp-57 alleles] are associated with IDDM also in Addison's patients.