Biomedical subjects

R Buelow

Publications and source records attributed to R Buelow.

67 records · Page 4Linked to original sources

PubMed1994

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)

Animals↗

PubMed1993

Comparison of three different forms of HLA-DR4Dw4 proteins.

The biochemical behavior and peptide binding properties of a soluble form of the human class II DR4Dw4 molecule (PI-DR4Dw4) were compared to DR4Dw4 molecules containing the transmembrane and cytoplasmic domains that were purified both from B and transfected chinese hamster ovary cells. Recombinant and B cell-derived DR4Dw4 molecules bound monoclonal anti-DR4Dw4 antibodies with different affinities and varied in their stability in the presence of sodium dodecyl sulfate. The three forms of DR4Dw4 bound peptides with a similar apparent affinity constant, but soluble class II molecules bound up to ten times more peptide than DR4Dw4 containing a transmembrane region. Peptide binding kinetics for soluble DR4Dw4 molecules were 10-20 times faster than for the other two forms of DR4Dw4 molecules. Finally, soluble PI-DR4Dw4/peptide complexes were shown to stimulate T cell proliferation.

Amino Acid Sequence↗

PubMed1993

Islet cell autoantigen 69 kD (ICA69). Molecular cloning and characterization of a novel diabetes-associated autoantigen.

We have identified a novel 69-kD peptide autoantigen (ICA69) associated with insulin-dependent diabetes mellitus (IDDM) by screening a human islet lambda gt11 cDNA expression library with cytoplasmic islet cell antibody positive sera from relatives of IDDM patients who progressed to the overt disease. The deduced open reading frame of the ICA69 cDNA predicts a 483-amino acid protein. ICA69 shows no nucleotide or amino acid sequence relation to any known sequence in GenBank, except for two short regions of similarity with BSA. The ICA69 cDNA probe hybridizes with a 2-kb mRNA in poly(A+) RNA from human pancreas, brain, heart, thyroid, and kidney, but not with skeletal muscle, placenta, spleen, or ovary. Expression of ICA69 was also detected in beta cells and cell lines, as well as in tumoral tissue of islet cell origin. The native ICA69 molecule migrates to 69 kD in SDS-PAGE as detected with specific antibodies. Serum samples from relatives of IDDM patients specifically reacted with affinity-purified recombinant ICA69 on Western blotting. The structural gene for ICA69 was designated ICA1. A homologue in the mouse, designated Ica-1 was mapped to the proximal end of chromosome 6 (within 6 cM of the Met protooncogene). ICA69 adds a novel autoantigen to the family of identified islet target molecules, and by the manner of its identification and characterization large amounts of antigen are available for development of quantitative, convenient predictive assays for autoantibodies and analysis of the role of this molecule in diabetes autoimmunity, as well as its physiologic function.

Amino Acid Sequence↗

PubMed1992

Antigen-specific (p30) mouse CD8+ T cells are cytotoxic against Toxoplasma gondii-infected peritoneal macrophages.

The importance of CD8+ T cells in immunity against Toxoplasma gondii is now well recognized. The mechanism by which these CD8+ T cells are able to confer this immunity is not yet understood. To examine the Ag specificity of this response, immune splenocytes from mice immunized with p30, a major surface parasite Ag, were evaluated for their ability to lyse peritoneal macrophages infected with three different strains of T. gondii. Macrophages infected with either the RH or P wild-type strain tachyzoites were lysed at varying E:T ratios by nylon wool nonadherent immune splenocytes whereas macrophages infected with a p30-deficient mutant (B mutant) of the P strain were not. The gene encoding p30 for the wild type and B mutant were amplified by the polymerase chain reaction. This revealed a nonsense mutation in the B mutant such that its primary translation product is predicted to be about two-thirds the size of the wild-type p30 molecule. mAb depletion studies indicate that the cytotoxic effect of the immune splenocytes is mediated by the CD8+ T cell population. Peritoneal macrophages infected with the three different strains (RH, P wild type, B mutant) from mice genetically restricted were not lysed by the immune CD8+ effector cell population. A cloned line (C3) of p30 Ag-specific CD8+ T cells exhibited significant cytotoxicity against syngeneic peritoneal macrophages infected with either the RH or P strain tachyzoites. There was no macrophage lysis observed by these CD8+ effector cells of either syngeneic macrophages infected with the B mutant or nonsyngeneic macrophages infected with the three different tachyzoite strains.

