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T Dyrberg

Publications and source records attributed to T Dyrberg.

At least 19 recordsLinked to original sources
PubMed1992

Production of epitope specific monoclonal IgG antibodies to HLA class II molecules by combining in vivo and in vitro immunization.

To study the expression of HLA-DQ beta chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 beta chain sequences. The splenocytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specific mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenocytes. Immunoblot analysis showed that several of the mAbs cross-reacted with DQ beta chain molecules. One mAb to a peptide representing DQw8 beta position [49-60] specifically recognised the DQw8 beta chain. Three mAbs to a peptide representing DQw8 beta position [39-52] specifically recognised an epitope consisting of Gly-Val-Tyr in position 45-47, i.e., all DQ beta alleles except DQw7 beta (position 45-47: Glu-Val-Tyr) and DQw2 beta (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound lymphocytes with the same specificity as found by immunoblotting analysis. Thus, by combining in vivo and in vitro immunization we have generated a number of epitope specific monoclonal IgG antibodies that distinguish closely related HLA-DQ beta chain alleles in predetermined positions.

Alleles↗
PubMed1992

Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.

From a plaque biopsy of a patient with mycosis fungoides, two different continuous cell lines were established by including both IL-2 and IL-4 in the culture medium. Both continuous cell lines appeared with characteristic chromosome markers after approximately 40 cell population doublings. The initial karyotype recognized in T cells from the skin biopsy was 46,XY and the karyotypes of the continuous cell strains were 46,XY, -18, + i(18q) and another with multiple chromosome aberrations as described in Sezary T-cell leukemia. Phenotyping with monoclonal antibodies and T-cell receptor analysis indicates that the latter cell strain represents a minority of T-cells in the plaque. Due to its many chromosomal aberrations it probably represents the malignant cell, which may be a central cell in the immune stimulation taking place in the skin.

Aged↗
PubMed1992

Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene.

Synthetic peptides representing unique sequences in rat proinsulin C-peptide I and II were used to generate highly specific antisera, which, when applied on sections of normal rat pancreas, confirm a homogeneous coexpression of the two C-peptides in all islet beta-cells. Insulin gene expression is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency of insulin-producing cells showed highly differential expression at the cellular level of the three proinsulin C-peptide immunoreactivities, as follows: C-peptide I greater than human C-peptide greater than C-peptide II. The fractions of cells expressing human C-peptide and C-peptide II decreased in time and were absent after more than 50 successive passages, while a C-peptide I-producing population was still present. Double-labeling experiments revealed a heterogeneous distribution of the three different C-peptides. Surprisingly, in the early passages a large fraction of cells would express only a single species of proinsulin-C-peptide immunoreactivity but still at high levels. However, rat C-peptide II and human C-peptide were often colocalized, even in later passages. In situ hybridization studies combined with the immunocytochemical data suggest that the differential expression occurs at the level of transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Recombinant glutamic acid decarboxylase (representing the single isoform expressed in human islets) detects IDDM-associated 64,000-M(r) autoantibodies.

GAD is an autoantigen in IDDM. Molecular cloning and specific antibodies allowed us to demonstrate that only the lower M(r) GAD64 isoform is expressed in human islets, in contrast to human brain, rat islets, and rat brain, all of which express both GAD64 and GAD67. Expression of the human islet GAD64 isoform in COS-7 and BHK cells resulted in an enzymatically active rGAD64, which is immunoreactive with diabetic sera comparable with that of the islet 64,000-M(r) autoantigen. Immunoprecipitation analyses showed that 21/28 (75%) IDDM sera had rGA D64 antibodies compared with only 1/59 (1.7%) of the healthy control sera. In immunoblot analyses, an SMS serum--but only 1/10 randomly selected IDDM sera--recognized the blotted rGAD64 without relation to immunoprecipitation titers. In conclusion, only the GA D64 isoform is expressed in human islets, in contrast to rat islets, which also express the GAD67 isoform. The immunological properties of human rGAD64 are comparable with the native 64,000-M(r) islet autoantigen, allowing further studies of the immunopathogenesis of IDDM.

Animals↗
PubMed1991

Cloning, characterization, and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus.

