Biomedical subjects
Publications and source records attributed to D Charron.
One hundred and four normal unrelated Chinese were typed for HLA-DPA1 and DPB1 alleles by polymerase chain reaction-restriction fragment length polymorphism. Increased frequencies of HLA-DPA1*0201 and DPB1*0501 were found in this Chinese population as compared with those detected in Caucasoids and blacks.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Skin biopsies were taken from 11 patients with morphoea, nine with acrosclerosis and 10 with diffuse systemic sclerosis and processed for immunohistochemical studies using a panel of monoclonal antibodies including antibodies to MHC class II antigens. A significantly higher percentage of HLA-DR positive dermal cells were observed in the reticular dermis in biopsies from patients with morphoea (44.1 +/- 16.2%), acrosclerosis (15.9 +/- 5.4%) and systemic sclerosis (39.5 +/- 2.3%) when compared with the controls (6.6 +/- 2%). A smaller percentage of dermal cells also expressed HLA-DP and -DQ. The degree of monocnuclear cell infiltrate in the biopsies, however, did not correlate with the percentage of HLA class II positive fibroblasts. In organ culture, the expression of the HLA class II antigens was almost totally lost after 3 days and was no longer detected on fibroblasts after 3 weeks of culture.
Analysis of intracellular localization of protein kinase C (PKC) in a lymphoblastoid B cell line shows that anti-human leucocyte antigen (HLA) class II antibodies induce an increase of cytosolic and membrane PKC activities. This phenomenon is both time- and dose-dependent. The maximal PKC activation was observed after exposure to 12.5 micrograms/ml antibody for 30 to 45 min. Unlike TPA, no translocation of the cytosolic PKC was observed at any time following exposure to the anti-HLA class II antibodies. We observed a good correlation between the [3H]phorbol dibutyrate binding activity and the enzymatic activity of PKC. Using a panel of antibodies specific for the HLA class II isotypes (DP, DQ, DR), we demonstrated that PKC activation via HLA class II molecules is not restricted to one isotype. We also showed by Western blot analysis that the increased PKC activity correlates with a quantitative increase of PKC. The increase of PKC activity induced by anti-HLA class II antibodies was completely abolished by the treatment with actinomycin D, a transcriptional inhibitor, or cycloheximide, a translational inhibitor. Finally, Northern blot analysis revealed that anti-HLA class II antibodies induce an increase of the PKC alpha and PKC beta mRNAs levels which are significant after 20 min of stimulation and rose to a maximum after 60 min. In summary, our results show that increased PKC activity induced by HLA class II antibody is regulated at the transcriptional level.
A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.
Signal transduction via HLA class-II antigens has been studied using human resting B lymphocytes and monoclonal anti-HLA class-II antibodies. An increased intracellular calcium flux, phosphatidylinositol biphosphate hydrolysis and activation of protein kinase C have all been observed following signal transduction via HLA-class-II molecules. The interaction of HLA-class-II-mediated signalling with sIg-mediated signalling has been studied using a non-mitogenic anti-sIg. This combination provides a model for T-cell-dependent antigenic stimulation. The results demonstrate that stimulation via HLA class-II antigens permits a proliferative response to an otherwise non-mitogenic anti-sIg and that this effect is not restricted to one HLA class-II isotype. These data suggest that HLA-class-II-mediated signalling is important in responses to T-cell-dependent antigens.
It has previously been demonstrated that interleukin-1 (IL-1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL-1 beta production by cultured human dermal fibroblasts. We have shown that IL-1 beta is constitutively expressed as a cell-associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) exerted a dose-dependent stimulation on the production of the cell-associated IL-1 beta, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL-1 beta production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 microgram/ml) of PGE2. In contrast, higher concentrations (0.1 and 1 micrograms/ml) of PGE2, as well as exogenous dibutyryl-cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL-1 alpha and TNF-alpha. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL-1 beta expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2-induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL-1 alpha and TNF-alpha, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL-1 beta induction by the two cytokines studied.
We have examined the activity and intracellular compartmentalization of protein kinase C (PKC) following activation of human B lymphocytes by anti-human leukocyte antigen (HLA) class II antibodies. 12-O-Tetradecanoylphorbol 13-acetate (TPA) treatment increased membrane-associated PKC (between five and nine times greater than the control value) and decreased cytosolic PKC (between 70% and 100% of the control value). In contrast, anti-class II antibodies induce an activation of PKC which results either in an increase of cytosolic activity or membrane-bound activity without redistribution of cytosolic PKC. The effect of TPA and HLA class II molecules on total PKC activity was comparable: when TPA induced an increase of total PKC activity so did HLA class II molecules and when TPA did not, HLA class II molecules did not. Measurement on SDS PAGE of histone phosphorylation confirmed the above results of PKC activity. Taken together, our results suggest that PKC might be implicated in HLA class II-induced B lymphocyte activation.