Amino Acid Sequence↗

PubMed1992

Localization of the immunologic activity in the superantigen Staphylococcal enterotoxin B using truncated recombinant fusion proteins.

The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate potent proliferation and induce anergy in T lymphocytes expressing the appropriate T cell Ag receptor V beta gene elements. Although T cell activation by the S. aureus enterotoxins requires the presence of accessory cells bearing class II Ag of the MHC, unlike the peptide fragments of nominal Ag, they contact the external surfaces of both the class II MHC and TCR molecules. This paper investigates the immunologically active domains of S. aureus enterotoxin B (SEB) using truncated fragments of rSEB expressed as a fusion protein with protein A. The results of the experiments reported here indicate that the minimal fragment of SEB able to stimulate and induce anergy in hemagglutinin-reactive human T cells expressing V beta 3.1 gene elements is located in the amino-terminal portion of the molecule within residues 1-138. Deletion of the first 30 amino acid residues renders rSEB unable to stimulate T cells expressing V beta 3.1, whereas polyclonal T cells still respond to this molecule. This implies that the stimulation of several TCR-V beta families may be caused by the interaction with different regions of the toxin. The localization of immunologically active sites in the bacterial enterotoxins is needed to investigate both their biology and potential application as immunomodulatory agents.

Antigens, Bacterial↗

PubMed1991

Staphylococcus aureus enterotoxin mediated specific non-responsiveness of human T cells.

Exotoxins produced by certain strains of Staphylococcus aureus are able to both stimulate and induce non-responsiveness in T cells expressing specific T cell antigen receptor V beta gene elements. The exposure of human CD4+ T cells to the appropriate enterotoxin rendered them anergic to restimulation with their natural ligand, although responsiveness to exogenous IL-2 remained intact. The loss of antigen-dependent proliferation was associated with the down-regulation of the TCR complex that was paralleled by enhanced cell surface CD2 and CD25. Further analysis of the phenotypic changes revealed that membrane levels of CD28 were increased only on activation, suggesting a differential expression of this protein on activated and anergic T cells. During the induction of anergy it was observed that the synthesis of the lymphokines IL-2, IL-4 and IFN-gamma was differentially regulated. IL-4 and IFN-gamma, but not IL-2 were detected in the supernatants of overnight cultures of T cells exposed to tolerising concentrations of toxin. Transcription of IL-4, as determined by polymerase chain reaction at selected intervals, was elevated during the induction of anergy and accounted for the presence of the protein in the supernatants. In contrast, no tight coupling was observed between protein and mRNA levels for IL-2, suggesting post-translational regulation.

Antigens, CD↗

PubMedDate not supplied

Induction of carbon monoxide in the donor reduces graft immunogenicity and chronic graft deterioration.

Chronic allograft dysfunction remains the major obstacle for long-term successful transplantation. To date there is no effective treatment. Overexpression of protective genes has provided increased graft function and survival. This mechanism has been implicated in the process of graft accommodation. One of these genes that has been shown to mediate protective effects decodes the enzyme heme oxygenase-1 (HO-1), and an HO-1 downstream product, carbon monoxide (CO). Using an established model of kidney chronic allograft rejection in the rat, we investigated the impact of methylene chloride (MC), a CO donor, as a therapeutic tool to reduce chronic graft deterioration. We showed that donor and long-term recipient treatment with MC improved graft function and reduced histological signs of chronic rejection. Carbon monoxide may be a promising agent to improve graft quality and long-term graft function.

Animals↗

PubMedDate not supplied