A 64-kDa islet protein is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Autoantibodies against the 64-kDa protein were recently shown to immunoprecipitate glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from brain and from islets. We present evidence that the autoantisera also recognize a hydrophilic islet protein of approximately 67 kDa in addition to the amphiphilic 64-kDa form. We have isolated a full-length rat islet GAD cDNA encoding a hydrophilic 67-kDa protein, which appears to be identical to rat brain 67-kDa GAD. A partial sequence of human insulinoma 67-kDa GAD was identical to human brain 67-kDa GAD. Allelic variations were observed in rat as well as in human 67-kDa GAD sequences. The expressed rat islet 67-kDa GAD protein is functional and is immunoprecipitated by IDDM sera; it comigrates electrophoretically with the 67-kDa islet autoantigen. The hydrophilic 67-kDa form of GAD in islets is an additional autoantigen in IDDM and is recognized by a different subset of autoantibodies than the 64-kDa autoantigen. Thus, mammalian cell lines expressing functionally active, recombinant GAD may become important tools to study the nature and the role of GAD autoreactivity in IDDM.

Alleles↗
PubMed1991

Growth hormone action in rat insulinoma cells expressing truncated growth hormone receptors.

Transfection of the insulin-producing rat islet tumor cell line RIN-5AH with a full length cDNA of the rat hepatic growth hormone (GH) receptor (GH-R1-638) augments the GH-responsive insulin synthesis in these cells. Using this functional system we analyzed the effect of COOH-terminal truncation of the GH receptor. Two mutated cDNAs encoding truncated GH receptors, GH-R1-294 and GH-R1-454, respectively, were generated by site-directed mutagenesis and transfected into the RIN cells. Both receptor mutants were expressed on the cell surface and displayed normal GH binding affinity. Whereas GH-R1-638 had a molecular mass of about 110 kDa, GH-R1-294 and GH-R1-454 showed molecular masses of 49 and 80 kDa, respectively. Cells expressing GH-R1-454 internalized GH to a similar extent as cells transfected with the full length receptor and the parent cell line, but GH-R1-294-expressing cells showed a markedly reduced capability of GH internalization. In contrast to cells transfected with GH-R1-638, none of the cell lines expressing truncated GH receptors exhibited any increase of the GH-stimulated insulin production. We conclude that domains within the COOH-terminal half of the cytoplasmic part of the GH receptor are required for transduction of the signal for GH-stimulated insulin synthesis, whereas cytoplasmic domains proximal to the transmembrane region are involved in receptor-mediated GH internalization.

Animals↗
PubMed1991

Interferon stimulates the expression of 2',5'-oligoadenylate synthetase and MHC class I antigens in insulin-producing cells.

The pathogenesis of type 1 diabetes involves autoimmune processes directed against the pancreatic beta-cells. The etiology is not known, but circumstantial evidence suggests a connection between virus infection and development of the disease. Therefore, because the interferon-(IFN) dependent 2',5'-oligoadenylate (2-5A) synthetase system constitutes an important part of the nonspecific immune defense against viral infections, the activity of the enzyme was examined in islets of Langerhans, RIN cells, and GH3 cells. First, the 2-5A synthetase was expressed constitutively in all cell types and, second, all cells were sensitive to stimulation with IFN-alpha. The 2-5A synthetase activity induced by 1,000 U/ml of IFN-alpha increased by 400% in pancreatic islets and by more than 1000% in GH3 and RIN cells. However, the IFN-alpha concentration needed to induce half-maximal 2-5A synthetase activity was nearly the same in the three cell types (i.e., ranging from 59 to 66 U/ml IFN-alpha). The 2-5A synthetase present in islets and RIN cells was highly sensitive to poly (I:C). In pancreatic islets and RIN cells, the 2-5A synthetase enzyme generated dimers and trimers of 2',5'-oligoadenylates. Furthermore, exposure of RIN cells to IFN-alpha showed an increase in MHC class I expression already at 5 U/ml and maximal expression at about 200 U/ml IFN-alpha. The examined endocrine cells express the 2-5A synthetase enzyme as well as MHC class I antigen constitutively, but also by stimulation with IFN in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗
PubMed1990

A novel affinity purification method to isolate peptide specific antibodies.

Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides.

Animals↗
PubMed1990

Immunological cross-reactivity between mimicking epitopes on a virus protein and a human autoantigen depends on a single amino acid residue.

Recently we identified a novel autoantigenic region in the alpha chain of the human acetylcholine receptor (HuAChR), residues 160-167 (P. L. Schwimmbeck, T. Dyrberg, D. B. Drachman, and M. B. A. Oldstone, J. Clin. Invest., in press). Antibodies to that sequence appeared in the sera of a subset of patients with myasthenia gravis (MG) and affinity-purified antibody was biologically active for an in vitro assay of the HuAChR. Sequence homology between the autoantigen and two separate regions of herpes simplex virus glycoprotein D (HSV-GpD) have been identified. These components are, respectively, the HuAChR, residues [160-167] (PESDQPDL), and residues [286-293] (PNATQPEL) and [381-388] (PEDDQPSS) of HSV-GpD. Antisera from rabbits immunized with synthetic peptides representing HSV-GpD [286-293] bound strongly to the AChR peptide. In contrast, antiserum to HSV-GpD [381-388] bound minimally, if at all, even though both virus peptides shared four amino acids with the receptor sequence. To investigate the molecular basis for the differential binding, we tested the reactivity of the HSV-GpD antisera to analogs of the [381-388] virus peptide containing single amino acid substitutions. The results demonstrated that the cross-reactivity between HuAChR [160-167] and HSV-GpD [286-293] predominantly depends on a single residue, the C-terminal leucine in HSV-GpD, position 293.