The molecular basis of eight DR4 subtypes resides in several nucleotide substitutions in the third hypervariable region of the DR beta 1 chain. The typing of DR4 subsets using the mixed lymphocyte culture (MLC) assay or allele-specific oligonucleotide hybridization is expensive, cumbersome, and requires the use of radioisotopes. We have therefore developed a rapid and safe procedure for subtyping DR4-alleles that involves selective amplification of the second exon of the DR4-DRBI gene followed by unambiguous subtype discrimination after digestion with five allele-specific endonucleases [polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)] and visualization of the polymorphic fragments with silver or ethidium bromide staining. Validity of this subtyping procedure was initially examined by the use of cell lines of known subtypes. Three groups of DR4 patients with insulin-dependent diabetes mellitus (IDDM) from Chinese, Tunisian, and Caucasian populations were subtyped and the prevalence of subtype associations with IDDM was compared.
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Interleukin 1 (IL-1) exerts biological properties on various immune and nonimmune cell types and tissues and thus may play an important role during chronic inflammatory processes. Here we have examined the IL-1 biosynthesis in adherent synovial lining cell (ASLC) cultures obtained from patients with rheumatoid arthritis (RA). We report that ASLCs in culture showed heterogeneous endogenous levels of IL-1 alpha and beta expression. Recombinant interleukin 1 (rIL-1) alpha or beta induced increases of IL-1 alpha and beta mRNA and proteins levels in ASLCs. Although IL-1 synthesis is enhanced by rIL-1 treatment, no soluble IL-1 alpha or beta could be detected by specific enzyme-linked immunosorbent assays. A pretreatment with recombinant IFN gamma (rIFN gamma) down-regulated the effect of rIL-1 on IL-1 synthesis in ASLCs. Actinomycin D suppressed the endogeneous and rIL-1-induced IL-1 mRNA expression Indomethacin, in the presence of rIL-1 alpha or beta, up-regulates the level of expression of IL-1 beta in ASLCs pretreated with rIFN gamma, but has the opposite effect in non-pretreated cells. The increase of IL-1 gene expression by rIL-1 in human ASLCs from RA patients may contribute as an amplification of the disease progress. These studies may also explain the beneficial effects of IFN gamma in experimental models of IL-1-induced bone and cartilage degradation and in patients with diseases involving IL-1.
Eighteen unrelated Chinese patients with insulin-dependent diabetes mellitus (IDDM) were analyzed for HLA Class II genes using a variety of molecular biological techniques including restriction fragment length polymorphism (RFLP), polymerase chain reaction with allele-specific oligonucleotides (PCR-ASO) and direct DNA sequencing. The high frequency of DR3/DR4 heterozygotes found in the Chinese with IDDM strengthens the importance of this combination of haplotypes in IDDM susceptibility since it is present in two genetically distant populations--Chinese and Caucasians. The frequency of DRw9, a rare allele in the Caucasian population, is much higher in the Chinese. Moreover, the DQ beta chain linkage of DRw9 was different in IDDM patients compared with control subjects. In contrast with previous results, codon 57 of the DQ beta chain was aspartic acid in DRw9 Chinese IDDM patients. Furthermore, one particular DRw9-DQw9 haplotype may be associated with IDDM susceptibility in the Chinese population.
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These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.
The spontaneous expression of HLA class-I and class-II molecules in 5 human breast carcinoma cell lines, MCF-7, T47D, ZR75-1, HSL-53, MDA-MB 231, and their modulation during IFN-gamma treatment, are reported. The expression of cell-surface determinants was examined by indirect immunofluorescence using monoclonal antibodies (MAbs) specific for HLA class-I and class-II (DR, DQ and DP) antigens. The biosynthesis and maturation of these molecules were analyzed by 2-dimensional gel electrophoresis analysis (2D-PAGE) of class I, DR alpha, beta and invariant immunoprecipitates. Transcription was analyzed by Northern blot hybridization with HLA class-I and -II cDNA-specific probes. In all cell lines, more than 80% of cells expressed HLA class-I antigens at their surface. 2D-PAGE and mRNA studies showed a variable basal level of HLA class-I biosynthesis and transcription with a constant increase after 1,000 U/ml IFN-gamma treatment. HLA class-II determinants were totally absent from the surface of MCF-7, MDA MB231, ZR75-1 and T47D but they were detected in a small subpopulation of HSL-53 cells (DR 6%, DQ 6%, DP 20%). Spontaneous biosynthesis of HLA-DR molecules in immunoprecipitates analyzed by 2D-PAGE or transcripts in Northern blot were not detected in the 5 cell lines. Treatment with 1000 U/ml IFN-gamma induced or increased the expression of HLA class-II molecules in all cell lines but DQ expression was variable. While T47D, ZR75-1 and HSL-53 increased their transcripts and antigen expression, MDA, MB231 and MCF-7 showed no DQ mRNA transcript. Biochemical analysis of the DR products revealed a classical alpha, beta and invariant (li) chain pattern, but indicated a constant glycosylation defect in the invariant chain in all cell lines, associated with weak expression of the beta chain and the presence of an extra spot of low molecular weight in the acidic part of the gel. Thus, post-transcriptional events did not appear to be totally controlled by IFN-gamma in the different cell lines. These differences in DQ expression and glycosylation process in different breast cancer cells may be important in the activation of the immune response among different individuals.