Amino Acid Sequence↗
PubMed1990

Cytotoxic T lymphocytes do not control lymphocytic choriomeningitis virus infection of BB diabetes-prone rats.

BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 x 10(7) p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies. In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.

Animals↗
PubMed1990

Characterization of the fine specificity of peptide antibodies to HLA-DQ beta-chain molecules.

In an attempt to produce epitope specific antisera which could distinguish two closely associated HLA-DQ beta-chain alleles, we immunized 20 rabbits with synthetic peptides representing sequences from the first domain of the HLA-DQw8 and -DQw7 beta-chain molecules, differing only by one amino acid in position 57. Several of the antisera in immunoblotting specifically recognized either the HLA-DQw7 or the HLA-DQw8 beta-chain allele as previously reported. The fine specificity of the antisera was tested in ELISA using synthetic peptides of varying length as solid phase antigen. Two out of the 20 antisera specifically recognized DQw7 beta peptides and two antisera bound only to DQw8 beta peptides from the region containing the amino acid in position 57. To analyze whether the antisera bound to native HLA-DQ beta-chain molecules, FACS analysis was carried out. Seven of the 20 antisera bound to intact EBV-transformed B-lymphocytes, and one antiserum bound preferentially to HLA-DQw8 positive cells.

Amino Acid Sequence↗
PubMed1990

Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2.

Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we have produced peptide antisera to an antigenic epitope which is unique to mouse and rat C-peptide 2. Antisera recognizing this epitope may be effective tools to study differential expression of the two insulin genes in mouse and rat.

Amino Acids↗
PubMed1989

Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes.

The HLA-DQ beta-chain gene shows a close association with susceptibility or resistance to autoimmune insulin-dependent diabetes mellitus (IDDM) and it has been suggested that the amino acid in position 57 may be of pathogenetic importance. To study the expression of the IDDM associated HLA-DQ beta-chain alleles, we immunized rabbits with 12 to 13 amino acid long peptides representing HLA-DQw7 and -DQw8 allelic sequences, differing only by one amino acid in position 57 being aspartic acid (Asp) and alanine (Ala), respectively. Immunoblot analysis of lymphoblastoid cells showed that several antisera recognized a 29-kDa protein, equivalent to the expected molecular size of the HLA-DQ beta-chain to yield two antisera specific for HLA-DQw7 (pos. 57Asp) and three antisera for HLA-DQw8 (pos. 57Ala) positive cells. Analysis of HLA-DR 3/4 positive IDDM patients (n = 24) and controls (n = 19) showed that all (100%) patients were positive for pos. 57Ala antiserum compared to 13 of 19 (68%) of the controls. The remaining six controls reacted with the pos. 57Asp antisera, whereas none of the patients did. We have therefore successfully been able to generate site-specific antibodies that distinguish single amino acid substitutions in predetermined positions of allelic HLA-DQ beta-chain gene products. Such sera should become useful to detect and investigate HLA associated susceptibility to autoimmune diseases in man.

Alleles↗
PubMed1989

Insulitis and diabetes are preceded by a decrease in beta cell volume in diabetes-prone BB rats.

Immunocytochemistry combined with morphometry was used to test the hypothesis that insulitis and diabetes are preceded by quantitative changes in the pancreas of diabetes-prone BB (DPBB) rats. Diabetes-resistant BB (DRBB) rats of the w-subline served as controls. In the first part of the study rats aged 15, 30, 45, and 60 days were studied. At 60 days preceding both insulitis and onset of diabetes, the DPBB rats demonstrated lower volumes of the entire pancreas, the parenchyma, the endocrine pancreas, and beta, A, and D cells. Body weights of the DPBB rats were lower than the DRBB rats from 15 days of age on. In the second part of the study, DP and DRBB rats were coreared by a foster mother to obtain weight-matched animals. Morphometric analysis at 70 days of age revealed a reduction in both the beta cell volume density and volume whereas no differences were seen in other pancreatic parameters. These results indicate that the appearance of insulitis and the later onset of insulin-dependent diabetes are preceded by a reduction in beta cell volume.

Animals↗